Program, Abstracts & Conference Information

19 – 24 JUNE 2016 | FAIRMONT SOUTHAMPTON | BERMUDA We wish to thank our corporate sponsors for their generous support Recovery of Biological Products XVII

FAIRMONT SOUTHAMPTON

On-Demand Affinity Purification Solutions BERMUDA

19 – 24 JUNE 2016 We wish to thank our corporate sponsors for their generous support Recovery of Biological Products XVII

FAIRMONT SOUTHAMPTON BERMUDA

19 – 24 JUNE 2016

AN INTERNATIONAL CONFERENCE Sponsored by: The American Chemical Society Division of Biochemical Technology

Conference Management Provided by: Precision Meetings & Events 301 N. Fairfax St., Suite 104 Alexandria, VA 22314 USA Table of Contents

Conference & Session Chairs ...... 3

Welcome Letter ...... 4

General Conference Information ...... 5

Activity Information ...... 7

Program Overview ...... 10

Daily Schedule ...... 12 Sunday ...... 12 Monday ...... 12 Tuesday ...... 13 Wednesday ...... 13 Thursday ...... 14 Friday ...... 14

Keynote Speakers ...... 15

Oral Abstracts ...... 21 The Origin of Impurities ...... 21 New Materials for Downstream Bioprocessing ...... 23 The Marriage of High Throughput Screening and Modeling ...... 26 Alternative Expression Systems: Strategic Impact for Biopharmaceuticals? ...... 28 Priorities in Cost and Performance Improvements ...... 30 Purification of Non-Protein Therapeutics ...... 32 Purification – from Platform to Diversity ...... 35

Process Challenges with Biosimilars ...... 38 Next Generation Unit Operations & Integrated Processes ...... 40 Biomolecular Modeling for Manufacturability ...... 43 New Developments in PAT and QbD ...... 45 Increasing Patient Access to Biopharmaceuticals ...... 47

Debates Session ...... 51

Poster Abstracts ...... 55

Participant List ...... 111

2 | Recovery of Biological Products XVII Conference Chairs Steven Cramer, Rensselaer Polytechnic Institute, United States Günter Jagschies, GE Healthcare Life Sciences, Sweden Phil Lester, Genentech, United States Keynote Speaker Moderator Charles Cooney, Massachusetts Institute of Technology, United States Oral Session Chairs Ashraf Amanullah, aTyr Pharma, United States Dorothee Ambrosius, Boehringer Ingelheim Pharma GmbH, Germany Ernst Broberg Hansen, NovoNordisk, Denmark Jon Coffman, Boehringer Ingelheim, United States John Curling, John Curling Consulting AB, Sweden Christopher Dowd, Genentech, United States Suzanne Farid, University College London, Great Britain Gisela Ferreira, MedImmune, United States Sanchayita Ghose, Biogen, United States Caryn Heldt, Michigan Tech, United States Michael Laska, Moderna Therapeutics, United States Thorsten Lemm, Roche, Germany Bruno Marques, GlaxoSmithKline, United States John Moscariello, CMC Biologics, United States Jill Myers, Fortress Biotech, United States Marcel Ottens, Delft University of Technology, Netherlands Lars Pampel, Novartis, Switzerland Michael Phillips, EMD Millipore, United States David Robbins, MedImmune, United States Jeff Salm, Pfizer, United States Peter Tessier, Rensselaer Polytechnic Institute, United States Nigel Titchener-Hooker, University College London, Great Britain Nihal Tugçu, Merck & Co . Inc ,. United States Ganesh Vedantham, Amgen, United States Suresh Vunnum, Amgen, United States Andrew Zydney, Penn State University, United States Debate Session Chairs Nigel Titchener-Hooker, University College London, Great Britain John Curling, John Curling Consulting AB, Sweden Poster Session Chairs Charles Haynes, University of British Columbia, Canada Abraham Lenhoff, University of Delaware, United States David Roush, Merck & Co ,. Inc ., United States

Recovery of Biological Products XVII | 3

The theme for RXVII is . The theme for RXVII

and the stimulating discussions . All our best, Lester Steve Cramer Guenter Jagschies Phil . raise the level of discourse to a new level are extremely grateful . We on the part of many individuals This Conference has required a huge effort program . Our poster session chairs in creating a state of the art to our oral session chairs for their help the Poster Sessions continue have done a wonderful job in assembling papers that will assure that are very grateful to our many corporate sponsors for their to be a centerpiece of the conference . We we thank the success . Finally, generosity in underwriting the expense of this program to ensure its years of planning and our two team at Precision Meeting and Events for their dedication throughout developing all the details related to this conference . beautiful venue, the activities hope you will enjoy the Conference, the to Bermuda . We Welcome . of new technologies and business decisions together the top academics, believe that this Conference is the premier forum for bringing We about how best to move our discussions and debate industrial scientists and engineers to stimulate high as evidenced by the remained field to the next level . Interest in the Recovery Conference has sponsors, and the number of the generosity of our corporate number of applications for attendance, to exceed your expectations have designed the program abstracts submitted for presentation . We provide opportunity for informal of topics, and to of scientific excellence, relevance and diversity we will have several world class poster sessions, discussion . In addition to the outstanding oral and debates which promise to new format, a series of three keynote addresses as well as an entirely Welcome to the 17th Recovery of Biological Products Conference to the 17th Recovery of Biological Products Welcome Series has been the Recovery Conference . For more than 32 years “Smart Bioprocessing” of the status, trends and forum for the presentation and discussion the premier international diagnostic, or therapeutic, with recovery of high value biological products opportunities in the including the implementation significant recent changes industrial use . Our field has experienced technologies, new disposable therapeutics, continuous bioprocessing, of biosimilars for protein to address these significant medicine . In order classes of biological products and personalized community must become truly bioprocessing, our challenges and opportunities for downstream to strategic implementation recovery science “smart” in all aspects ranging from fundamental 4 | Recovery of Biological Products XVII A Warm Welcome Welcome A Warm Chairs the Conference from

Conference Information Conference Information

General Conference Information

Welcome Hospitality Policy on Publication LOCATION: Frangipani Room, Mezzanine Level The Conference does not publish Proceedings . Participants should obtain individual permission from presenters if they On Saturday and Sunday, all participants and guests arriving wish to have copies of slides, posters, or other materials . early to the hotel that are unable to check in are welcome to relax in the hospitality suite . Enjoy a beverage and a Recording and Photography chance to put your feet up . The Conference Chairs would like to remind participants Guest Hospitality that both audio and visual recording of any session during the Conference is not permitted . Photography at oral LOCATION: Rose Room, Mezzanine Level sessions and photographic documentation of posters is On Sunday from 3:00 – 6:00 PM, registered guests of not permitted unless by express permission from the participants are welcome to gather to meet one another presenting author . and enjoy a glass of wine and light fare . Name Badges Oral Presentations Please wear your name badge during the Conference . LOCATION: Mid Ocean Amphitheater, Lower Lobby Badges will be checked upon entrance to all technical Level (same location as general session) sessions and social events . Speakers, you may upload and preview your presentations prior to your scheduled talk time . Please make every Conference Registration attempt to hand in your presentation to the conference desk Conference Registration will be open Sunday, 19 June from staff via a flash drive no later than the evening before your 12:00 PM – 6:00 PM in Poinciana Foyer on the lobby level scheduled time so that it may be loaded to the presentation of the hotel . The Conference Staff is here to assist you with computer in advance . Should you need to transfer your anything you need . Please do not hesitate to contact a staff presentation another way, please let the staff know and member if you have a question regarding the schedule, we will provide other options . Note that there will only be activities, attire or any other aspect of the program . one presentation computer . We are unable to accommodate requests for use of your own laptop . Hotel Direction Information The daily general sessions will take place in Mid Ocean Poster Presentations Amphitheater located on the lower lobby level . Poster LOCATION: Poinciana Ballroom I & II sessions will take place in Poinciana Ballroom I & II on the lobby level . Breakfasts and the Sunday Welcome Dinner will Posters should be in place no later than 8:00 AM, Monday, be on the Great Sound Lawn under a tent . Lunch and dinner 20 June and removed by 10:00 AM, Thursday, 23 June . locations vary so please check your schedule for locations . Posters remaining after 12:00 PM Thursday will be There will also be staff present to direct you . discarded .

Recovery of Biological Products XVII | 5 .fairmont .com/fpc/enroll/ to enroll . visit https://www Hotel Check-OutHotel and Payment 23 – Thursday, 19 June from Sunday, Hotel accommodations staying fee . If you are in your registration June are included the after the conference, nights prior to and/or additional of your arrival and departure dates . hotel has been notified credit card to the front desk clerk upon Please present your that the charges for those additional arrival (please note your personal folio) . Any personal nights will appear on the hotel, such as telephone, fax, expenses incurred at bar bills, use of recreational facilities, Internet access fees, scheduled conference meals), are the and food (other than attendee and/or their guest(s) and responsibility of each must be paid upon check-out . Club President’s Fairmont strongly encourage you to join the Fairmont President’s We prior to arriving in Bermuda . Joining Club if you didn’t will have the recognition club is the only way you to the fitness center complimentary wifi access, and access the hotel will not grant access to and spa . Unfortunately, the program . Please these amenities unless you sign up for . . If you have 6 | Recovery of Biological Products XVII Typical low temperatures are in the mid low temperatures are in the mid (°F)/high 20s (°C) . Typical (°C .) Please bring sunscreen and hats so (°F)/mid 20’s 70’s weather that you may safely enjoy the beautiful . technical sessions will not be interrupted Attire high Dress during the conference is casual . Typical or low 80’s 70’s temperatures in Bermuda in June are high Messages board located in Poinciana Foyer There will be a message during Desk . Please check the board near the Registration and breaks . Messages will not be personally delivered Please be sure materials . Please be in your registration be included with you to each activity to bring those tickets would like to make changes to your not pre-registered or have the opportunity to do so at the reservation, you will registration desk . Tour and Recreational and Recreational Tour Information tickets will activities, your tour pre-registered for If you have

Conference Information Conference Information

Activity Information We have scheduled activities for Monday and Wednesday. Please see below for descriptions.

Activity Voucher The Conference Chairs are providing each participant with a credit worth $75 .00 to apply towards the RXVII activities . If you have pre- registered for an activity prior to the conference, your credit card was charged the total amount minus the $75 .00 credit . If you haven’t pre- registered, please visit the Conference Registration desk in Poinciana Foyer to sign up for an activity of your choice .

The activities in or near the hotel, and the ferry boat to Hamilton (the ferry boat is complimentary to all hotel guests) are also wonderful options . Please visit the conference registration desk for assistance . We will assist with applying your $75 voucher to these activities as well .

Monday | 20 June 2016 Water-based activities depart from the Waterlot Dock at the Fairmont Southampton. The St. George tours and the Railway Bike Tour depart from the main entrance of the resort. You will be returned to the resort with time to prepare for dinner.

ST. GEORGE WALKING TOUR KAYAKING PRICE: $60.00 per person PRICE: $85.00 per person TIME: 1:15 pm – 4:45 pm TIME: 1:30 pm – 5:00 pm Guests will be guided on a walking tour of St . George by one of Kayaking in Bermuda is a great way to explore the coves, wildlife, the Towne’s most well-known residents . Stops include the World marine life, coral and more . Kayakers will depart from the Heritage Center, Ordinance Island, Perfumery, forts and boutique Waterlot Dock at the Fairmont Southampton . museums . It also includes private personalized tours of the State House . Stories told by well-known residents and stops at quaint GLASS BOTTOM & local eateries and shops . SIGHTSEEING TOUR ST. GEORGE BIKE TOUR PRICE: $40.00 per person TIME: 1:45 pm – 4:00 pm PRICE: $68.00 per person TIME: 1:15 pm – 4:45 pm Boat will depart from the Waterlot Dock at the Fairmont Southampton and cruise across the Great Sound, under Watford Covers over 10 historic sites, beaches, eateries, museums, Bridge, past King’s Point and Somerset Long Bay . Commentary cemeteries and coastline in a comfortable, scenic area loop . will be provided . Once at Daniels Head, group will go down below Includes guide, bike, helmet, locks and baskets . to view coral and the Vixen shipwreck through the glass bottom . A relaxing cruise back to the Fairmont . Cash bar available (not RAILWAY BIKE TOUR included in price .) PRICE: $85.00 per person TIME: 1:30 pm – 5:00 pm BEACH DAY Explore the history and beauty of Bermuda by joining a Enjoy the beautiful pink sandy private beach at the Fairmont guided tour of the historic Railway Trail by bicycle . Includes Southampton . Relaxing beach amenities include lockers, towels, transportation from the Fairmont Southampton to and from the showers, chaise lounge chairs, Cabana Bar & Grill & Oceanclub trail, guide, mountain bike, helmet, bottled water and a 30 minute Restaurant . The Dive Bermuda shop is also on the beach . Should beach break . you wish to rent a snorkel kit, please visit the registration desk and we will assist in the arrangement to cover the cost .

Recovery of Biological Products XVII | 7 . It . Commentary . Since this snorkel is offshore and $40.00 per person $70.00 per person 1:45 pm – 4:00 pm 2:30 pm – 5:30 pm

Take an hour cruise to the shipwreck site where you will plunge an hour cruise to the shipwreck site where you will Take shipwrecks in into crystal blue water and view the most popular will see some of the most spectacular coral reef Bermuda . You and marine life along the way swimmers only in deeper waters, it is recommended for strong Dock is subject to weather conditions . Departs from the Waterlot at the Fairmont Southampton . BEACH DAY Enjoy the beautiful pink sandy private beach at the Fairmont Southampton . Relaxing beach amenities include lockers, towels, showers, chaise lounge chairs, Cabana Bar & Grill & Oceanclub Restaurant . The Dive Bermuda shop is also on the beach . Should you wish to rent a snorkel kit, please visit the registration desk and we will assist in the arrangement to cover the cost . GLASS BOTTOM & TOUR SIGHTSEEING PRICE: TIME: at the Fairmont Dock the Waterlot Boat will depart from across the Great Sound, under Watford Southampton and cruise Long Bay Point and Somerset Bridge, past King’s below will be provided . Once at Daniels Head, group will go down shipwreck through the glass bottom . to view coral and the Vixen A relaxing cruise back to the Fairmont . Cash bar available (not included in price .) SHIPWRECK BERMUDA SNORKEL PRICE: TIME: 22 June 2016 22 | $85.00 per person $85.00 per person $68.00 per person 8 | Recovery of Biological Products XVII

$60.00 per person 1:30 pm – 5:00 pm 1:30 pm – 5:00 pm 1:15 pm – 4:45 pm 1:15 pm – 4:45 pm

Kayaking in Bermuda is a great way to explore the coves, wildlife, marine life, coral and more . Kayakers will depart from the Dock at the Fairmont Southampton . Waterlot beach break . KAYAKING PRICE: TIME: TIME: by joining a Explore the history and beauty of Bermuda by bicycle . Includes guided tour of the historic Railway Trail to and from the transportation from the Fairmont Southampton water and a 30 minute trail, guide, mountain bike, helmet, bottled Covers over 10 historic sites, beaches, eateries, museums, Covers over 10 historic sites, beaches, eateries, scenic area loop . cemeteries and coastline in a comfortable, . Includes guide, bike, helmet, locks and baskets TOUR BIKE RAILWAY PRICE: local eateries and shops . GEORGEST. BIKE TOUR PRICE: TIME: Guests will be guided on a walking tour of St . George by one of Guests will be guided . Stops include the World most well-known residents the Towne’s boutique forts and Ordinance Island, Perfumery, Heritage Center, personalized tours of the State museums . It also includes private residents and stops at quaint House . Stories told by well-known Tour depart from the main entrance of the resort. You will be returned to the resort with time to prepare for dinner. resort with time will be returned to the You entrance of the resort. depart from the main Tour GEORGEST. WALKING TOUR PRICE: TIME: Wednesday and the Railway Bike St. George tours Southampton. The Dock at the Fairmont the Waterlot activities depart from Water-based

Conference Information Conference Information Notes

Recovery of Biological Products XVII | 9 Departure Day Departure | 24-Jun-16 Friday Breakfast LAWN SOUND GREAT 7:00 Time

Jon Coffman, Jeff Salm, Gisela Ferreira, Thursday 23-Jun-16 Session 12: Increasing Patient Access to Biopharmaceuticals SESSION CHAIRS: Bruno Marques MID OCEAN AMPHITHEATER End of Scientific Program Closing Reception BEACH PRIVATE FAIRMONT comfortable shoes. Wear will be in the sand! You Closing Dinner BEACH PRIVATE FAIRMONT comfortable shoes. Wear will be in the sand! You End of Conference Breakfast LAWN SOUND GREAT Session 9: Next Generation Unit Operations & Integrated Processes SESSION CHAIRS: John Moscariello MID OCEAN AMPHITHEATER in the Global Burden of Trends Disease: Early Results from the Global Burden of Disease 2015 Study and Forecasts to 2040 Institute for Christopher Murray, Health Metrics and Evaluation MID OCEAN AMPHITHEATER Session 10: Biomolecular Modelling for Manufacturability SESSION CHAIRS: Christopher Dowd, Peter Tessier MID OCEAN AMPHITHEATER Lunch Break WINDOWS ON THE SOUND RESTAURANT Session 11: New Developments and QbD in PAT SESSION CHAIRS: Thorsten Lemm MID OCEAN AMPHITHEATER 7:00 8:00 16:20 17:55 19:00 20:00 22:00 10:35 11:20 12:55 14.15 Time ) MID OCEAN Charles Haynes, Nihal Tugçu, Nihal Tugçu, Suresh Vunnum, 22-Jun-16 Wednesday and depart for activities örg Thömmes, Biogen boxed lunch grab-n-go in Dinner POOLSIDE Poster Session 2 SESSION CHAIRS: Abraham Lenhoff, David Roush POINCIANA BALLROOM I & II End of Day Breakfast Great Sound Lawn from Session 7: Purification Platform to Diversity SESSION CHAIRS: Dorothee Ambrosius MID OCEAN AMPHITHEATER The future of Biomanufacturing J MID OCEAN AMPHITHEATER Session 8: Process Challenges with Biosimilars SESSION CHAIRS: Jill Myers MID OCEAN AMPHITHEATER Lunch break ( FOYER Activity Afternoon 7:00 8:00 Time 18:45 20:00 22:00 10:35 11:20 12:55 13:25

Nigel Titchener Nigel Titchener Suzanne Farid, David Robbins, Michael Laska, Tuesday 21-Jun-16 Bacci, Newport Room, Ocean Club, (in series) SESSION CHAIRS: John Curling Hooker, MID OCEAN AMPHITHEATER End of Day Lunch break WINDOWS ON THE SOUND RESTAURANT Session 6: Purification of non- Protein Therapeutics SESSION CHAIRS: Caryn Heldt MID OCEAN AMPHITHEATER Dine-Around (HOTEL RESTAURANTS) Waterlot Debates Session I / II / III Breakfast LAWN SOUND GREAT Session 4: Impact from Systems Alternative Expression SESSION CHAIRS: Lars Pampel MID OCEAN AMPHITHEATER to Targeting A Holistic Approach Disease with Polymeric Nanoparticles Mark Saltzman, Yale W. University MID OCEAN AMPHITHEATER Session 5: Priorities in Cost & Performance Improvements SESSION CHAIRS: Sanchayita Ghose MID OCEAN AMPHITHEATER 7:00 8:00 22:00 12:25 14:25 17:30 19:45 10:05 10:50 Time

) MID OCEAN Charles Haynes, Andrew Zydney, Andrew Zydney, Marcel Ottens, Monday 20-Jun-16 and depart for activities boxed lunch grab-n-go in SESSION CHAIRS: Abraham Lenhoff, David Roush POINCIANA BALLROOM I & II End of Day Lunch break ( FOYER Activity Afternoon Dinner POINCIANA BALLROOM III Poster Session 1 Breakfast LAWN SOUND GREAT for Session 2: New Materials Downstream Processing SESSION CHAIRS: Michael Phillips MID OCEAN AMPHITHEATER and Bioprocess Research Practice in the Coming Age of Personalized Medicine Charles Haynes, University of British Columbia MID OCEAN AMPHITHEATER Session 3: The Marriage of HTS & Process Modelling SESSION CHAIRS: Ernst Broberg Hansen MID OCEAN AMPHITHEATER 7:00 8:00 20:00 22:00 18:45 11:20 12:55 13:25 10:35 Time

Ganesh Arrival day Arrival | 19-Jun-16

Sunday Sunday 10 | Recovery of Biological Products XVII End of Day Repertoire in Humans George Georgiou, University of Texas Opening Dinner SOUND LAWN GREAT Vedantham, Ashraf Amanullah Vedantham, MID OCEAN AMPHITHEATER Reception Welcome SOUND LAWN GREAT Blood? in Your What’s The Circulating Antibody OPENING REMARKS Günter Jagschies, Steve Cramer, Phil Lester MID OCEAN AMPHITHEATER Session 1: The Origin of Impurities SESSION CHAIRS: Guest Hospitality ROSE ROOM Conference Opening Poster Set-Up POINCIANA BALLROOM I & II Registration Opens POINCIANA FOYER FRANGIPANI ROOM FRANGIPANI Welcome Hospitality Hospitality Welcome

9:00 Time 22:00 20:00 18:30 19:15 16:20 15:00 16:00 12:00 Program Overview Program

Schedule Sunday | Arrival day Monday Tuesday Wednesday Thursday Friday | Departure Day Time 19-Jun-16 Time 20-Jun-16 Time 21-Jun-16 Time 22-Jun-16 Time 23-Jun-16 Time 24-Jun-16 7:00 Breakfast 7:00 Breakfast 7:00 Breakfast 7:00 Breakfast 7:00 Breakfast GREAT SOUND LAWN GREAT SOUND LAWN Great Sound Lawn GREAT SOUND LAWN GREAT SOUND LAWN Schedule 9:00 Welcome Hospitality 8:00 Session 2: New Materials for 8:00 Session 4: Impact from 8:00 Session 7: Purification from 8:00 Session 9: Next Generation FRANGIPANI ROOM Downstream Processing Alternative Expression Systems Platform to Diversity Unit Operations & Integrated SESSION CHAIRS: Andrew Zydney, SESSION CHAIRS: Suzanne Farid, SESSION CHAIRS: Nihal Tugçu, Processes Michael Phillips Lars Pampel Dorothee Ambrosius SESSION CHAIRS: Jeff Salm, MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER John Moscariello MID OCEAN AMPHITHEATER 10:35 Bioprocess Research and 10:05 A Holistic Approach to Targeting 10:35 The future of Biomanufacturing 10:35 Trends in the Global Burden of Practice in the Coming Age of Disease with Polymeric Jörg Thömmes, Biogen Disease: Early Results from the Personalized Medicine Nanoparticles MID OCEAN AMPHITHEATER Global Burden of Disease 2015 Charles Haynes, University of W. Mark Saltzman, Yale Study and Forecasts to 2040 British Columbia University Christopher Murray, Institute for MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER Health Metrics and Evaluation MID OCEAN AMPHITHEATER 12:00 Registration Opens 11:20 Session 3: The Marriage of HTS 10:50 Session 5: Priorities in Cost & 11:20 Session 8: Process Challenges 11:20 Session 10: Biomolecular POINCIANA FOYER & Process Modelling Performance Improvements with Biosimilars Modelling for Manufacturability Poster Set-Up SESSION CHAIRS: Marcel Ottens, SESSION CHAIRS: David Robbins, SESSION CHAIRS: Suresh Vunnum, SESSION CHAIRS: POINCIANA BALLROOM I & II Ernst Broberg Hansen Sanchayita Ghose Jill Myers Christopher Dowd, Peter Tessier MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER 12:55 Lunch break 12:25 Lunch break 12:55 Lunch break 12:55 Lunch Break (boxed lunch grab-n-go in MID OCEAN WINDOWS ON THE SOUND (boxed lunch grab-n-go in MID OCEAN WINDOWS ON THE SOUND FOYER and depart for activities) RESTAURANT FOYER and depart for activities) RESTAURANT 15:00 Guest Hospitality 13:25 Activity Afternoon 14:25 Session 6: Purification of non- 13:25 Activity Afternoon 14.15 Session 11: New Developments ROSE ROOM Protein Therapeutics in PAT and QbD SESSION CHAIRS: Michael Laska, SESSION CHAIRS: Gisela Ferreira, Caryn Heldt Thorsten Lemm MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER 16:00 Conference Opening OPENING REMARKS Steve Cramer, Günter Jagschies, Phil Lester MID OCEAN AMPHITHEATER 16:20 Session 1: The Origin 16:20 Session 12: Increasing Patient of Impurities Access to Biopharmaceuticals SESSION CHAIRS: Ganesh SESSION CHAIRS: Jon Coffman, Vedantham, Ashraf Amanullah Bruno Marques MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER 18:30 Welcome Reception 18:45 Dinner 17:30 Dine-Around 18:45 Dinner 17:55 End of Scientific Program GREAT SOUND LAWN POINCIANA BALLROOM III (HOTEL RESTAURANTS) POOLSIDE 19:15 What’s in Your Blood? Bacci, Newport Room, Ocean Club, 19:00 Closing Reception Waterlot The Circulating Antibody FAIRMONT PRIVATE BEACH Repertoire in Humans Wear comfortable shoes. George Georgiou, University of Texas You will be in the sand! 20:00 Opening Dinner 20:00 Poster Session 1 19:45 Debates Session I / II / III 20:00 Poster Session 2 20:00 Closing Dinner GREAT SOUND LAWN SESSION CHAIRS: Charles Haynes, (in series) SESSION CHAIRS: Charles Haynes, FAIRMONT PRIVATE BEACH Abraham Lenhoff, David Roush SESSION CHAIRS: Nigel Titchener Abraham Lenhoff, David Roush Wear comfortable shoes. POINCIANA BALLROOM I & II Hooker, John Curling POINCIANA BALLROOM I & II You will be in the sand! MID OCEAN AMPHITHEATER 22:00 End of Day 22:00 End of Day 22:00 End of Day 22:00 End of Day 22:00 End of Conference

Recovery of Biological Products XVII | 11

12:55 Dinner 7:00 – 8:00 Breakfast 8:00 – 10:05 11:20 – 12:55 13:00 – 18:30 18:45 – 19:45 20:00 – 22:00 10:05 – 10:35 10:35 – 11:20 Refreshments POINCIANA FOYER MID OCEAN FOYER Charles Haynes, GREAT SOUND LAWN GREAT New Materials for New Materials for Activities (optional) POINCIANA BALLROOM III KEYNOTE ADDRESS MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER POINCIANA BALLROOM I & II Downstream Processing Boxed Lunch (Grab-N-Go) Poster Session 1 & Dessert University of British Columbia Monday | 20 June 2016 20 June | Monday The Marriage of HTS & Process Modelling The Marriage of HTS & Process

12:00 15:00 ROSE ROOM 9:00 – 17:00 20:00 – 22:00 18:30 – 19:15 19:15 – 20:00 16:00 – 16:20 16:20 – 18:00 Poster Set-Up POINCIANA FOYER FRANGIPANI ROOM FRANGIPANI GREAT SOUND LAWN GREAT GREAT SOUND LAWN GREAT SOUND LAWN GREAT Welcome Dinner Welcome Guest Hospitality Opening Remarks Registration Opens KEYNOTE ADDRESS MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER The Origin of Impurities Poinciana Ballroom I & II Cocktails & Entertainment Hospitality Suite for early arrivals Hospitality Suite We invite participant’s guests to enjoy guests to invite participant’s We George Georgiou, University of Texas 12 | Recovery of Biological Products XVII Sunday | 19 June 2016 19 | Sunday a glass of wine and get to know one another a glass of wine and get to know one

Schedule Tuesday | 21 June 2016 Wednesday | 22 June 2016

7:00 – 8:00 7:00 – 8:00 Breakfast Breakfast Schedule GREAT SOUND LAWN GREAT SOUND LAWN

8:00 – 9:35 8:00 – 10:05 Impact from Alternative Purification from Platform Expression Systems to Diversity MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER

9:35 – 10:05 10:05 – 10:35 Refreshments Refreshments POINCIANA FOYER POINCIANA FOYER

10:05 – 10:50 10:35 – 11:20 KEYNOTE ADDRESS KEYNOTE ADDRESS W. Mark Saltzman, Yale University Jörg Thömmes, Biogen MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER

10:50 – 12:25 11:20 – 12:55 Priorities in Cost & Process Challenges Performance Improvements with Biosimilars MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER

12:25 – 14:25 12:55 Luncheon Boxed Lunch (Grab-N-Go) WINDOWS BY THE SOUND RESTAURANT MID OCEAN FOYER

14:25 – 16:30 13:00 – 18:30 Purification of non-Protein Therapeutics Activities (optional) MID OCEAN AMPHITHEATER 18:45 – 19:45 17:30 – 19:30 Dinner Dine-Around POOLSIDE HOTEL RESTAURANTS: BACCI, NEWPORT ROOM, OCEAN CLUB, WATERLOT 20:00 – 22:00 Poster Session 1 & Dessert 19:45 – 22:00 POINCIANA BALLROOM I & II Debate Sessions MID OCEAN AMPHITHEATER

Recovery of Biological Products XVII | 13 Breakfast 7:00 – 10:00 GREAT SOUND LAWN GREAT Friday | 24 June 2016 | 24 Friday Notes

7:00 – 8:00 8:00 – 10:05 Breakfast Luncheon 16:20 – 17:55 19:00 – 22:00 14:15 – 15:50 15:50 – 16:20 10:35 – 11:20 11:20 – 12:55 12:55 – 14:00 10:05 – 10:35 Refreshments Refreshments POINCIANA FOYER POINCIANA FOYER Closing Banquet GREAT SOUND LAWN GREAT Keynote Address Biopharmaceuticals FAIRMONT PRIVATE BEACH PRIVATE FAIRMONT MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER MID OCEAN AMPHITHEATER & Integrated Processes for those that prefer not to walk) (bus transportation will be provided Increasing Patient Access to Health Metrics and Evaluation WINDOWS BY THE SOUND RESTAURANT Christopher Murray, Institute for Christopher Murray, Next Generation Unit Operations Next Generation New Developments in PAT and QbD New Developments in PAT 14 | Recovery of Biological Products XVII Thursday | 23 June 2016 | 23 June Thursday Biomolecular Modelling for Manufacturability Biomolecular Modelling for Manufacturability

Schedule Keynote Speakers

George Georgiou University of Texas Sunday, 19 June, 19:15 – 20:00 GREAT SOUND LAWN Keynote Speakers

Charles Haynes, Ph.D., P.Eng., FCIC, FRSC University of British Columbia Monday, 20 June, 10:35 – 11:20 MID OCEAN AMPHITHEATER

W. Mark Saltzman Yale University Tuesday, June 21, 10:05 – 10:50 MID OCEAN AMPHITHEATER

Jörg Thömmes Biogen Wednesday, June 22, 10:35 – 11:20 MID OCEAN AMPHITHEATER

Christopher Murray Institute for Health Metrics and Evaluation Thursday, June 23, 10:35 – 11:20 MID OCEAN AMPHITHEATER

Recovery of Biological Products XVII | 15

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different antibodies, yet only a relatively small set (which we different antibodies, yet only a relatively 12 ABSTRACT BIOGRAPHY George Georgiou What’s In Your Blood? What’s In Your George Georgiou George Remarkably, of an individual at any time . Remarkably, antibody proteins) are present in the blood 4 The Circulating Antibody Repertoire in Humans Antibody Repertoire The Circulating How he antibody repertoire informs on strategies to improve the seasonal influenza vaccine . seasonal influenza How he antibody repertoire informs on strategies to improve the disease have overcome Identification of therapeutic antibodies directly from patients that or inflammation How is antibody immunity shaped by age and by persistent infection 16 | Recovery of Biological Products XVII 1 . 2 . 3 . We have developed a suite of proteomic, microfluidic, protein engineering and informatics technologies that has of proteomic, microfluidic, protein engineering have developed a suite We of antibodies in biological fluids and the delineation enabled the deconvolution of the identities and relative amounts B cell immunological mechanisms . This unique of the relationships between antibody production and the relevant issues related to human health and the research toolset has enabled us to address a plethora of fundamental that will be discussed in this talk will include: development of therapeutics and vaccines . Topics estimate is of the order of 2x10 to determine whether an individual has an more than 100 years since the discovery of antibodies, it is only possible HIV or strep tests) but not the number of distinct antibodies, their amino acid antibody response to a pathogen (say, antibodies produced in that person . sequences, relative amounts or biological functions of the pathogen-specific is very important for therapeutic and Understanding the nature of antibodies elicited by disease or vaccination prophylactic purposes . Antibodies are present in blood at high concentrations (about 10 mg/ml) and are critical for defense against pathogens . concentrations (about 10 Antibodies are present in blood at high nearly all approved vaccines is the production of to infection elicited by the main mechanism of protection Notably, neutralize the pathogen . circulating antibodies that bind to, and well over 10 The human immune system can generate He founded GGMJD (1999; acquired by Maxygen in 2000), Aeglea Biotherapeutics (2013-Present; NASDAQ: Maxygen in 2000), companies . He founded GGMJD (1999; acquired by a Director and Chairman of the SAB for both as AGLE) and Kyn Therapeutics Inc . (2015-Present) and currently serves Nature Biotechnology Researchers by of the top 20 Translational companies . In 2013 Georgiou was selected as one Professor Georgiou was elected to the National Academy of Engineering (2005), National Academy of Medicine (2011) (2005), National Academy Engineering was elected to the National Academy of Professor Georgiou and of the American Institute for Biological a Fellow of Arts and Sciences (2016) . He is also and the American Academy for the Advancement and the American Association the American Academy of Microbiology Medical Engineers (AIMBE), of 87 issued or pending US patents, and co-inventor >240 research publications of Science (AAAS) . He is the author of have been licensed to 27 pharma & biotech distinct technology suites) more than 65% of which (comprising 24 from the University of He received his B .Sc . from the University Austin . a Professor at the University of Texas, George Georgiou is antibody repertoire on understanding the serological .D . from Cornell . His research is focused U .K . and Ph Manchester, and on the discovery and preclinical development disease and TCR repertoires) in human health and (as well as the BCR of metabolism . errors therapeutics for cancer and for inborn of enzyme and antibody

Keynote Speakers Charles Haynes, Ph.D., P.Eng., FCIC, FRSC

BIOGRAPHY Dr . Charles Haynes is a Professor and holds the Canada Research Chair in Interfacial Biotechnology within the Michael Smith Laboratories and the Department of Chemical and Biological Engineering of the University of British Columbia . The central objective of Dr . Haynes’ research program is to improve our understanding of the interfacial and binding- recognition behavior of biomolecules and cellular systems, and to use this fundamental knowledge to invent new technologies and instruments for isolating and analyzing biological analytes from complex samples .

From that work, he has pioneered and licensed a number of technologies to efficiently localize, purify, or analyze biomolecules that have achieved widespread use in the biotechnology and healthcare industries . Companies created

based on those inventions include EIChrom Inc ., CBD Technologies Inc ., and, more recently, AbCellera Inc ., which Keynote Speakers uses novel microfluidic enabling technologies developed in the Haynes lab to discover and characterize monoclonal antibodies, and 3Ci Diagnostics Inc ., a new personalized medicine company that markets digital PCR-based tests for genetic variations prognostic or theranostic of life-threatening diseases, including a range of cancers . Dr . Haynes is a Fellow of the Royal Society, and recipient of several awards, including the Winnaker Technology Transfer Award, the Les Shemilt Award, and the BC Gold Medal in Innovation .

ABSTRACT Bioprocess Research and Practice in the Coming Age of Personalized Medicine Charles (Chip) Haynes Michael Smith Laboratories University of British Columbia

The potential for predictive or precision medicine to improve patient care and speed development of new biologic treatments has only begun to be realized . Fueled by major initiatives such as the Precision Medicine Initiative, ‘omics science is now rapidly discovering genes harboring polymorphic or somatic variations that contribute to human disease, linking genetic variations to patient response to dozens of treatments, and identifying the molecular causes of major diseases, particularly certain forms of cancer . This is allowing development of powerful diagnostic tests of genetic and gene-product variations that are prognostic or theranostic of patient response to targeted therapy . The opportunities provided by personalized medicine are tremendous to improve health and health outcomes, but the challenges to its effective implementation are also tremendous . Adoption of personalized medicine is challenged by the lack of a comprehensive strategy to deliver its benefits to patients . That strategy must include ways to rapidly and cost-effectively develop and manufacture new targeted therapies that actually improve treatment efficacy without complications, and to optimize treatment by steering patients to the right drug at the right dose at the right time . Initiatives in our and other laboratories aimed at addressing needs within this strategy are under way, and I will describe those needs and their importance to overcoming current challenges to realizing the personalized medicine concept, and then present engineering-based technologies in development that might serve to help realize the promise of personalized medicine to improve care while keeping costs in check . These include creation of clinically suitable technologies for multiplexed multi-loci testing for genetic variant signatures for common, complex diseases, assembly of an ensemble of existing and new classes of biologic drugs and new ways to more rapidly and effectively screen them to identify promising leads, and creation of scaled-down platforms that greatly accelerate bioprocess design, including clonal selection and testing, culture protocols, and challenging elements of the downstream process .

Recovery of Biological Products XVII | 17

2004),

He has served as a . He has served as a

Saltzman was appointed . Saltzman was appointed Tissue Engineering ( Engineering Tissue At Yale, Dr . At Yale, (Second Edition, 2015), (Second Edition, 2015), ABSTRACT BIOGRAPHY W. Mark Saltzman Mark W. His research has impacted the fields of drug delivery, biomaterials, the fields of drug delivery, . His research has impacted Department of Biomedical Engineering Biomedical Engineering Yale University, New Haven, CT 06511, USA University, Yale W. Mark Saltzman Mark W. with Polymeric Nanoparticles with Polymeric . A Holistic Approach to Targeting Disease to Targeting A Holistic Approach Paradoxically, as the field has matured, the notion of ‘targeting’ has been minimized to the as . Paradoxically, (2001) . Drug Delivery 18 | Recovery of Biological Products XVII Mark Saltzman is an engineer and educator . Mark Saltzman is an Saltzman has been recognized widely for his excellence in research and teaching . He has received the Camille his excellence in research . Saltzman has been recognized widely for Here, I present examples of alternate approaches, in which where this kind of targeting is difficult to achieve . Here, I present delivery, treatment of brain tumors, targeted correction of nanoparticle design is used to achieve a positive outcome in the DNA damage in skin, and antibody-mediated long-lasting, safe prevention of UV-mediated a single-gene disorder, nanoparticles, but with unique designs that targeting of inflamed endothelium . These examples all involve targeted guide their biological activity The primary goal of nanomedicine—using materials such as polymer nanoparticles—is to improve clinical outcomes . The primary goal of nanomedicine—using materials such as polymer non-productive distribution while improving diagnostic For example, targeted nanoparticles can be engineered to reduce and therapeutic efficacy applications have focused on intravenous concept of increasing affinity of a nanoparticle for its target, and most of the Biomedical Engineering Society (2010); a Member of the Connecticut Academy of Science & Engineering (2012); a Member of the Connecticut of the Biomedical Engineering Society (2010); of the US National Academy of Medicine (2014) . (2013), and a Member a Fellow of the National Academy of Inventors Dr Outstanding (1990); the Allan C . Davis Medal as Maryland’s Award and Henry Dreyfus Foundation Teacher-Scholar Progress in (1996); the Professional Award Investigator Young Engineer (1995); the Controlled Release Society Young from Iowa State University (2013) Awards Citation in Engineering Engineering (2000) and Professional Achievement Medical and Biological Engineering (1997); a Fellow Institute for (2000) . He has been elected a Fellow of the American in chemical engineering (1981), He with a B .S . in chemical engineering University with distinction from Iowa State Mark Saltzman graduated engineering (1987) from MIT . in medical (1984) and a Ph .D received an S .M . in chemical engineering University and Yale Cornell University, Hopkins University, faculty member at Johns served Engineering in July of 2002, and Professor of Chemical and Biomedical as the Goizueta Foundation Department of Biomedical Engineering in 2003-2015 . as the founding chair of Yale’s W patents . in more than 300 research papers and this work is described and tissue engineering: nanobiotechnology, of three textbooks: He is also the author and

Keynote Speakers Jörg Thömmes

BIOGRAPHY Dr . Jorg Thommes is currently the Senior Vice President of Technical Development at Biogen . This function is responsible for development of Biogen’s manufacturing processes at lab and pilot scale for all therapeutic modalities ranging from proteins to oligonucleotides and small molecule pharmaceuticals . Previous positions include Senior Vice President of Operations Technology and Innovation, Vice President of Global Engineering and Facilities, and Vice President of Biopharmaceutical Development . Jorg graduated from University of Bonn, Germany with a degree in Chemistry (Diplom-Chemiker) and holds a doctorate in natural sciences from the same university . Post doctorate, Jorg spent nine years at the Institute of Enzyme Technology at University of Dusseldorf, Germany and received an advanced research and teaching degree (Habilitation) in Biochemical Engineering . Jorg is currently the chairman of the board

of the Recovery of Biological Products Conference series . Keynote Speakers

ABSTRACT Manufacturing of Biological Products – Lessons from the Past and Visions for the Future Jorg Thommes

From the point of view of a process development scientist, the development of biopharmaceutical manufacturing over the past four decades is nothing short of amazing . From the time when manufacturing of gram quantities of recombinant proteins required very large volume bioreactors and purification was considered an art to today’s reality with more than 10 metric tons of annual therapeutic protein manufacturing, our industry has experienced a more than 1000 fold increase in productivity . However, if one were to analyze this history from an outsider’s point of view, then the conclusion could be that nothing has changed over four decades: the fundamental technology is still based on large stirred tank reactors followed by chromatography . From afar it may appear that innovation has been remarkably absent, which an outsider could explain by the absence of any market pressures .

Both these observations are true in the context of the observer’s environment; the question is, what can we learn from both interpretations of our industry’s history and can we make some predictions of what the future holds?

Significant debate has occurred in literature and at conferences on whether small volume flexible manufacturing will replace traditional fixed manufacturing infrastructure . Many have postulated that disruptive innovation is a must in biomanufacturing, while others have found that existing infrastructure and technologies can be refined to deliver on the promise of biotherapeutics . Continuous protein manufacturing, while around for decades without receiving much attention, has come into focus both in industry and at regulatory agencies; it is discussed oftentimes as the next generation of manufacturing processes . What role can these different approaches play in the future of biotherapeutics?

This presentation will try to analyze how our industry reached the present state of biotherapeutics manufacturing and attempt to draw some conclusions on what the next steps for biomanufacturing might be . We will explore how different therapeutic fields may pose different challenges to manufacturing and which manufacturing solutions could meet them . From localized small volume manufacturing for rare diseases where accessibility is the driver, to very large volume manufacturing for millions of patients with affordability drivers, to biosimilars manufacturing with sales price in mind, solutions will vary from flexible factories to operational excellence of traditional facilities and approaches employed in the manufacturing of industrial enzymes . We will also try to expand the discussion from protein manufacturing to novel (or not so novel) modalities .

Biomanufacturing translates the promise of science into products for patients . The full breadth of therapeutic opportunities in our industry will most likely not be met by a one size fits all future manufacturing approach . Solutions will have to be developed in the context of the therapeutic area, its market size, and economic pressures . This presentation will attempt to span the breath of challenges and potential solutions for our industry .

Recovery of Biological Products XVII | 19

Murray is a founder of . Murray is a founder

. Alan Lopez of the GBD . Gro Harlem Brundtland was . Countries around the world continue to undergo an ABSTRACT BIOGRAPHY Murray worked at the World Health Organization (WHO), where he . Murray worked at the World Murray has authored or edited 14 books, many book chapters, and more than . Murray has authored or edited 14 books, Study and Forecasts to 2040 Study and Forecasts Christopher J L Murray J Christopher Trends in the Global Burden of Disease: Burden in the Global Trends Murray focused on tuberculosis control and the development with Dr and . Murray focused on tuberculosis control Early Results from the Global Burden of Disease 2015 of Disease 2015 the Global Burden from Results Early 20 | Recovery of Biological Products XVII future disease burden scenarios that will create a better understanding of population and country disease profiles over of population future disease burden scenarios that will create a better understanding the ability to propose what-if scenarios the next 25 years . Once baseline scenarios are constructed, users will have a new drug or analyze the impact of introducing to model future trends . For example, health system managers could for this work have clear implications for technology on the future burden of disease of a population . The possibilities drug, vaccine, and technology development . The GBD is a valuable resource for understanding trends and patterns of disease burden and allows users of disease . The GBD is a valuable resource for understanding trends . Consequently policy officials, government leaders, to understand and visualize the disease profile of a given population need for resources in order to target and reduce the and other public health advocates are better able to articulate the causes of death and disability in that population . will develop University of Washington (IHME) at the New research from the Institute for Health Metrics and Evaluation The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) is the largest and most comprehensive effort to and Risk Factors Study (GBD) is The Global Burden of Diseases, Injuries, of the GBD study show major progress in results date to measure epidemiological levels and trends worldwide . The causes of death and disability that could be reduced combating infectious disease, but also significant gaps in tackling with increased access to drugs, vaccines, and primary health care as the major contributors to the burden diseases epidemiological transition from communicable to non-communicable He holds Bachelor of Arts and Science degrees from . He holds Bachelor of Arts and Science publications 250 journal articles in internationally peer-reviewed from and a medical degree Oxford University, from a DPhil in International Health Economics Harvard University, Harvard Medical School . In his earlier work, Dr methods and applications . From 1998 to 2003, Dr Policy Cluster while Dr Evidence and Information for served as the Executive Director of the for Global Health and the Harvard Center for Initiative . He went on to become Director of the Harvard Director-General Professor of Public Policy at the Harvard School well as the Richard Saltonstall Population and Development Studies, as of Public Health, from 2003 until 2007 . Dr health and medical care systems, and assess the cost effectiveness of health technologies . Dr care systems, and assess the cost effectiveness health and medical of health loss to quantify the comparative magnitude effort Disease (GBD) approach, a systematic the Global Burden of of almost over time . He led the collaborative and risk factors by age, sex, and geography due to diseases, injuries, Factors Study 2010 of Diseases, Injuries, and Risk Burden 50 countries that produced the Global 500 researchers from (GBD 2010) . Murray, MD, DPhil, is a Professor of Global Health at the University of Washington and Institute Director and Institute at the University of Washington Global Health MD, DPhil, is a Professor of Christopher J .L . Murray, health for everyone has focused on improving career Metrics and Evaluation (IHME) whose of the Institute for Health of a his work has led to the development economist, health evidence . A physician and health worldwide by improving public analyze the performance of health measurement, and empirical studies to strengthen range of new methods

Keynote Speakers Oral Abstracts

Session 1 | The Origin formation is observed in many manufacturing process steps from cell culture, purification, and formulation of Impurities unit operations as well as during process holds . This presentation will incorporate understanding of aggregate SESSION CHAIRS: formation to illustrate and explain how manufacturing conditions turn into the source of the aggregate formation . Ganesh Vedantham, Amgen The work was built on both well-established principles and Ashraf Amanullah, ATyr Pharma experiences in the field . These conditions include 1) cell culture medium components and feed strategy, 2) interfacial stresses such as foaming and mixing condition, 3) sensitivity 1. The Sources of mAb Aggregate to pH, temperature and duration during the low pH viral and Process Control Strategy inactivation step; 4) elution pH for Protein A and CEX chromatography; 5) sensitivity to photostress under process *Min Zhu, MedImmune, United States relevant conditions; and 6) duration and temperature for Guillermo Miro-Quesada, MedImmune, USA in-process hold . Several analytical techniques, such as Oral Abstracts Sanjeev Ahuja, MedImmune, USA mass spectrometry (MS), differential scanning calorimetry Tizita Mammo, MedImmune, USA (DSC) and circular dichroism (CD) are utilized to connect Adrian Man, MedImmune, USA process observation with aggregate formation mechanism . Karim Nakuchima, MedImmune, USA Each mAb sub-class shows different level of the sensitivity Allen Bosley, MedImmune, USA to specific manufacturing conditions and aggregation Kara Shafer, MedImmune, USA mechanisms . In addition, through empirical and molecular Erica Hackner, MedImmune, USA modeling analyses, the inputs on the design of the molecule David Robbins, MedImmune, USA to avoid inherent aggregation will be discussed . The artificial aggregation phenomena due to sample handling, Protein aggregate is a critical quality attribute (CQA) that specifically for cell culture fluid samples will be covered . needs to be adequately controlled during manufacturing In the end, a control strategy as a holistic and integrated of monoclonal antibody (mAb) product . The types of approach is proposed based on the in-depth understanding the aggregate can be covalently formed oligomers (e .g . of the sources of the aggregate formation . The strategy disulfide-linked), or reversible or irreversible non-covalent begins with molecular design, balancing an appropriate dimer, trimer and eventually oligomers, typically mediated level of the risk at each unit operation; eventually by hydrophobic interaction or mAb denaturing . Aggregate demonstrates the aggregate formation is in control .

Recovery of Biological Products XVII | 21

. Key feature of bispecific Penzberg, Germany Unassembled heavy chains are actively chains for IgG . Unassembled heavy chains are actively (ER) . Buchner et retained in the endoplasmatic reticulum chain is al . showed, that the CH1 domain of the heavy it apart from intrinsically disordered in vitro, which sets other antibody domains . It folds only upon interaction with the light chain CL domain . Therefore, in natural IgGs to be secreted into lacking a light chain cannot be expected . In contrast, the medium during the fermentation process to show a bispecific CrossMAbs have been observed assembled “¾ different behavior and the incompletely antibody” fragments can be detected in the fermentation supernatant . In addition the correct folding of the CH1 3. Isolation and Characterization and Characterization 3. Isolation Assembled of Incompletely AntibodyBispecific Variants Roche Innovation Center *Thomas von Hirschheydt, bispecific antibodies The formation of unsymmetrical engineering to establish the requires specific protein these protein chains . However, right assembly of the often come along with new types novel antibody formats caused by the protein of product related byproducts not be addressed by generic engineering which may represent a . Bispecific CrossMAbs purification protocols new member of the IgG family Fab region . CrossMAbs is a domain crossover in the behave While the fully assembled bispecific CrossMAbs side product like standard IgGs, new types of possible assembled were found for CH1-CL-crossing: incompletely light chain, (3/4 CrossMAb fragments lacking the crossed (heavy chain dimers) . antibodies) or lacking both light chains prerequisite This observation is surprising because a into a defined for antibody secretion is their assembly heavy and two light quaternary structure, composed of two .

. The strain was capable of post- 22 | Recovery of Biological Products XVII United Kingdom *Darren N. Nesbeth, University College London, *Darren N. Nesbeth, John Ward, UCL, UK John Ward, Desmond, Schofield, UK Desmond, Schofield, UK Ernestas Sirka, UCL, UCL, UK Eli Keshavarz-Moore, We conclude that exploitation of different translocation We pathways can improve industrial performance of engineered E . coli production strains that feature recombinant proteins intended to effect impurity removal in addition to recombinant protein products . 5L scale fermentation experiments, SRP-routed nuclease 5L scale fermentation experiments, SRP-routed leakage relative co-expression coincided with reduced Fab’ to the SEC-routed nuclease strain . Mathematical modelling the rate of of periplasm filling and overload predicted the onset intracellular accumulation of Fab’ and predicts occupancy of Fab’ leakage from cells at 6% periplasm We sought to improve to the surrounding growth medium . We using a different Fab’ retention in the engineered strain by expression . We translocation route for periplasmic nuclease signal recognition directed translocation of nuclease by the influenced particle (SRP) route and tested if route choice Fab’ fragment . In periplasmic leakage of the recombinant in which exogenous Fab’ and staphylococcal nuclease are in which exogenous Fab’ and staphylococcal via translocation both expressed in the periplasmic space by the SEC pathway with a resultant homogenisation clearance of genomic DNA, in reduction in feedstock viscosity and improvement Fab’ leakage clarification performance, but also increased We previously used cell-engineering approaches to construct to construct approaches used cell-engineering previously We production strain . coli) Fab’ fragment an Escherichia coli (E DNA Impurities from Fab’ Fragment Fragment Fab’ DNA from Impurities Streams Process 2. Optimising Cell Design Steps for Design Steps Cell 2. Optimising E. of Removal Efficient Host Cell coli

Oral Abstracts domain of the natural heavy chain can be supported by the but also resulted in substantial increases in process engineered heavy chain to form a stable heavy chain dimer and product related impurities . Due to the diverse without any light chains . Independent chromatographic physicochemical properties of these impurities, there is methods for depletion of “¾ antibodies” and the targeted an ever increasing need for new technologies that offer expression and isolation of heavy chain dimers will be increased productivity and improved economics without presented as well as biochemical characterization and sacrificing process robustness required to meet final drug structure elucidation by transmission electron microscopy . substance specifications . This work examined the use of new generation hybrid filters modified with a high binding capacity of quaternary amine (Emphaze™ AEX) and novel salt tolerant biomimetic (Emphaze™ ST-AEX) ligands for Session 2 | New Materials host-cell protein (HCP), residual DNA, soluble aggregate removal and viral clearance . It combines three technologies: for Downstream anion-exchange (AEX) functional polymers, fine-fiber Bioprocessing nonwoven materials, and multizone membranes to deliver an all-synthetic clarifying product containing both an AEX ligand and a bioburden-reduction membrane . Binding SESSION CHAIRS: isotherms using bovine serum albumin (BSA) were obtained Andrew Zydney, Penn State University to develop adsorption isotherm models as a function of ionic Michael Phillips, EMD Millipore strength for hybrid filters and compared against traditional positively charged depth filters . The ionic capacity of these depth filters was measured and correlated with their 1. Evaluation of Next Generation ability to remove impurities for multiple molecules . The Hybrid Filters for Simultaneous AEX capacity of hybrid filters significantly exceeded that Oral Abstracts Clarification and Purification of conventional depth filters, providing substantially higher reduction of soluble anionic impurities including DNA and of Biologics anionic HCPs . Implementing Emphaze™ AEX hybrid filter *NRIPEN SINGH, Bristol Myers Squibb, United States with quaternary amine functionality at the clarification stage significantly enhanced the performance of Protein Michael Peck, Bristol Myers Squibb, United States A chromatography capture, easing the challenges faced Abhiram Arunkumar, Bristol Myers Squibb, United States by downstream polishing steps . Furthermore, Emphaze™ Michael Borys, Bristol Myers Squibb, United States ST-AEX filter with guanidinium functionality demonstrated Zhengjian Li, Bristol Myers Squibb, United States improved process related impurity removal and viral Alexei Voloshin, 3M, United States clearance at both low and high conductivity post Protein Jonathan Hester, 3M, United States A chromatography . The consequences of this radically enhanced process performance are far reaching as it Recent progress in mammalian cell culture process permits restructuring and simplification of the downstream has resulted in significantly increased product titers, polishing train .

Recovery of Biological Products XVII | 23

. The static protein nces in in the field of Pankaj Maharjan, Monash University, Australia Pankaj Maharjan, Monash University, Australia Eva M Campi, Monash University, Australia Monash University, Roshanak Sepehrifar, Australia Reinhard I Boysen, Monash University, Australia William R Jackson, Monash University, Biological Products Biological Products Australia *Milton Hearn, Monash University, Over the last decade, adva biochromatography as it impacts on the analytical and preparative capabilities of downstream processing of cytochrome C and bovine serum albumin as model basic albumin as model C and bovine serum cytochrome of yeast in the presence proteins, respectively, and acidic as expanded bed hybrid systems such cells . Unlike other and simple, requiring operation was stable adsorption, or equipment compared with no special procedures despite the chromatography, conventional packed-bed present optical and electron presence of cells . We to show the fidelity between the final microscope images designs and the computer-aided prints and the original respectively solid-phase pore structures, are given as Langmuir isotherms adsorption capacities conditions on dynamic binding and the effects of operating peak shapes are described . capacity and loading/elution studies, including cell passage/ Residence time distribution retention, are also presented . The results show that 3D for producing printing has significant potential as a method columns with preparative-scale monolithic chromatography geometries at a fine control of solid- and mobile-phase the presence of resolution suitable for protein capture in a cell suspension . in 3. Application of Smart Polymers of Processing the Downstream functionality and the monolith was then used to separate was then used to separate and the monolith functionality 24 | Recovery of Biological Products XVII Suhas Nawada, University of Canterbury, New Zealand of Canterbury, Suhas Nawada, University New Zealand of Canterbury, Don Clucas, University Anne Gordon, University of Canterbury, New Zealand of Canterbury, Anne Gordon, University New Zealand of Canterbury, Simone Dimartino, University New Zealand of Canterbury, University Huber, Tim Moreover, the geometric features were identical in the x, Moreover, y and z directions . The agarose solid-phase was modified with 6-aminohexanoic acid to impart cation exchange designed to capture proteins while allowing passage of designed to capture proteins while allowing .0 cm in suspended solids . The printed monoliths were 1 with a series of designed lengths of the order diameter, solid-phase 10 .0 cm, and the geometric features of the with and the mobile-phase flowpaths were identical, 200 microns . characeristic diameters of approximately Clearly, this implies corresponding control over media . Clearly, thus both solid the mobile-phase channel geometry and a priori . and mobile phase geometries can be designed performance In this presentation, we demonstrate the monoliths with of 3D-printed, agarose, cation exchange geometries, uniform mobile-phase flowpath and solid-phase . Furthermore, channels that are complex and ill-defined solids-laden packed beds are unsuitable for processing . In contrast, feed streams such as fermentation broths the ability to additive manufacturing (3D printing) offers and spatial exert control over the size, shape, orientation within porous placement of solid-phase geometric elements Conventional preparative-scale chromatography media Conventional preparative-scale of randomly packed, spherical are typically comprised size and present mobile-phase flow particles that vary in Capture from Solids-Laden Feeds Solids-Laden from Feeds Capture New Zealand of Canterbury, *Conan Fee, University 2. 3D-Printed Cation- Agarose for Protein Monoliths Exchange

Oral Abstracts biological products have largely been driven by development of new separation materials and better understanding of 4. Novel Ultrafiltration Membranes the underlying separation mechanisms . The aim here has Produced by Electrospinning been to achieve greater selectivity, higher resolution, faster speed, enhanced handling capabilities and overall improved *Mikhail Kozlov, MilliporeSigma, United States productivity . Of these factors, control over selectivity has Alex Xenopoulos, MilliporeSigma, USA remained the single most challenging objective . A highly William Cataldo, MilliporeSigma, USA desirable approach to manipulate selectivity and scalability Clif Ngan, MilliporeSigma, USA at the same time as achieving multi-dimensionality in separation-based adsorption phenomena is to use ‘smart’ We report the first ultrafiltration membranes based on stationary phases that have tunable surface properties . In electrospun nanofibers of extremely small diameter . this manner, separation selectivity can be better adjusted Highly uniform, composite nanofiber mats were produced to accommodate variation in feedstock composition or by electrospinning a polyamide solution, with the fiber operational conditions, thus providing improved access to diameter of retentive layer on the order of 10 nm . The separation systems that serve as more efficient platform nanofiber mat samples were characterized by SEM imaging, technologies with lower levels of waste generation . This water permeability, liquid-air porometry, macromolecule presentation examines recent progress addressing these rejection, and performance in model tangential flow objectives from the perspective of design, synthesis and filtration processes . The results show that these use of several new classes of stationary phases that act electrospun nanofiber membranes have the properties of as stimuli-responsive polymeric (SRPs) adsorbents . Also, open ultrafiltration membranes . The performance of the the basis of the prevailing separation mechanisms will be nanofiber membranes in ultrafiltration and diafiltration contrasted for several new SRP adsorbents[1], formed from applications was evaluated using a series of dextran the immobilisation onto suitable porous support materials

feeds and compared to conventional solution-cast Oral Abstracts of pre-formed block co-polymers or alternatively in situ grafted polymeric systems, which exhibit changes in their polyethersulfone (PES) UF membranes of similar nominal properties in response to an external stimulus . Results will molecular weight cut-off (NMWCO) . The nanofiber UF be presented based on batch binding studies, packed bed membranes had about two-fold advantage in water investigations in different formats and larger scale process permeability and ultrafiltration process flux compared to applications as well as insights into the controlling capture the benchmarks . These encouraging first results may and release mechanisms where the potential of these novel pave the way for the next generation UF membranes with stimuli-responsive chromatographic materials has been enhanced performance in purification of biological products documented for energy efficient purification of biological such as vaccines and recombinant proteins . products generated by recombinant DNA/cell culture methods or during process stream recovery . [1] Hearn, M .T .W ., Woonton, B .W ., Maharjan, P ., De Silva, K ., & Jackson, W .R . US Patent 8,877,477 B2 and patents in other national jurisdictions .

Recovery of Biological Products XVII | 25 HTPD and molecular datasets . HTPD datasets are to inform mined independent of molecular information possible future high throughput activity and understand mechanisms for process performance . At a first level, buffer species, protein solubility mining shows that certain solubility almost pH, and counterion concentrations drive independent of structure . Next, the biological molecular understanding descriptors are added to enable further for process of biochemical and molecular level drivers the outcomes . These approaches will further demonstrate utility of HTPD datasets to build smarter processes and biological molecular design . These high throughput techniques throughput techniques molecules . These high biological with each of the methods to assist can be effective rapid and to ensure the challenges above development also the development of candidates but progression have been . Several key workflows of robust processes of biological molecules at introduced in the development multiple methods have . Specifically, Bristol-Myers Squibb the solubility of a candidate evolved to directly measure of solution conditions, while other molecule as a function understanding chromatographic techniques focus on now . These workflows are separations and filtration a rapid progression of mature enough to support . As such, for each of molecules through development have compiled large datasets for these workflows, we molecular multiple biological molecules . In a similar way, computational modeling of biologicals has improved with level descriptors . power and the ability to mine molecular molecular dynamics Advanced computational tools such as understanding simulations have enabled a more detailed then previous approaches . All of this has led to an that can be readily additional store of molecular level data mined . In this work, we demonstrate novel approaches of data mining and exploration for the comprehensive used in the cell culture, recovery, and purification of and purification cell culture, recovery, used in the

The Marriage Marriage The | 26 | Recovery of Biological Products XVII of Technology Zheng Ouyang, Bristol-Myers Squibb, United States Zheng Ouyang, Bristol-Myers Squibb, United States Joseph Lomino, Bristol-Myers Squibb, United United States Nate Domagalski, Bristol-Myers Squibb, United States Stanley Krystek, Bristol-Myers Squibb, Bristol-Myers Squibb, United States Michelle Wang, States LeLand Paul, Bristol-Myers Squibb, United Joseph Calzada, Bristol-Myers Squibb, United States Joseph Calzada, Bristol-Myers Squibb, Ensuring each of these aspects are addressed patient) . Ensuring each of these aspects are addressed with equal rigor is paramount . High throughput techniques have evolved for most, if not all, of the unit operations The development of biological therapeutics continues The development of biological therapeutics . These include to pose significant technical challenges but also the early traditional challenges such as scale-up building quality by design assessment of manufacturability, rapid delivery of (QbD) into the process, and ensuring the (speed to biological candidates to the clinic and market Datasets for Smarter Process and Datasets for Smarter Process Molecular Design Bristol-Myers Squibb, United States *Gregory Barker, Ernst Broberg Hansen, NovoNordisk Ernst Broberg The Application of High 1. Development Process Throughput Screening and Modeling and Modeling Screening SESSION CHAIRS: Delft University Marcel Ottens, Session 3 Session Throughput of High

Oral Abstracts on scientific understanding rather than mathematical 2. Mechanistic Chromatography algorithms . This lean approach enables the adaptation of a Modeling for Industrial Applications generic chromatography model for a given biomolecule in – A Lean Approach to a less than 1 week in most cases . The ease with which this approach can be applied by novice modelers is a further Complicated Tool advantage . Expanding pH ranges, adding quality attributes, *Chris Williams, Genentech, United States and other phase appropriate modifications to model complexity are only applied when needed . Mechanistic chromatography modeling, with the right approach, can potentially provide significant savings in the time and resources required for optimization and 3. Experimental and Computational characterization of a chromatography operation . However Lysate Characterization for Fast through an industrial perspective, time is invaluable Bioprocess Optimization in and the opportunity costs of informative but inefficient Tailored Manufacturing chromatography modeling activities can be high . A balance between the value that a mechanistic model brings to *Pascal Baumann, Karlsruhe Institute of Technology (KIT), a controlled biopharmaceutical manufacturing process Germany and the cost to build in additional complexity is critical . Tobias Hahn, Karlsruhe Institute of Technology (KIT), The preferred approach would provide the most essential Germany phase appropriate information for industrial applications Kai Baumgartner, Karlsruhe Institute of Technology (KIT), from the fewest resources, even if not achieving academic Germany expectations . One lean approach enables the very rapid Juergen Hubbuch, Karlsruhe Institute of Technology (KIT),

development of models that provide accurate predictions Germany Oral Abstracts of key process indicators and quality attributes within controlled manufacturing ranges . The approach begins by With biopharmaceutical industry moving away from large using a high throughput batch binding screen to assess quantity blockbuster production of therapeutic agents (‘one the binding behavior of a mAb or other biomolecule on the drug fits all approach’) to a more personalized medicine chromatography resin of interest . Binding behavior is used (‘tailored manufacturing’), finding a generic approach to design small-scale or RoboColumn-scale experiments for purification process development is a key challenge . that can be used for model calibrations . The calibration Straightforward routines, like antibody platform processes, itself focuses on matching experimentally observed key might not be applicable anymore and thus all stages process indicators (KPIs) and quality attributes of interest, of process development for biopharmaceuticals might as opposed to chromatogram curve fitting . In this approach, require individual and tailored productions . Conventional tools have been developed to couple parameters with approaches based on heuristics, high-throughput shared interactions in order to allow the efficient navigation experimentation (HTE) and statistical modeling alone of a multivariate parameter space with direction built hardly keep pace with this trend . Expert knowledge,

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Alternative Alternative | Chapman Wright, Biogen, USA USA Catie Bartlett, MIT, Natarajan, Biogen, United States Venkatesh Heather Saforrian, Biogen, USA USA J. Christopher Love, MIT, *Matthew Westoby, Biogen, United States *Matthew Westoby, workhorse CHO-based manufacturing has been the for biopharmaceutical production . Despite its success, including production with CHO has several challenges, Both approaches are . Both approaches lysate characterization on in silico and process development fast and effective qualified for minimal while requiring of critical impurities identification effort . and experimental sample consumption Session 4 Systems: Expression Impact for Strategic Biopharmaceuticals? SESSION CHAIRS: London Suzanne Farid, University College Lars Pampel, Novartis Alternate Reality: Alternate 1. and the Future Systems Expression of Biomanufacturing on deterministic experimental lysate scoring, the other lysate scoring, the experimental on deterministic

28 | Recovery of Biological Products XVII we propose two advanced strategies for integrated up- we propose two advanced strategies for and downstream process optimization . The first is based integrated fashion . The evaluation and characterization by of a large set of different feed stocks generated by in silico high-throughput cultivations was performed lysate characterization . This methodology was used in an a phase II liver integrated process optimization study for enzyme produced in Escherichia coli . In summary, solubility, shown for different proteins of acidic, neutral shown for different proteins of acidic, solubility, and alkaline isoelectric point . Besides experimental hybrid approach lysate characterization, we developed a and in silico combining high-throughput experimentation and lysate scoring . By merging these two technologies, up- in an entirely downstream processes could be optimized and the physical and chemical nature of the product or and the physical and chemical nature of impurities . The pH was found to be the central parameter shown in for ion-exchange chromatography optimization supernatant . a case study of a complex Pichia Pastoris hydrophobic interaction chromatography Analogously, driven by protein processes were determined to be mostly To that end, we first developed . To downstream performance designs for chromatography based deterministic process characterization . In this context, on experimental lysate serve as a basis to generate characteristic key parameters for any complex feed stock new optimized processes of the feed composition without any prior knowledge processing and product formation is hard to predict with is hard to predict and product formation processing model which is why this step still a physico-chemical screening using high-throughput requires experimental large number the . Subsequently, cultivation experiments need to be scored in terms of of candidate lysates HTE and mechanistic modeling constitute a good constitute a good modeling HTE and mechanistic process development for fast and effective foundation be potential can only but their full for new products, procedures . Upstream a combination of these realized in

Oral Abstracts limits to productivity, high media costs and extensive The emergence of novel classes of biotherapeutics creates purification requirements . Recent work has focused new challenges for the development of purification on improving productivity and cost by adapting CHO processes meeting aggressive development timelines . to perfusion-based cell culture and running in single- For domain antibodies (dAb) derived from VH or VL domains use, continuous manufacturing formats . While these and expressed in E . coli, a toolbox approach was developed efforts improve flexibility, they result in only incremental during the last five years to accelerate early phase process improvements in productivity and cost . In addition, these development . After an initial risk assessment, a pre- efforts have not been readily transferable to high volume established decision tree was used to define a first intent production . To achieve the next level in biopharmaceutical purification sequence based on the molecular construct . production, alternate high productivity expression systems The results from the small scale evaluation were used to should be considered . Biomanufacturing 2020 is an select pre-established high throughput (HTP) and small initiative at Biogen to create a disruptive, high productivity scale column experiments to establish a final process . biomanufacturing platform . Within this initiative, we are The initial capture was performed using either Protein A, identifying and integrating new technologies and processing Protein L or mixed mode chromatography followed by either paradigms that result in at least a 10-fold increase in one or two chromatographic steps . The rapid optimization productivity and significant cost reductions compared to of the capture step was enabled by a fundamental current processes . A critical component of this project is understanding of the dAb’s binding capacity on protein A the selection and development of an expression system . In and protein L resins . The differences in the polishing steps’ this talk, we describe our process of identifying expression resin binding properties were characterized using a HTP systems of interest, developing selection criteria, and plate approach . Gradient elution was adopted by first intent . defining a development path for these non-mammalian Lastly, the observed variability in HCP levels was addressed systems . In addition, we illustrate how an alternate host by adopting a comprehensive HTP approach to characterize could enable a smarter, streamlined downstream process HCP elution profiles to guide polishing step optimization . Oral Abstracts and lead to novel manufacturing platforms . In all cases, pre-defined work packages leveraged the same raw materials . As a result, raw materials for GMP campaigns could be ordered shortly after the initial small 2. Lessons Learned from Developing scale evaluation removing process development from the a Platform Purification Approach for critical path . This approach was implemented successfully Domain Antibodies for several projects under very aggressive development timelines, demonstrating its ability to address the *Andre C. Dumetz, GSK, United States development challenges due to the diversity in Jeff T. Kurdyla, GSK, USA the dAbs . Gerald J. Terfloth, GSK, USA

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ters have increased Priorities in Priorities | Key options to further increase throughput include . Key options to further increase throughput Asha Radhamohan, Genentech, United States Asha Radhamohan, Genentech, United Benjamin Sackett, Genentech, United States Chris Williams, Genentech, USA Juno Therapeutics, United States Mary Mallaney, Rachel Specht, Genentech, USA Genentech, USA Yigzaw, Yinges Over the last two decades, antibody ti increased the by almost a 100-fold which has significantly biotechnology throughput of manufacturing plants in the industry of continuous continued increase in titers and/or pursuit manufacturing . Downstream process bottlenecks have further increase in been highlighted as a key challenge with have thus pivoted titers and the industry conversation may . Benefits in the direction of continuous manufacturing better process controls such as robust product quality, and ease of scaling have also been highlighted with continuous manufacturing . While continuous bioprocessing in principle offers several advantages, its broader Session 5 Session and Performance Cost Improvements SESSION CHAIRS: MedImmune David Robbins, Biogen Sanchayita Ghose, for Approach A Downstream 1. Titer Recoverable Maximizing Throughput and Facility *Amit Mehta, Genentech, United States . 30 | Recovery of Biological Products XVII Netherlands Netherlands Netherlands Netherlands Danielle van Wijk, Synthon Biopharmaceuticals BV, Biopharmaceuticals BV, Danielle van Wijk, Synthon Synthon Biopharmaceuticals BV, Gerard Rouwendal, Biopharmaceuticals BV, Gerry Ariaans, Synthon Meng Liu, Synthon Biopharmaceuticals BV, Netherlands BV, Meng Liu, Synthon Biopharmaceuticals Netherlands Biopharmaceuticals BV, Guy de Roo, Synthon Netherlands Biopharmaceuticals BV, Kim Burgers, Synthon Netherlands Biopharmaceuticals BV, Bram Kamps, Synthon abundant impurities by acidic precipitation (4) clarification abundant impurities by acidic precipitation by affinity by depth filtration and TFF (5) purification and (6) chromatography followed by polishing steps formulation in a stable buffer is the lack of established methods for production and is the lack of established methods for production purification . Here we present Lemna duckweed as a antibodies . promising platform for production of SIgA of SIgA The production process comprises (1) expression of SIgA in stably-transformed duckweed, (2) extraction naturally by disruption of the plant material (3) removal In recent years, secretory IgA (SIgA) antibodies have In recent years, secretory IgA (SIgA) antibodies therapeutics attracted increased attention as potential for infectious and malignant diseases . Despite this, SIgA advanced . One of antibodies have not been commercially SIgA antibodies the main problems hampering work with *Michel Eppink, Synthon Biopharmaceuticals BV, BV, Synthon Biopharmaceuticals *Michel Eppink, 3. Purification of Secretory IgA from IgA from of Secretory 3. Purification Minor (duckweed)Lemna

Oral Abstracts adoption will likely be hindered by several technical, flow through column step . First, the ionic capacity of operational and near-term economic challenges . Some five commercially available depth filters was measured of these challenges include retooling of an established using a unique counterion displacement method . Small manufacturing base, fitting complex processes for novel scale virus spiking experiments showed that parvovirus drug formats into continuous manufacturing platform, clearance by depth filters may be influenced by process development of continuous processes in an environment conditions (pH, conductivity) as well as the filter media that is shooting for higher productivity and ever shortening charge characteristics . However, complete retrovirus clinical development timelines . This talk will focus on a clearance was observed on all five filters . A PCR-based downstream process concept that meets the industry desire assay was developed to directly measure the reduction for higher throughput by maximizing recoverable titer and in Type C retrovirus-like particles across the depth leverages recent innovations in protein purification and filters in actual process streams . Finally, a tandem anion separation media to overcome downstream bottlenecks . exchange-hydrophobic interaction chromatography column This process concept in combination with recent facility was designed for an antibody fragment molecule with design innovations and single-use technologies can also unique challenges related to HCP clearance and product- reduce the capital expenditure by providing high throughput at a reduced scale . Data will be presented with several related variants . By combining the anion exchange and molecules including molecular format diversity . Overall hydrophobic interaction chromatography unit operations, this transformative but not operationally disruptive process a highly non-platform process was fit more easily into the concept can lead to higher return on manufacturing assets, manufacturing schedule without compromising product is broadly implementable, accommodates a diverse and quality . Both the tandem flow through chromatography novel molecule portfolio, syncs with the ever shrinking step as well retrovirus clearance on depth filters have been development timelines and furthermore has the potential demonstrated at scale in GMP clinical manufacturing . to be semi-continuous for additional facility throughput Oral Abstracts increases . 3. Meeting Challenges in Productivity and Product Quality 2. GMP: Get the Most Out of Your Plant Improvements for the Manufacture (by integrating unit operations) of a Recombinant Enzyme Ron Kowle, Eli Lilly, USA *Michaela Wendeler, MedImmune, United States Dayue Chen, Eli Lilly, USA Xiangyang Wang, MedImmune, USA Samantha Streicher, Eli Lilly, USA Purbasa Patnaik, Eli Lilly, USA Economical process design can be challenging for non- *Jace Fogle, Eli Lilly, United States monoclonal antibody products, especially when low titers conflict with high product demands, and complex This talk will highlight two novel approaches for reducing separations of product-related impurities are required . downstream process cycle time and cost – the use of This case study illustrates process development for a depth filters for clarification as well as retrovirus clearance recombinant enzyme that had to address significant and a tandem anion exchange/hydrophobic interaction challenges to meet cost and quality targets . The initial

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. The matrix was The main aim of this . The main aim of this uman mesenchymal stem cells (hMSC) in stem cells uman mesenchymal therapy applications . Paula M. Alves, iBET, Portugal iBET, Paula M. Alves, Manuel T. Carrondo, iBET, Portugal iBET, Carrondo, Manuel T. Furthermore, we also showed that EBA bed rate (1 .4) . Furthermore, we also showed that EBA on the already chromatography can be efficiently integrated processing (DSP) of established process for the downstream efficiency more than hMSC, where it improved the washing of cells after total 10-fold, recovering approximately 70% viability processing . This strategy showed not to impact cell characteristics in terms of (> 95%), neither hMSC’s and adhesion immunophenotype, proliferation morphology, potential . Overall, capacity and multipotent differentiation the beginning negative mode chromatography represents applications, where of a promising platform for cell therapy new adsorbents can be designed to have affinity with target impurities (e .g . BSA) and not to the final product itself, the cells . This means that the methodologies herein developed can be adopted to other type of cell products relevant for cell *Cristina Peixoto, IBET, Portugal IBET, *Cristina Peixoto, h The use of has been increasing over the last clinical applications characteristics, as decade . Their immunomodulatory bioactive molecules with well as capacity in secreting regenerative features, have been anti-inflammatory and candidates for autologous and making them attractive in a clinical to be applied . However, allogeneic therapies to comply with specific requirements in setting hMSC need potency and purity terms of identity, tangential flow filtration work is to improve established strategies, further increasing hMSC (TFF)-based washing (EBA) using negative mode expanded bed adsorption purity, prototype matrix chromatography with a new multimodal based on core-shell bead technology could be obtained characterized and a stable, expanded bed what is used for using standard equipment adapted from processes . The conventional packed bed chromatography recovery yield effect of different expansion rates on cell . The best and protein removal capacity was assessed protein clearance trade-off between cell recovery (89%) and expansion (67%) was achieved using an intermediate

. Purification of Purification | n vastly insufficient productivity due productivity n vastly insufficient 32 | Recovery of Biological Products XVII Uppsala, Sweden Uppsala, Sweden Uppsala,, Sweden Uppsala, Sweden Jean-Luc Maloisel, GE Healthcare Bio-Sciences AB, Jean-Luc Maloisel, GE Healthcare Bio-Sciences John Clachan, GE Healthcare Bio-Sciences AB, Anna Åkerblom, GE Healthcare Bio-Sciences AB, Bárbara Cunha, iBET, Portugal Bárbara Cunha, iBET, Portugal Ricardo J.S. Silva, iBET, Portugal Margarida Serra, iBET, AB, John Daicic, GE Healthcare Bio-Sciences 1. Improving Washing Strategies Strategies Washing Improving 1. Stem Cells of Human Mesenchymal Expanded Mode Using Negative Bed Chromatography SESSION CHAIRS: Michael Laska, Moderna Therapeutics Caryn Heldt, Michigan Tech productivity and product quality productivity and product Session 6 Therapeutics Non-Protein advancements in product understanding paved the way for paved the in product understanding advancements and downstream optimization . We integrated upstream and opportunities presented by the discuss the challenges upstream process, the evaluation of change to a perfusion options, and the implementation of continuous downstream process with significantly improved a robust manufacturing process resulted i process resulted and enzyme stability, level, poor to low expression protein variants that of closely related the presence . Systematic low efficiency separations necessitated

Oral Abstracts as purification potential and yield but also on their ease to 2. Challenges in the Development be performed aseptically, with a premium placed on ready- and Scale-up of an Attenuated to-use, single-use technologies . However, in one instance, Live Virus Vaccine Candidate a ready-to-use, single-use format was not available for a promising resin-based chromatography, therefore requiring *Marc Wenger, Merck & Co., Inc., United States us to adapt this step to the sterile processing environment . Adam Kristopeit, Merck & Co., Inc., United States The final purification process enables a scalable path for Janelle Konietsko, Merck & Co., Inc., United States commercialization, while exceeding targets for purity Justin Ma, Merck & Co., Inc., United States and yield . Tung Nguyen, Merck & Co., Inc., United States Katherine Phillips, Merck & Co., Inc., United States 3. Use of Ultrafiltration for Matthew Woodling, Merck & Co., Inc., United States the Purification of Conjugated Joseph Joyce, Merck & Co., Inc., United States Polysaccharide Vaccines Prophylactic live attenuated vaccines (LAV) have been *Andrew Zydney, Penn State University, United States successfully developed for multiple viral disease targets, Mahsa Hadidi, Penn State University, United States offering an advantage over subunit vaccine approaches by John Buckley, Pfizer, United States simultaneously stimulating innate, humoral and cellular immune responses . However, the development of robust Capsular polysaccharides from pathogenic bacteria manufacturing processes for LAVs at commercially have been used to produce vaccines against important viable scales can be challenging, particularly because diseases such as pneumonia and meningitis . Traditional of the need to use novel and/or adherent cell lines, the polysaccharide vaccines are largely ineffective for inefficient performance of conventional chromatography Oral Abstracts children < 2 years of age and elderly people . Effective for processing large viral particles, the presence of similarly sized host-cell microvesicle contaminants, and vaccination can be provided using a conjugate vaccine the complexity of product characterization . Further adding generated by chemically coupling the polysaccharide to these challenges, closed-system aseptic processes are to a highly immunogenic protein (e .g ., CRM197, a non- required for those viruses too large for terminal sterile toxic mutant of diphtheria toxin) . The development of filtration, thereby limiting processing options . Highlighting multivalent conjugated vaccines requires the coupling these challenges, we present here on the development of of each purified polysaccharide with the immunogenic a scalable, fully sterile purification process for a candidate protein, with any unreacted (free) polysaccharide removed LAV vaccine . In the process development of this vaccine, in the purification process . This purification can be a a variety of purification approaches were evaluated major challenge in the commercialization of conjugated including membrane and monolith supports, newly designed vaccines . The objectives of our work were to evaluate bead-based media for viruses and large biomolecules the potential of using membrane ultrafiltration for the (e .g . Capto Core 700), and non-chromatographic methods purification of these conjugated vaccines and to develop such as selective precipitation and large-pore tangential a fundamental understanding of the factors controlling flow filtration . The final purification unit operations were the transmission of both free polysaccharides and their selected based not only on usual performance criteria such corresponding conjugates through commercially available

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. . Details Ratnesh Joshi, Biogen, United States Ratnesh Joshi, Biogen, United States Kris Ruanjaikaen, Biogen, Fillon, Biogen, United States Yannick United States Amgen, Carson Tran, United States Firoz Antia, Biogen, . Overall, this a second, orthogonal step is employed purity, key product- process design achieves control of several over earlier related impurities and provides robustness platforms without sacrificing yield or productivity . of the optimized process will be presented 4. Re-designing Purification Purification 4. Re-designing Oligonucleotides; for Processes Achievable? What’s United States *Robert Gronke, Biogen, oligonucleotides has traditionally Purification of antisense that a single step been based on the philosophy along with precipitation and chromatography process, deliver an detritylation steps would be sufficient to sufficient purity active pharmaceutical ingredient (API) of are paying more attention to product- Regulators, however, once considered an related impurities that in the past were we have developed acceptable part of the API . Recently, of antisense a much improved downstream processing based capture oligonucleotides by employing an aqueous of product- step that gives surprisingly high resolution related impurities . Then, in an effort to further enhance Ultrafiltration data were obtained in a . Ultrafiltration data 34 | Recovery of Biological Products XVII factors controlling the ultrafiltration behavior of bacterial factors controlling the ultrafiltration behavior design of effective polysaccharides and a framework for the of polysaccharide- membrane processes for the purification based vaccines . conjugates as determined by size exclusion chromatography conjugates as determined by size exclusion using the different buffer solutions . Ultrafiltration results separation showed significant opportunities for enhanced concentration by exploiting both solution conditions and of the membrane polarization to maximize the selectivity into the process . These results provide important insights were also strong functions of solution ionic strength due were also strong functions of solution ionic electrostatic and intra-molecular to a combination of inter- and the interactions between the charged polysaccharides charged membrane . Additional insights into the effects of the changes in solution ionic strength were obtained from and effective hydrodynamic volume of the polysaccharides to concentration polarization effects, with the data in good effects, with the data in good to concentration polarization stagnant film model . Polysaccharide agreement with the at high filtrate flux and when fouling became significant solutions, consistent with the using more concentrated wall concentration for fouling for presence of a critical transmission each serotype . The flux and polysaccharide using both dynamic light scattering and size exclusion and size exclusion dynamic light scattering using both chromatography regenerated cellulose and stirred cell using composite . Polysaccharide transmission polyethersulfone membranes a strong function of filtrate flux due in dilute solutions was Experiments were performed were performed membranes . Experiments ultrafiltration polysaccharide serotypes pneumococcus using several of charge) over a range size and electrical (with different were characterized strength . Polysaccharides solution ionic

Oral Abstracts are multiple modules to eliminate aggregates based on Session 7 | Purification – their interaction type . Although the HCP load is highly dependent on upstream conditions and the choice of a cell from Platform to Diversity line, we created a toolbox to quickly reduce HCP content . Furthermore we achieve cost savings by implementing generic buffer systems that are applicable in all purification SESSION CHAIRS: steps . I will present the current status of our modular Nihal Tugçu, Merck & Co., Inc. approach to transfer well proven elements from platforms Dorothee Ambrosius, Boehringer into downstream processes of non-antibody molecules . I Ingelheim Pharma GmbH will show case studies from our highly diverse portfolio of complex molecules ranging from enzymes and fusion proteins to vaccines, addressing the issues mentioned 1. Modular Approaches for above and exemplifying our strategy . Diverse Molecules: Reinventing Smart Bioprocessing 2. Leveraging HTPD to Streamline Implementation of Non-platform *Stefan Schmidt, Rentschler Biotechnology, Germany Therapeutics to Minimize Impact to The biopharmaceutical industry is in the middle of a Development Timelines revolution as novel complex protein based therapeutics are threatening the dominance of monoclonal antibodies . *Jennifer Pollard, Merck & Co, United States These designer molecules are difficult to purify as in many John Welsh, Merck & Co, United States cases they lack a common motif that would enable the Michael Rauscher, Merck & Co, United States Oral Abstracts installation of a platform process based on affinity . In order Sandra Rios, Merck & Co, United States to accommodate this molecular diversity we have chosen an David Roush, Merck & Co, United States optimization strategy focusing on individual characteristics Nihal Tugçu, Merck & Co, United States of the respective proteins . Our concept does not necessarily rely on sequential brute force high throughput process External pressures on the industry have challenged research development only, but on the intelligent deconvolution of and development to accelerate molecules to the clinic . general purification issues in manageable chunks that are Historically, these accelerated timelines were achieved systematically rearranged to form a logical sequence of through the use of purification platforms, most readily for minimally required steps to achieve the intended quality full length monoclonal antibodies (mAbs) . As a wider range and purity profile . Multiple modules are then assembled of novel biologic targets are pursued, a more diverse set of according to the specifics of each new molecule or class therapeutic modalities such as antibody fragments, fusion of molecules to redesign a quasi-platform downstream proteins, peptides and nanobodies provide challenges to process . Irrespective of the molecule type, the challenges of downstream development . These challenges can be related enabling a fast and cost effective capture, HCP reduction, to unique impurities such as product fragments or more virus inactivation and aggregate removal remain the typical impurities such as residuals or aggregates . As more same . For instance we can chose from several individual non-platform molecules are desired, a different approach virus inactivation steps, taking into account the different is needed to address the time and resource challenge . sensitivities of protein molecules . Another example

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The assembly approach results in . The assembly approach This presentation will describe the evolution of molecules . This presentation will describe the evolution for assembled a standard downstream purification process knob-in-hole bispecific antibodies . Select case studies will from the standard be presented including some “deviations” challenges process due to molecule and/or format-specific as well as related to undesirable assembly byproducts to expression high levels of host cell proteins related host differences . years due to their ability to bind to two different targets, to two different to their ability to bind years due with targeting strategies the door to unique thus opening otherwise success that are for therapeutic greater potential . antibodies with traditional monospecific unavailable antibody technology employs Knob-in-hole bispecific domains of the half antibodies engineering of the CH3 heavy chain heterodimerization to to preferentially drive minimizing homodimer formation . form the bispecific while the the possibility of light-chain mispairing, minimize To are produced separately in two intact half antibodies cultures and assembled to form the different production bispecific antibody impurities such as unassembled unique product-related homodimers that must be removed by half antibodies and the downstream purification process . The conventional be unsuitable for mAb purification process was found to development this purpose, necessitating further process . Employing high for early knob-in-hole bispecific antibodies the optimization throughput process development tools for development of the assembly process as well as for the bispecific of downstream purification steps for multiple a generic bispecific antibodies enabled the identification of production process for CHO and E .coli host derived Bispecific antibodies have gained a lot of interest in recent a lot of interest antibodies have gained Bispecific engagement due to its ability to utilize engagement due to 36 | Recovery of Biological Products XVII Michelle Butler, Genentech, Inc., United States Michelle Butler, Genentech, United States Philip Lester, Kimberly Kaleas, Genentech, United States Glen Giese, Genentech, United States Amit Mehta, Genentech, United States Josefine Persson, Genentech, United States Chris Dowd, Genentech, United States 3. Evolution of a Standard 3. Evolution of a Standard for Process Purification Downstream Knob-in-Hole Bispecific Antibodies *Asif Ladiwala, Genentech, United States applied to develop an additional polishing chromatography applied to develop an additional polishing the final step for aggregate removal as well as optimize ultrafiltration conditions . For the fusion protein, HTPD was step to clear used to successfully identify a chromatography product related fragments . which may be needed, as early in development, impurity which may be needed, will be case studies . In this paper, characterization is lacking . the impact on time and resources employed to demonstrate will show the impact of HTPD The case studies presented of a non-platform mAb and a on both the development was fusion protein . For the non-platform mAb, this workflow a platform and a non-platform molecule and allow for more molecule and allow and a non-platform a platform fixed amount of resources . HTPD allows experiments with a for earlier program enabling the use of transiently small amounts of material, facilitates a more produced protein . This workflow also to development and optimization, empirical approach The utilization of high throughput process development process development of high throughput The utilization to provides a strategy non-platform molecules (HTPD) for throughput . This high impact to the timelines mitigate the between in development time can reduce the gap workflow

Oral Abstracts inclusion body wash procedure . Optimizing the cell 4. A Miniaturized Process on μl- disruption via parameters such as shaking frequency scale Representing the Entire and biomass concentration led to high comparability of Process Chain from Upstream to this small scale method to the bench scale high pressure Downstream Processing homogenization in respect to DNA, HCP and product release . Using this high throughput platform the correlation *Cornelia Walther, BOKU & Boehringer-Ingelheim-RCV, of upstream conditions for up to 24 fermentations with Austria recovery properties of IBs can be determined in parallel per Martin Kellner, Boehringer-Ingelheim RCV, Austria 96 well plate . In the end, the inclusion bodies can be further Matthias Berkemeyer, Boehringer-Ingelheim RCV, Austria processed in high-throughput methods to set up optimized Cecile Brocard, Boehringer-Ingelheim RCV, Austria solubilization, refolding and capture conditions . This fast Astrid Duerauer, BOKU & Austrian Centre of Industrial and material saving platform method provides a high-quality Biotechnology, Muthgasse 18, Austria evaluation of fermentation screenings and reduces the initial barrier for IB processes opening potential for new Process optimization should be carried out in a holistic way products . The principle of operation of the platform will be taking the whole chain of unit operations into consideration . demonstrated with two examples – an affinity scaffold and Integration of DoE will lead to numerous experiments a single-chain antibody . and thus such optimization can be only managed in μl- scale . The so-called “new formats” are extremely diverse molecules compared to antibodies and require flexibility for 5. A Molecular Properties process development . These formats are often expressed Perspective to Aid in the in E . coli and the low cost production with high yield can Development of Chromatographic be realized especially when the product is expressed as Strategies for the Purification for Oral Abstracts inclusion body . Harvesting of inclusion bodies is simple and the product is already present in high purity . IB formation Bispecific Antibodies and their characteristics depend on multiple parameters . *Tiago Matos, Downstream Technology, Global Research, During process development the interpretation of upstream Novo Nordisk A/S, Denmark optimization DoEs is often based solely on fermentation Hanne Sophie Karkov, Downstream Technology, Global titers and not on the entire process chain . High titers do not Research, Novo Nordisk A/S, Denmark warrant success of the subsequent downstream processes . Gorm Andersen, Downstream Technology, Global Research, A laboratory platform was developed which connects Novo Nordisk A/S, Denmark extensive DoE setups in upstream development to recovery Lars Sejergaard, Mathematical Modelling Department, of active protein from inclusion bodies at a very early stage CMC, Novo Nordisk A/S, Denmark of process development . Here, we present a miniaturized Haleh Ahmadian, Downstream Technology, Global and parallelized inclusion body recovery process consisting Research, Novo Nordisk A/S, Denmark of mechanical cell disruption in a bead mill and subsequent Steven Cramer, RPI, United States

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Process Process | for all, best illustrated by making HIV/ARV medicine for all, best illustrated by making HIV/ARV is developing a available worldwide for $1/day in 2002, which biosimillars class world equivalent $1/day of portfolio . Cipla will be approved and commercialized worldwide Class Quality”, “World is implementing a “World BioTec to biosimilars . Class Economics”, “Be Local” approach overall efforts The presentation will outline Cipla Biotec’s and and approach including some of the innovations treatment for partnerships used by Cipla to deliver $1/day and how this learning is being applied to Cipla BioTec HIV, focus on Engineering Optimization to improve Cipla BioTec’s production of Biopharmaceuticals . Session 8 Session with Challenges Biosimilars SESSION CHAIRS: Amgen Suresh Vunnum, Biotech Jill Myers, Fortress Care: Cancer Transforming 1. and Accessible Affordable Biosimilars United States Cipla BioTec, *Steven Lehrer, less than 8% of diagnosed cancer patients have Worldwide frequently access to Standard of Care treatment which includes biopharmaceuticals . In the US and EU, only about of therapy as the 25% of the population is treated, with cost continuing Cipla’s major barrier to treatment . Cipla BioTec medicine tradition of ensuring affordable and accessible tal screening and in-silico analysis bodies (bsAbs) have emerged as a promising have emerged bodies (bsAbs) 38 | Recovery of Biological Products XVII general not sufficient for developing efficient downstream general not sufficient for developing efficient bioprocesses . In contrast, multimodal (MM) resins, shown to exhibit particularly Capto MMC ImpRess were capacity and recovery for the desired bsAb good selectivity, a generic process for the purification of molecules . Finally, resin is proposed . bsAbs using this MM chromatographic associated bsAbs, we were able to identify some of associated bsAbs, we were able to identify trends . The the underlying reasons for these selectivity results of the experimen interaction indicated that while traditional hydrophobic (IEX) exhibited (HIC) and ion-exchange chromatography results were in some selectivity for these mixtures, the trends obtained with these various mixtures and resin trends obtained with protein surface property analysis was systems, a detailed of novel molecular descriptors carried out using a range in our lab . By examining the protein recently developed associated with each key molecular surface property maps mAbs and the descriptor for a wide range of parental mAbs from the bsAbs requires the development of the development of the bsAbs requires mAbs from due to the similarity of the new purification strategies variety of chromatographic molecules . In this work, a wide were examined to determine the resins and conditions systems for several bsAbs separation selectivity of these selectivity in order to understand the challenges . Further, Bispecific anti Bispecific . In of complex diseases for multiple targeting alternative of monoclonal the downstream bioprocessing contrast to of the two parental (mAbs), the separation antibodies

Oral Abstracts 2. Challenges and Approaches in 3. Advances and Challenges in the Development of a Biosimilar the Analytical Characterization of Purification Process Biosimilar Products *Russell Shpritzer, Pfizer, United States *Jeff Allen, Pfenex Inc, United States Kim Sterl, Pfizer, United States Priscilla Jennings, Pfizer, United States Biosimilar development is substantially based on demonstrating analytical similarity to the reference Brian Korniski, Pfizer, United States product with abbreviated clinical studies utilized to remove Daniel LaCasse, Pfizer, United States residual uncertainty . Pfenex Inc, a leader in biosimilar Timothy Iskra, Pfizer, United States development, routinely deploys its extensive in house Richard Wright, Pfizer, United States analytical infrastructure in support of product development . Significant challenges arise in the process of demonstrating While many of the important product quality attributes of the comprehensive analytical similarity of a given biosimilar a potential biosimilar are driven by the upstream process, to its reference product . In some cases, reference products the downstream process plays a critical role in the removal contain excipients such as Human Serum Albumin (HSA) of product- and process-related impurities and the control that interfere with analytical similarity assessment due to of product-related isoforms . Pfizer’s platform purification the large amount of this protein excipient relative to the process served as the starting point for the purification of active drug protein . In order to enable analytical similarity all five of our Phase 3 monoclonal antibody biosimilars . analysis between Pfenex’s interferon beta-1b biosimilar In addition to streamlining the development process and candidate and the marketed HSA-formulated reference enabling facility fit at the commercial scale, the platform

product, an HSA-depletion method was developed by Oral Abstracts process routinely reduces process-related impurities to Pfenex to remove interferences from this excipient and acceptable levels with minimal development . However, allow additional characterization at all levels of protein additional strategies may be required when considering the structure, including hydrogen/deuterium exchange (H/DX) levels of product-related impurities and isoforms that may to elucidate higher order structural (HOS) forms . Exploration be present in the innovator’s drug product . This presentation and characterization of possible protein complexes, protein will discuss some of the approaches that were taken, form distribution, and activity for both formulations was beyond the platform process, to meet the requirements performed by 2-dimensional analysis across the product of each unique biosimilar . profile . The data from these experiments demonstrate that Pfenex’s biosimilar candidate shows high similarity to the reference product based on the analytical methods developed and employed .

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Jeffrey Salm, Pfizer, United States Jeffrey Salm, Pfizer, United States Pfizer, Bob Kottmeier, States United Marcus Fiadeiro, Pfizer, States United Jill Kublbeck, Pfizer, States Raquel Orozco, Boehringer Ingelheim, United States Boehringer Ingelheim, United Scott Godfrey, States Jon Coffman, Boehringer Ingelheim, United This talk will describe a multi-stage hybrid integrated This talk will describe a multi-stage hybrid ~1 kg batch of downstream system for manufacturing monoclonal antibody (mAb) from a 100 L continuous perfusion bioreactor over a 5 to 15 day period . The key to this design is integrated operational effectiveness, where each stage and sequence of the system maximizes its assumption, the entire process can be characterized in can be characterized the entire process assumption, for the time . Case studies and quality over terms of purity to will be employed of a monoclonal antibody purification residence of unit operation the establishment demonstrate propagation times throughout the times and deviation not only . The case studies will continuous bioprocess to characterize connected process highlight the ability the ability to utilize the extracted performance, but also, control strategies that will tolerate information to design and material segregation . This major process deviations multivariate data analysis coupled with methodology, lead to dynamic process control techniques will ultimately reliably deliver protein with highly strategies that will purity profiles from continuous consistent quality and bioprocesses . 2. A Multi-Stage Hybrid Integrated Processing for Downstream System a of Monoclonal Antibodies from Bioreactor Continuous Perfusion United States Pfizer, *Rob Fahrner, purification train and applying a temporal steady state a temporal steady train and applying purification Next Generation Next | . By connecting the permeate stream from a To address this challenge, we present here . To 40 | Recovery of Biological Products XVII Finn Hung, Merck & Co., Inc., United States States Adrian Gospodarek, Merck & Co., Inc., United Nuno Pinto, Merck & Co., Inc., United States States David Pollard, Merck & Co., Inc., United the holistic downstream process performance through the holistic downstream process performance perturbation analysis where response to an upstream stimulus is measured at several unit operations simultaneously perfusion bioreactor directly to a continuous downstream continuous protein production processes operating at continuous protein production processes periodic steady states . One obstacle for implementation of characterization of continuous bioprocessing is the lack linked tools for unit operations that are truly dynamically to one another characterizing on the development of a methodology for with sophisticated automation strategies is within reach . with sophisticated automation strategies operations Advances in technology for traditional unit cell culture, such as cell-retention devices in perfusion and single- continuous multi-column chromatography to pilot-scale pass tangential flow filtration have led and fully- demonstrations of both semi-continuous Implementation of continuous bioprocessing integrated Implementation of continuous bioprocessing John Moscariello, CMC Biologics John Moscariello, Steady States in of Perturbations 1. Continuous Bioprocessing Merck & Co., Inc., United States *Mark Brower, Integrated Processes Integrated SESSION CHAIRS: Jeff Salm, Pfizer Session 9 Session & Operations Unit

Oral Abstracts individual advantages while minimizing any disadvantages . concentrate system built in a cube format . Data presented We will present how the system has been engineered from prototype pilot runs will demonstrate the ability to for efficient use of space, time, equipment, and reduced successfully operate such a system with product quality, operational complexity . Highlights of the system include including HCP, leached ProA, DNA, dimer, and aggregate the ability to operate under a wide range of bioreactor that is comparable to product produced in batch mode . The parameters, including a bioreactor productivity of 1–4 g/L/ pilot runs were able to produce purified mAb with a product day, which accounts for variation of Qp, sieving decay, and concentration of ≥ 60 g/L at ≥ 70% downstream yield reactor VVD . The system has been developed as a hybrid of through the SPTFF in the periodic stage, turbidity <6 NTU three operational stages: continuous, periodic, and batch . at all unit operations, and consistent achievement of pH During the continuous stage: The bioreactor permeate is targets . The principles of the design and operation of this continuously harvested by TFF and is alternately loaded integrated, hybrid system provide an effective method for onto two 1 Liter protein A columns, where the loading downstream processing of continuous perfusion bioreactors . phase switches between the two columns and does not pause . It has the ability to self-adjust for variable product concentration and flow rate at the system inlet . During the 3. Alternative Downstream periodic stage (which occurs every 4 to 12 hours): After Processing Based on Continuous loading, the first protein A column is eluted (while the Coupled Precipitation-Filtration second protein A column is being loaded in parallel) and Capture Operations runs continuously through 1) a tankless hold, 2) a low pH virus inactivation chamber, 3) a 0 .5 Liter weak partitioning Qin Gu, Carnegie Mellon University, United States anion exchange column, 4) an SPTFF unit, 5) in line pH Zhao Li, Penn State University, United States and conductivity conditioning, and 6) is collected into a *Todd Przybycien, Carnegie Mellon University, United States single use mixing bag . While the product stream moves Andrew Zydney, Penn State University, United States Oral Abstracts continuously through this section, each individual unit operation is taken offline when not containing product, Capture via continuous coupled precipitation-filtration offers and is regenerated and sanitized in parallel with the other a new, alternative paradigm for the downstream processing unit operations, which permits significant flexibility for of recombinant proteins . In this process, target protein is variable product streams and maximizes the periodic stage captured from pre-concentrated clarified cell culture fluid, productivity . The periodic stage then pauses until the with concentration >10 to 100 g target/L, in a series of second protein A column is ready for elution . During the paired tubular mixer-hollow fiber filter stages in which batch stage: After the complete 1 kg batch is processed the target is preferentially precipitated, the precipitate through the periodic stage, it is taken through VRF, UF/ is de-watered and washed to remove soluble entrained DF, and filtration operations for final formulation . This contaminants, and the purified precipitate is re-dissolved . allows a clear definition of a batch for release testing and Re-dissolution conditions can be set to achieve target disposition . The overall process operation is simplified concentrations up to 100 to 250 g/L, depending on target so that dynamic control is minimized, and the automation solubility and viscosity limitations . Keys to efficient process is integrated across all stages . The complete integrated operation include: (1) pre-concentration of the target stream system occupies a footprint of 8 m x 5 m, including a buffer to enhance the yield and purity of the target precipitate

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90% continuous ≥ continuous bioprocessing of biological drugs, included the continuous bioprocessing of biological drugs, single-pass TFF Cadence™ Inline Concentrators within the continuous (SPTFF) platform, the BioSMB® multicolumn separation chromatography platform, acoustic wave a disruptive cell culture clarification technology, (AWS), to address and novel continuous diafiltration strategies, solution for the the innovation gap to provide a simplified continuous final formulation step . By utilizing a 20L CHO density range of fed-batch cell culture bioreactor with cell 90% cell viability, 25x10^6 – 30x10^6 cells/mL and 65 to multiple in-house feasibility runs were conducted through a novel integrated continuous bioprocessing train of unit operations . For instance, while achieving materials usage, simplicity, and cost relative to packed and cost usage, simplicity, materials has . This process capture bed chromatography-based in of biotherapeutics implications for production particular on total with stringent limits environments or low-resource cost-of-goods . of an End-to-End4. Implementation Platform Continuous BioProcessing Technologies Using Novel Corporation, United States Pall *Engin Ayturk, the biopharmaceutical industry The next frontier for continuous is the widespread adoption of integrated . The key to its bioprocessing for biologics manufacturing is the availability of novel upstream success, however, not only reduce and downstream technologies that will cost of facility footprint, capital expenses and product productivity, goods (CoGs), but also will increase process of single- flexibility and further facilitate the utilization use and/or disposable technologies . In this context, the evaluated suite of cutting-edge technologies we have of to enable cost effective and reliable implementation and to offer significant advantages in performance, raw in performance, significant advantages and to offer 42 | Recovery of Biological Products XVII continuous protein A chromatography-based process and continuous protein A chromatography-based with the current protein A-based platform process . We expect the envisioned process to be generalizable to a wide range of protein- and proteinaceous particle-based products during precipitation; and we have connected precipitate during precipitation; and we have connected and fouling morphologies with filtration performance used process simulation have further mechanisms . We and water/reagent software to compare the CAPEX, OPEX usage for a fully continuous precipitation-filtration-based with a fully monoclonal antibody purification process effects of proteins in solutions of Zn2+, a reversible cross- effects of proteins in solutions of Zn2+, and poly(ethylene glycol) or PEG, a reversible volume linker, of mock excluder; we have formed Zn2+/PEG precipitates in the presence of targets with similar yields and purities morphologies, clarified CHO culture fluids, but with different conditions to demonstrate the importance of contacting tangential flow filtration pre-concentration operation and tangential flow filtration pre-concentration operations, with subsequent polishing chromatography slurry-based e .g ., using membrane adsorbers or continuous to result in a fully continuous purification chromatography, have conducted preliminary work to realize process . We the synergistic solubility have modeled this process . We de-watering and washing stages; and (5) augmentation de-watering and washing re-dissolution stages with additional of the washing and use of multiple washing and/ pairs to permit mixer-filter and to allow counter-current or re-dissolution solutions buffer requirements . This capture operation to reduce with an initial single-pass operation can be integrated experiences the same steady state Camp number, or state Camp number, the same steady experiences time and average shear rate, to product of residence precipitate morphologies; (4) tailoring produce consistent including size distribution of the precipitate morphology, optimize filtration performance during and “stickiness”, to phase; (2) use of synergistic pairings of precipitants, here of precipitants, use of synergistic pairings phase; (2) well, excluders work cross-linkers and volume reversible flow (3) use of a plug total precipitant needs; to reduce that each fluid element to ensure format in precipitation

Oral Abstracts clarification yield for the processing of a batch with 1 .25 g/L titer, 25x10^6 cells/mL & ~70% viability, aforementioned, 1. Synthesis of Computational and process platform was able to deliver ≥ 2 g/h mAb for Experimental Developability to the continuous purification train utilizing a stable 4-fold Support Protein Engineering continuous concentration step for the integration of continuous clarification and continuous capture trains . With *David Roush, Merck, Sharp and Dohme, Inc., United States the coupling of the novel continuous polishing, continuous Francis Insaidoo, Merck, Sharp and Dohme, Inc., viral clearance and continuous final formulation steps, such United States platform, with the current PD-scale bioreactor capacity, Suvrajit Banerjee, Rensselaer Polytechnic Institute, will generate ≥ 1 g/h mAb . Coupled with the process United States economics modeling, this presentation will provide a risk- Steven Cramer, RPI, United States based and data-driven overview of an integrated continuous bioprocessing platform and highlight its subsequent A wealth of possibilities exists for computational requirements, challenges and opportunities for product modeling of proteins including atomistic, coarse-grain and development, process monitoring, validation, control macroscopic Quantitative Structure Retention Relationships and automation . (QSRR) . The key to successfully developing a therapeutic protein from Discovery to Commercialization is the synthesis of the appropriate computational and experimental strategies to rapidly achieve this goal . Protein ligand Session 10 | Biomolecular interactions can significantly influence the biophysical Modeling for properties of the protein . For biologics development, this perturbation can influence the purification profile, solubility

Manufacturability properties, and stability profile of the protein . The focus Oral Abstracts in early development is on affinity and selectivity of the SESSION CHAIRS: therapeutic protein to its cognate ligand, followed by developability measured through experimental techniques . Christopher Dowd, Genentech Currently, in pharmaceutical development strategies, Peter Tessier, Rensselaer the goal is to rapidly assess the impact on platform fit or Polytechnic Institute manufacturability . Under these conditions, the goal is to discern subtle changes in the protein properties (ex . charge

Recovery of Biological Products XVII | 43 . . The computed binding strength binding is sufficient to produce an extensive mAb network binding is sufficient to produce an extensive order 100 mg/ in solution for solution concentrations of predicted mL . In order to investigate the validity of the model predictions high-affinity binding configuration, the for which are used to generate in silico mAb dimers . SANS data taken scattering spectra are calculated a priori mAb show clear on solutions of the strongly-associating resolution capability evidence of mAb clusters, and the high predicted and of SANS allows direct comparison of the these methods together, measured scattering spectra . Taken provide considerable insight into mAb solution behavior that can be utilized along with standard mutagenesis approaches to develop mAbs with both biological efficacy and desirable processing and formulation properties . of residues; such diversity has also been studied explicitly also been studied such diversity has of residues; interest are Of special mutagenesis . by site-directed readily may aggregate mAbs, which strongly-associating viscosities in concentrated anomalously high and may have of the formulations . A more complete understanding such behavior would allow molecular molecular origins of the problematic behavior, designs that could eliminate of as little as a single residue potentially by mutation region (CDR), remote from the complementarity-determining would not be impaired . In this so that biological affinity discuss the synergistic use of atomistic presentation we will neutron scattering (SANS) to simulations and small-angle of mAb-mAb association . We determine the configuration mechanics calculations to study use atomistic molecular number of a set of three mAbs differing by only a small high-viscosity point mutations that appear to control the behavior observed in one of them . A small number of high- were identified affinity mAb-mAb binding configurations and electrostatic and refined, with both non-electrostatic contributions accounted for conditions in isolated configurations under experimental exceeds 30 kT corresponding to high-viscosity behavior to show that such A statistical mechanical model is used encountered even for mAbs differing in only a small number in only a small even for mAbs differing encountered . This G to selectivity) .  G to k’ or differences in G to k’ or differences  44 | Recovery of Biological Products XVII Norman Wagner, University of Delaware, United States University of Delaware, Norman Wagner, David Rosenman, University of Delaware, United States David Rosenman, University of Delaware, University of Delaware, United States Amit Vaish, United States Douglas Godfrin, University of Delaware, P. United States Daniel Greene, University of Delaware, Genentech Inc., United States Sandeep Yadav, Isidro Zarraga, Genentech Inc., United States *Abraham Lenhoff, University of Delaware, United States The large number of monoclonal antibody (mAb) therapeutics that have been developed has revealed the great diversity of biophysical properties that may be 2. Molecular Basis of Strong 2. Molecular Basis of Strong Association in Monoclonal Computations Antibodies: Atomistic and Small-Angle Scattering and statistical assessments of the multiple interactions and statistical assessments of the multiple . The presentation that define purification and formulation approach will provide an overview of the strategic through defined to span Developability from Discovery Commercialization . defined, the focus shifts to optimization of productivity defined, the focus shifts from the a translation requires development stage of second into macroscopic parameters atomistic level of information (ex . linkage of of the second stage requires a Successful development coarse-grain analysis, QSRR different toolset including Ultimately these biophysical tools these biophysical essential . Ultimately analytics is define the impact of primary liabilities can be employed to molecule, which is the endpoint for and to select the best in the Discovery phase . Once the field Protein Engineering for analytics and process has been of potential modalities distribution) on stability, ligand binding (type of ligand) ligand binding on stability, distribution) combination of atomistic . Hence, the and formulation with High Throughput simulations coupled scale molecular and purification, formulation (HTS) encompassing Screening

Oral Abstracts 3. Mechanistic Model Of Excipient Session 11 | New Exchange During Protein Formulation by UF/DF Developments in PAT *Roger A. Hart, Amgen, Inc., United States and QbD

The basis for the compositional design of a parenteral SESSION CHAIRS: drug formulation is focused on the concentration of the active protein ingredient, dosage volume per injection, Gisela Ferreira, MedImmune product stability, and injection tolerability; the later being Thorsten Lemm, Roche largely controlled by the viscosity, osmolality, and excipient species and concentrations . Ultrafiltration and diafiltration is typically employed for the manufacture of protein drug 1. From First Principles products to control the formulation pH, osmolality, and Chromatography Modeling to excipient properties . During UF/DF, complex interactions Process Control Strategy between and among the high-concentration protein molecules and formulation excipients, coupled with volume *Cenk Undey, Amgen, United States exclusion effects, cause divergence of the drug substance Oliver Kaltenbrunner, Amgen, United States pH, excipient concentration, and osmolality from that of the diafiltration buffer . This paper describes a UF/DF unit Traditional process characterization to support a biological operational model which accounts for the effect of these marketing application typically starts with risk-based interactions on excipient retention or rejection throughout parameter prioritization followed by the development of the initial concentration, diafiltration, final concentration, an empirical model based on designed experiments that Oral Abstracts and flush recovery . Electrostatic interactions are described evaluate the effects of the selected parameters . This using the Poisson-Boltzmann theory with full accounting for empirical model is used to understand process responses electro-neutrality, protein contribution to buffer capacity, within the characterized ranges for unit operations in the protein charge titration, zwitterion excipient speciation, process . While very effective, these techniques still require excipient activity coefficients, and protein residue activity . considerable amount of resources and time and do not take Protein molecular weight and net charge are calculated advantage of the available fundamental understanding from the amino acid sequence and excipient properties of unit operations . It is desirable to utilize the available utilize NIST traceable constants; a single “charge bias” first principles understanding of the process for improved constant is used to calibrate the otherwise entirely mechanistic model . Performance was assessed and robustness, predictability and optimization and to integrate qualified for four monoclonal antibodies through statistical this knowledge in process control strategies and regulatory comparisons between model predictions and experimentally applications . We have been successfully advancing first measured data from DOE multi-factorial characterization principles modeling in the area of cation exchange (CEX) studies . Following model assessment, additional process chromatography to mimic the results of wet experiments . scenarios were modeled to demonstrate the model’s The focus of this presentation will be a review of our potential as a tool for process design and optimization . methodology of model development, the development of

Recovery of Biological Products XVII | 45 This leads to a situation . This leads ii) predictive models to predict drug substance process ii) predictive models to predict drug substance drug substance performance based on resin RM data and analysis and its process data; iii) chromatogram feature consistency; iv) correlation with process performance and believe this type resin cycle performance prediction . We drug substance of process analytics collaboration between will more than manufacturers and raw material suppliers compared to each double the power of smart bioprocessing party acting alone . with the production process itself, leaving significant leaving significant process itself, with the production understand and control to better room for improvement (RM) variability raw material place is usually the first manufacturing where commercial resulting in of RM variability, to experience the impact manufacturing investigations and consequences of lengthy this conundrum, this batch rejection . In an attempt to solve a synergistic life cycle approach to case study will illustrate RM variability through data sharing understand and control analytics between a biologics and collaborative process and a chromatography resin raw manufacturer (Biogen) Healthcare) . The essence of this material supplier (GE assess RM variability through approach is to continuously by analyzing combined data from RM the process lifecycle RM risk by and drug substance processes, and to retire control improvement . continuous process understanding and enable this approach, data aggregation systems, To open collaboration multivariate data analysis capability and . A pilot case between both sides are critical elements multivariate analytics encompassing end-to-end study, on a commercial applied across multiple unit operations demonstrate manufacturing downstream process, will proof-of-concepts of i) resin variability assessment resin RM data; through multivariate analysis of expanded QbD paradigm have mostly focused on variability associated on variability have mostly focused QbD paradigm

. 46 | Recovery of Biological Products XVII Roger Lundqvist, GE Healthcare, Sweden Dave Kolwyck, Biogen, United States Biogen, United States Sarah Yuan, Robert Guernard, Biogen, United States Mattias Ahnfelt, GE Healthcare, Sweden Per Lidén, GE Healthcare, Sweden all critical sources of variability are identified and explained; all critical sources of variability are identified and, (3) product (2) variability is managed by the process; reliably predicted quality attributes can be accurately and biologics process over the design space established . So far, even under the development and characterization efforts A process is generally considered well understood when (1) A process is generally considered well Collaborative Process Analytics Analytics Process Collaborative *Gunnar Malmquist, GE Healthcare, Sweden *Canping Jiang, Biogen, United States CEX chromatography Life Cycle Synergistic 2. Smart2: A to Understand and Control Approach through Variability Material Raw pharmaceutical process to improve process robustness and process robustness process to improve pharmaceutical wet experiments and development control while reducing leveraging advanced cycle time . This opportunity of for efficient process development is process understanding case studies of demonstrated on industrial a regulatory filing strategy, and an overview of feedback of feedback and an overview filing strategy, a regulatory will articulate . We regulatory interactions received from can be of a unit operation understanding how first principles of a bio/ and regulatory filing in the development integrated

Oral Abstracts 3. Raman Spectroscopy-based Session 12 | Increasing Multivariate Analysis as a PAT Tool for Impurity Detection and Patient Access to Adaptive Process Control in Biopharmaceuticals Downstream Bioprocessing *Siddharth Parimal, Biogen, United States SESSION CHAIRS: Sanchayita Ghose, Biogen, United States Jon Coffman, Boehringer Ingelheim John Pieracci, Biogen, United States Bruno Marques, GlaxoSmithKline

The call for increased understanding and better control of biopharmaceutical processes by the regulatory authorities 1. Broad World Access Requires under the QbD framework has paved the way for various a Broad Approach to Process PAT tools to be used for monitoring and analysis of critical Application: Process, Equipment, quality attributes at various steps in the biomanufacturing process . The work presented herein proposes a paradigm Automation and New Facility shift in the way product quality is typically measured in Designs a GMP facility . Currently, the analysis of downstream *Joseph Shultz, Novartis Pharma AG, Switzerland intermediates is performed at-line or on-line . In contrast, we Lars Pampel, Novartis Pharma, Switzerland use a combination of Raman spectroscopy and multivariate data analysis as a potential enabling technology for Mesbah Crietz, Novartis Pharma AG, Switzerland fast, non-invasive, in-line analysis during downstream Urs Wernli, Novartis Pharma AG, Switzerland Oral Abstracts bioprocessing . We develop correlations of Raman spectra Spencer Fisk, Novartis Pharma AG, Switzerland with different critical quality attributes (e .g ., high molecular weight species, acidic isoforms) for multiple monoclonal To sustainably deliver biopharmaceuticals to both antibodies, demonstrating a PAT tool which is easy to use industrialized and emerging markets, requires that the and has minimal manual intervention . We provide examples entire development cycle must be economically feasible where in-process measurements of the intermediates can on a global scale . Thus, we must look far beyond yield drive feedforward control for the subsequent purification maximization and raw materials cost reduction . The step, resulting in flexible processes where input parameters entire Development and Manufacturing spectrum must be are modulated to deliver consistent product quality . The addressed, from molecule design to process development approach presented in this strategies, to the clinical program and manufacturing facility work can provide opportunities for real-time, adaptive design . The measure is not as simple as targets like COGM control strategies to be implemented for robust, continuous of 1% of the selling price, but rather total costs low enough control of downstream operations . to serve emerging markets, while also serving industrialized markets and making biosimilar entry financially

Recovery of Biological Products XVII | 47 Günter Jagschies, GE Healthcare Life Sciences, Sweden Günter Jagschies, GE Life Sciences, Sweden John Joseph, GE Healthcare is tremendous pressure to fill up the excess capacity with is tremendous pressure to fill up the excess contract manufacturing . Then, if market demand increases to begin with, or if the factory was too small significantly, build or outsource the cycle repeats and we are forced to is manufacturing . Even if capacity is just right, capital cost a barrier to change . New technologies that require different significant equipment are difficult to industrialize when have to be writeoff of equipment is required, or if buildings seem particularly smart . Therefore, modified . This doesn’t 2. A Simple GPS2. A Simple Lowering for High, Is Too Cost, Which Capital High Margins Even With United States *John Erickson, GSK, industry has debated Cost of Goods The biopharmaceutical time, but that measure alone can Sold (COGS) for a long assumes that capital be misleading . The COGS calculation this over a long time . Usually, cost is fully depreciated expense comparable to labor and makes the depreciation when you have to ask raw materials . That is fine, except the factory in for hundreds of millions of dollars to build the the first place . As price pressures continue to mount in this problem should get worse . Compounding the industry, about taken traditionally have plants that fact the is problem . This means 3 or more years to design, build and validate be made well in that major investment decisions need to even advance of actual market demand and sometimes before products are approved . Because market forecasts are that are either too inexact, this leads to building factories big or too small . If the factory turns out to be too big, there

48 | Recovery of Biological Products XVII discuss what we feel are the major economic impactors discuss what we feel are the major economic key aspects that will enable broader global access and and advanced of how continuous/integrated processes automation, allows us to implement commercial a broad range manufacturing facilities that will serve of molecule formats for the future . broad range of molecule-types . This will be facilitated span from cellular with flexibly integrated processes that and distribution . production through drug product packaging – process, In fact, the future requires that all components integrated as one, equipment, automation and facility – be will profile . We to unlock the optimal flexibility and cost we are not already burdened with a large underutilized we are not already burdened with a large to redefine how we infrastructure, we have the opportunity . Development develop and manufacture biopharmaceuticals newer technologies will utilize a balance of traditional and masses in small, to enable the production of large-facility can produce a easily built and replicated facilities that gram produced, regardless of how we design our processes of how we design our processes gram produced, regardless to unexpected demand changes • Ability to rapidly respond drug product presentation costs • Management of flexible attrition and sunk development • Exposure to program by a few commercial products costs that must be recouped time under patent coverage As • Limited commercial broader markets and efficient distribution chains may be distribution chains and efficient broader markets are driving to a new development more important . We that can be applied anywhere and manufacturing paradigm, historical challenges, like: in the world and address and labor that adds cost to every • Underutilized capital Thus, the scope of process development scope of process development . Thus, the unappealing the containment across to also enable cost must expand drug substance already capable of . Novartis is organization to sales price, but access at less than 1% of production

Oral Abstracts no matter whether COGS in a particular market is a large or small fraction of sales, everyone should be concerned about 3. Manufacturing gp120 Based reducing capital cost . Standardization of equipment design Novel HIV Vaccine Candidates: across the industry should eliminate redundant engineering Simultaneously Meeting the costs and decrease leadtime and can be done without any Demands of Cost and Rapid new technological innovations, but requires us all to work together . To guide the journey to even lower and lower Progression From Bench To Bedside capital cost, we propose using the total volume of process *Abhinav Shukla, KBI Biopharma Inc., United States equipment as a surrogate for capital cost . If the size of the Leslie Wolfe, KBI Biopharma Inc., USA equipment gets smaller, it should become less expensive Carnley Norman, KBI Biopharma Inc., USA and easier to install . The building should be smaller Niket Bubna, KBI Biopharma Inc., USA as well . Process volume can be calculated easily from process parameters for individual unit operations or by an Sigma Mostafa, KBI Biopharma Inc., USA overall mass balance for an entire factory . To map out the Jimmy Smedley, KBI Biopharma Inc., USA endpoints of the journey, we calculated the process volume Munir Alam, Duke Human Vaccine Institute, USA of an existing factory by adding up the process volume of Thomas Denny, Duke Human Vaccine Institute, USA each major piece of equipment . We then used a simple Barton Haynes, Duke Human Vaccine Institute, USA mass balance to calculate a theoretical, smallest possible plant, where all the process equipment had 100 mg/mL Development of a vaccine for HIV has long been a holy of product in it at all times, which is a practical solubility grail for immunologists . The complications of dealing with limit . Assuming a cycle time of 8 hours for the entire a rapidly mutable virus with multiple mechanisms evolved downstream process in this theoretical plant, compared to evade the human immune system have led to the failure with one week for the existing facility, the theoretical of multiple vaccine candidates in clinical trials . However, Oral Abstracts minimum plant volume, normalized by output, is 5 orders recent progress made at the NIAID funded Duke Human of magnitude less than that of the existing facility . Since Vaccine Institute is paving the way for possible solutions for buffer makeup and hold tanks constitute the vast majority this devastating disease . Scientists at DHVI have created of current downstream process volume, we start with ways a series of protein sequences [1,2] based upon the HIV- to decrease buffer tankage and will show results of process 1 trimeric vaccine envelope glycoprotein (ENV) that are modeling for various scenarios where buffers are made up capable of inducing a broad neutralizing antibody (bNAb) continuously . We will also provide simple models showing response that might finally form a viable pathway to protect the effects of resin capacity, residence time, buffer usage against this virus that has had devastating consequences and product concentration on process volume . These models around the world . Introduction of a novel HIV vaccine will can make process development scientists smart enough to have significant consequences for global health and serve develop processes that reduce capital cost, which in turn patients across economic and geographic spectra . The will make it easier to adapt to whatever the future development and manufacturing of these novel candidates may bring . is a key bottleneck in introducing and testing these vaccine

Recovery of Biological Products XVII | 49 Kepler, S . Harrison . Affinity .Kepler, B-cell lineage immunogen design in . B-cell lineage immunogen . Gao, H . Liao, T . Kepler culture process development – reproducibility, scalability culture process development – reproducibility, 718-727, and control, Biotechnology Progress, 30(3), 2014 . 5) U . Gottschalk, A . Shukla . Single-use disposable Trends technologies for biopharmaceutical manufacturing, 31(3), 147-154, 2013 . in Biotechnology, Finally, seamless transition seamless bioreactors [4] . Finally, cell culture was achieved clinical manufacturing into cost optimal production a single-use manufacturing by leveraging some of the will highlight train [5] . This presentation leveraged to create this family immunological pathways and describe the rapid development of vaccine antigens platforms that have taken these of process and analytical to bedside . The economics of rapidly candidates from bench clinical trial candidates will developing and producing driver throughout the development be shown to be a key . 1) B . Haynes, G . Kelsoe, S . and manufacturing process Harrison, T Nature study, as a case with HIV-1 vaccine development . 2) D . Fera, A . Schmidt, 2012, 30(5), 423-433 Biotechnology, B . Haynes, F B-cell lineage maturation in an HIV broadly neutralizing PNAS, 2014, through re-orientation of variable domains, S . Mostafa, . Barringer, C 111(28), 10275-10280 . 3) L . Wolfe, A . Shukla . Multimodal chromatography: characterization through of protein binding and selectivity enhancement A, mobile phase modulators, Journal of Chromatography . Miller, 1340, 151-156, 2014 . 4) S . Rameez, S . Mostafa, C for cell A . Shukla . High-throughput miniaturized bioreactors was achieved by leveraging high throughput miniaturized throughput miniaturized by leveraging high was achieved . 50 | Recovery of Biological Products XVII assay to monitor effective binding to selected bNAbs assay to monitor effective binding to selected for each vaccine glycoprotein . Rapid cell culture process glycan profile development to produce an optimally active provided a unique capture step that simultaneously cleared provided a unique capture step that simultaneously product stability host cell protein species and improved steps and The development of dedicated viral clearance and weak polishing steps based on ceramic hydroxyapatite guided by the cation exchange chromatography were based binding use of a surface plasmon resonance (SPR) cell proteins, heavy glycosylation and the requirement to cell proteins, heavy glycosylation and the needed to maintain binding activity to key bNAb antibodies lay be overcome . The solution to these diverse challenges process in the form of a well evolved platform downstream steps . based purely upon non-affinity chromatographic wash steps [3] Multimodal chromatography with selective consideration due to the number of candidates that need to the number of candidates that need to consideration due to was found trials . A platform approach progress into clinical to meet with this dual challenge of to be a key necessity multiple challenges including However, speed and low cost . proteolytic clipping, low expression the susceptibility to of some difficult to remove host levels, the presence replicate the pathways by which some resistant patients some resistant patients pathways by which replicate the . One-off process development render the virus ineffective approaches are clearly efforts using conventional the needs in this space . Costs for inadequate to serve are another simultaneous development and manufacturing These trials are an . These trials in human clinical trials candidates against preventative vaccine in perfecting a essential step requiring iterative process will eventually be an HIV which can sequences that of multiple, evolved the production

Oral Abstracts The Recovery Conference Debates

Continuous Processing Mathematical Modelling vs. DoE Disruptive Technologies Debate Session

Recovery of Biological Products XVII BERMUDA, JUNE 19-24, 2016 Debate Session: Tuesday June 21 at 19 .45 Session Chairs: Nigel Titchener-Hooker, John Curling

Recovery of Biological Products XVII | 51

John Curling

Jonathan Coffman and Brian Kelley Jonathan Coffman MODERATOR: SUT! Choose… ≠ I: Continuous Processing I: Continuous DEBATERS: DEBATERS: Connected ≠ Perfusion history is checkered, continuous purification is yet unproven… why use continuous DSP? perfuse, then If you don’t Production flexibility is limited (CMOs, >1 site in an internal network) What problem(s) are we solving with continuous processing? very complicated … continuous USP + DSP = a flying car! It’s Investment in continuous processing technologies mitigates perceived risk mitigates perceived Investment in continuous processing technologies Continuous processing can make more Kgs and products in the same facility Kgs and products in the Continuous processing can make more for its complexity assembly line was also criticized Henry Ford’s and fast facility construction Continuous processing enables flexible critical path with a launch to get off the It allows development and clinical manufacturing capable process 52 | Recovery of Biological Products XVII • Continuous Is there a strong business case or are we looking for things to do!? • • • AGAINST: • • • Are we afraid of new things? • • • • Is the increased investment and additional risk worth the reward? Is the increased investment FOR: Continuous bioprocessing is all the rage! A fully continuous, scalable commercial cGMP process would be scalable is all the rage! A fully continuous, Continuous bioprocessing or benefits does it offer vs . established . But what problems tour-de-force unprecedented, a technological to be valuable? we really need a process to be fully continuous batch processing? Do unit distinctly different topics – disposable and disentangle related but behind the hype, discuss what’s Let’s etc . operations, connected vs . continuous, PAT,

Debate Session II: Mathematical Modelling vs. DoE MODERATOR: Jürgen Hubbuch DEBATERS: Karol Łaçki and Arne Staby

Process understanding is crucial to modern bioprocessing . Design-of-Experiments (DoE) and other statistical methods are the industrial workhorses behind process development and process validation efforts for they are intuitive, well described and easy to implement . However do they provide a real process understanding or just give a false perception of being in control? In contrast, mechanistic models based on first principles are seen by many as the ultimate process understanding tool . Advances in basic research and in computational power of standard computers seem to support this expectation, but is it really realistic or does it make sense? While a first principle model of a scalable commercial cGMP process would be unprecedented, it is feasible but what problems or benefits does it offer vs . established DoE?

An industry wide discussion, including both academia and industry, on pros and cons for both approaches and their applicability to process control, trouble shooting, and other related areas is required .

To Do Experiments or not to Do (many) Experiments? That is the question!

FOR: Mechanistic Modelling

• It’s complicated… or is it?

• Regulatory agencies understand it (not) Debate Session • Why settle for less?

• Methods may be extrapolated outside experimental areas

As long as you know why it really works then...... ??

FOR: DoE

• It’s simple – everyone can do it!

• Regulatory agencies understand it

• Accuracy good enough

• Who needs to extrapolate anyway!

As long as it works, you really don’t need to know why!

Recovery of Biological Products XVII | 53 . Why Manufacturing costs must come down . Todd Przybycien Todd Hanne Bak and Jörg Thömmes Hanne Bak and MODERATOR: MODERATOR: DEBATERS: III: Technologies Disruptive . COGs not an issue, don’t waste my time COGs not an issue, don’t is my key driver and time to market Therapeutic protein manufacturing is a high margin business works great and has a lot of upside The CHO platform combined with packed bed chromatography I upset the apple cart? regulated environment, why would are working in a highly We resolving power of column It is about patient safety: purity rules and nothing can touch the Titers are going up and up, making it hard for column chromatography to keep up and making alternative it hard for column chromatography are going up and up, making Titers really simple and robust Alternative processing equipment can be lately? Have you purchased 500L of protein A resin Healthcare costs are a real issue, not only campaign rhetoric . Healthcare costs are a real issue, not only manufacture can’t populations . We with very large patient Proteins are moving into therapeutic areas risk delaying launch on all these unknowns? optimize this first potential, let’s chromatography proteins for tens of millions of patients the way we manufacture for hundreds of thousands the way we manufacture for proteins for tens of millions of patients processes more attractive What is the driver here? • • • • • Are we afraid of new things? AGAINST: • • • FOR: • • purifying therapeutic proteins by column chromatography forever? Or will our industry learn from industrial chromatography forever? purifying therapeutic proteins by column protein refineries? enzymes and purification trains will become versus New (Old) School. Mano a mano. pick this apart. Old School Let’s In therapeutic protein manufacturing the CHO based platform and column chromatography rules . Will it and manufacturing the CHO based platform In therapeutic protein platforms be by-passed by disruptive traditional therapeutic protein manufacturing be ever thus? Or will computers were made obsolete? mainframe large integrated steel manufacturing or innovation the way and mobile like the PC by mobile productive be replaced by something more Will the current platforms is no way to compare steel mills and cell lives in Healthcare, there are saving people’s computing? But we . Or is there? If so, will we be in pharmaceutical manufacturing phones with innovative medical research 54 | Recovery of Biological Products XVII

Debate Session Poster Abstracts

the present work we have developed a new pre-sequence Session 13 | Poster Session with alkaline pI that will allow the use of cation exchange chromatography as capture step when producing a low – Oceans of Innovation pI protein in E .coli . As model protein we have used human growth hormone, a protein of 22 kDa with a pI of SESSION CHAIRS: approximately 5 [3], similar to a large number of the proteins Charles Haynes, University of originating from E .coli . In order to be able to remove the pre-sequence, hence allowing production of a mature British Columbia protein, a prerequisite for development of the pre-sequence Abraham Lenhoff, University of Delaware was that it should be possible to remove it enzymatically David Roush, Merck & Co., Inc. using diamino peptidase during the downstream purification process . Another requirement was soluble protein expression and preferably high expression level . In order 1. Development and Lab to Pilot to obtain an increase in pI of approximately one pH unit, Scale Testing of a New High pI it was calculated that the pre-sequence should contain at least three lysine or arginine residues . Previous experiments Presequence Useful for Purifying had shown that diamino peptidase does not work well with Low pI proteins from E.coli peptides containing arginines [2, 4] and hence the tests Fermentation focused on lysine rich peptides . Subsequently, a series of pre-sequences were tested containing 2, 3, 6 or 7 lysine *Christine Bruun Schjødt, Novo Nordisk A/S, Denmark residues named SEQ1, SEQ2, SEQ3, SEQ4, SEQ5 and SEQ6 . Gong Wei, Novo Nordisk A/S, China As expected from the theoretical calculations, it was found Yu Mingrui, Novo Nordisk A/S, China that three lysines indeed provided an increase of pI by one Chen Jianhe, Novo Nordisk A/S, China unit and also rendered soluble protein at high expression level . Unfortunately, approximately 10% of the expressed Xia Wenjuan, Novo Nordisk A/S, Denmark protein was N-terminally formylated, leaving the N-terminus He Xugang, Novo Nordisk A/S, China blocked for DAP activity . To mitigate an inherent productivity Christian Tihic Rasmussen, Novo Nordisk A/S, Denmark loss, the pre-sequence was improved by inserting a serine Anja K Pedersen, Novo Nordisk A/S, Denmark residue as the second residue, thereby enabling removal of the N-terminal methione by E .coli enzymes during protein The large majority of E coli. proteins have slightly acidic expression [1] . The resulting pre-sequence SEQ7 was Poster Abstracts pI values, presenting a purification challenge when subsequently used for experiments in 1 L, 20 L and 100 producing recombinant proteins using E .coli as host . In L pilot scale to investigate scalability of fermentation as

Recovery of Biological Products XVII | 55 . In our work, however, we we . In our work, however, target protein is captured as the intein segments assemble target protein is captured as the intein into an active complex . Although the Npu intein has been researchers, it developed as a self-cleaving tag by other thiol compounds was shown that the intein still required in order to cleave effectively segments of have made several point mutations to both this intein . These mutations provide a highly effective with only pH in means for controlling the assembled intein the intein we have modified the absence of thiol . Further, be covalently segments so that one intein segment can onto a chromatographic specific orientation in a immobilized backbone . The other intein segment, which is only 35 amino acids in length, then becomes the target protein tag . Once assembled and washed, our modified Npu intein is complex glycoproteins expressed in mammalian and other in mammalian and expressed complex glycoproteins premature reason for this is hosts . The primary eukaryotic protein tags self-cleave during pH-sensitive cleaving, where pH and temperature due to the permissive expression cell culture . Although required for mammalian conditions tags have been developed, the thiol-induced self-cleaving compounds break disulfide bonds required thiol-containing and are economically prohibitive at in the target proteins a truly convenient and large scale . Thus, the promise of system has yet to materialize for effective self-cleaving in mammalian systems – until now complex glycoproteins have extensively re-engineered the In our recent work, we from the Npu dnaE protein, which has naturally split intein well as separate segments, and been shown to express with extraordinary speed . An important assemble and splice split intein aspect of split inteins is that the separated unable segments are inactive alone, and are therefore . Once to cleave prematurely during protein expression the assembled segments recombined in vitro, however, to generate form an active complex, and can be used self-cleaving tags . Thus, one segment of a split intein onto a solid can be expressed, purified and immobilized used to tag a support, while the other segment can be target protein . Strong and specific association of the tags resin, where the effectively mimics a conventional affinity . mple

. This platform More importantly, however, tags generally cannot however, . More importantly, 56 | Recovery of Biological Products XVII USA Atefeh Alizadehbirjandi, Ohio State University, USA Atefeh Alizadehbirjandi, Ohio State University, Tzu-Chiang Han, Ohio State University, USA Tzu-Chiang Han, Ohio State University, USA Ohio State University, Joseph Taris, USA Ohio State University, Merideth Cooper, USA Fan, Ohio State University, Yamin Changhua (Steven) Shi, University of California at Irvine, Changhua (Steven) Shi, University of California In our previous work, we have developed highly desirable . In our previous work, we have developed self-cleaving tags by combining conventional affinity tags with modified inteins . Although this approach has been highly effective for proteins produced in bacterial expression hosts, these methods have been ineffective for remain regarding impacts of the tag on the target protein remain regarding impacts of the tag on activity due to the be used for therapeutic protein manufacture the expense and potential immunogenicity of the tag and removal . For these process complexity associated with tag tag removal is reasons, a cheap and effective means for The ability to purify arbitrary target proteins using a si The ability to purify arbitrary target proteins in biological universal platform has long been a goal research and the biopharmaceutical industry the use of affinity is provided at laboratory scale through occasional concerns tags, although even at laboratory scale 2. A Self-cleaving Tag System System Tag 2. A Self-cleaving Glycoproteins for Complex USA Ohio State University, Wood, *David W. scale showed that the process was indeed very robust and was indeed very robust that the process scale showed development and the pre-sequence . Data from reproducible scales will be presented in the poster the tests in various 86, 8247-8251 2 . References: 1 . Hirel et al (1989) PNAS and purification 76, 59- et al (2011) Protein expression Yang . US07858747 B2 et al (1993) 3HHR .PDB 4 64 3 . De Vos well as recovery process and subsequent removal of the subsequent removal process and well as recovery capture of expression, recovery, . Comparison pre-sequence to 100 L from lab removal of the pre-sequence and finally

Poster Abstracts induced to rapidly cleave in response to a small pH shift, small-scale model experiments to produce aggregates of releasing the target for collection . Importantly, the affinity monoclonal antibodies by controlled shifts in pH or ionic resin can then be regenerated, where the small tag intein strength as well as by exposure to various interfaces . segment is removed to prepare the column for the next Aggregate secondary and tertiary structure was analyzed by target protein . We have successfully used this system spectroscopic methods . Aggregation kinetics was shown to to purify several proteins from several expression hosts, be highly reproducible and rate constants of nucleation and including fully glycosylated and active secreted alkaline growth were extracted from kinetic traces . In an additive phosphatase (SEAP) and tissue plasminogen activator (tPA) screen including compounds from three different substance expressed in HEK and CHO, without any trace of premature classes, molecular descriptors were correlated with both cleaving . Further, the availability of a reusable affinity aggregation rate constants and thermal stability changes resin, akin to Protein A for non-antibodies, suggests a high using a quantitative structure activity relationship (QSAR) potential impact for these methods at both laboratory and approach . The analysis reveals a correlation between manufacturing scales . Finally, we have also incorporated certain molecular properties such as relative and absolute this technology as the first capture step of our DARPA surface polarity and the protective effect of additives BioMOD device, which will also be discussed . tested . Negative correlation with some descriptors, i .e . destabilizing properties, were also identified . This approach 3. QSAR Analysis of Additive will be extended to a larger compound set with the aim to Effects on the Aggregation of obtain more detailed information, eventually enabling the Monoclonal Antibodies design of improved additives . Olubukayo Oyetayo, Biberach University of Applied 4. Using Quantitative Structure- Sciences, Germany Fabian Bickel, Biberach University of Applied Sciences, Property Relationship Analysis Germany and High-Throughput Methods Martina Merg, Biberach University of Applied Sciences, as Tools for Molecular Assessment Germany & Process Development Oscar Mendez Lucio, Cambridge University, United Kingdom Lydia Beasley, Genentech, Inc., United States Andreas Bender, Cambridge University, United Kingdom Brian Connolly, Genentech, Inc., United States *Hans Kiefer, Biberach University of Applied Sciences, Tom Patapoff, Genentech, Inc., United States Germany Paul McDonald, Genentech, United States *Benjamin Tran, Genentech, Inc., United States Aggregates of biopharmaceutical proteins that form upon exposure to various stress conditions can adopt High-throughput partition coefficient (Kp) determination widely different molecular structures . Depending on the enables the rapid mapping of antibody binding behavior mechanism of formation, they also behave differently with on different chromatography resins . This information can respect to aggregation reversibility . We have established

be used to inform chromatographic process development Poster Abstracts

Recovery of Biological Products XVII | 57 . All these studies are ., Williams, D . R . & Liddell, . It was shown that over all . Y The major part of this work presents an . The major part of this work presents an are then correlated with aggregation data obtained from are then correlated with aggregation data size-exclusion chromatography especially be found test conditions, good correlations could between B22 and aggregation rate . Finally a few selected both good solution conditions were chosen that indicated the validity of B22 and poor stability to further investigate as an indicator for protein stability the mechanisms performed enabled a better insight into of SIC- and of aggregation via an improved understanding . REFERENCES CIC- protein-protein chromatograms obtained Hedberg, S . H . M ., Heng, J . Y J . M . (2015) . Self-Interaction Chromatography of Mabs: . Pharmaceutical Accurate Measurement of Dead Volumes describes protein-protein interactions solution, which can solution, which protein-protein interactions describes the aggregation propensity tool for predicting be a useful propensity of predicting aggregation . One way of proteins (SIC), which recently chromatography is self-interaction promising tool for better understanding have shown to be a cross- of proteins . Another technique, of phase behaviour (CIC), has shown to be an interaction chromatography technique than its predecessor even more high-throughput here was . The work reported with the same capabilities as well as a polyclonal IgG . performed on two mAbs SIC immobilisation strategy a In order to find the best chromatographic resins and solution number of different to established the most effective conditions were screened of these . For the best employment immobilisation method from techniques a scale-down study was performed columns, laboratory scale macro-columns to micro-scale process by which will enable more efficient screening of a mAbs for a only needing micrograms or milligrams full stability study pH and salt, extensive formulation study of mAbs, varying as well as as well as the presence of different stabilisers aggregation . The different external factors known to induce formulation study B22 and B23 values determined from the (B22) is a fundamental physiochemical property that property that fundamental physiochemical (B22) is a

58 | Recovery of Biological Products XVII United Kingdom John Liddell, Fujifilm Diosynth Biotechnologies, John Liddell, Fujifilm Diosynth Biotechnologies, United Kingdom Daryl Williams, Imperial College London, Jerry Heng, Imperial College London, United Kingdom Jerry Heng, Imperial College London, United proteins such as mAbs . Much effort has been employed to gain a better understanding of aggregation, however the mechanisms leading to protein aggregation are still not fully understood . The osmotic second virial coefficient Protein-protein molecular interactions are known to be Protein-protein molecular interactions are behaviour and involved in protein solution aggregation of therapeutic are a common issue for the manufacturing Aggregation for mAbs for during Aggregation Bioprocessing United Kingdom *Sarah Hedberg, Imperial College London, We show resin . We commonly used cation- and anion-exchange high-throughput how this data can be used in concert with to accelerate batch binding and robocolumn methods purification process development . and Controlling 5. Predicting molecular dynamics modeling using antibody primary amino modeling using antibody primary amino molecular dynamics were then calculated acid sequences . Molecular descriptors outputs including, but not limited using specific generated charge, solvent-accessible surface to, residue position, then used to . These descriptors were area, and trajectories model to predict antibody Kp values on train a QSPR-based antibodies on commonly used resins from their amino resins from their amino on commonly used antibodies during early . This is advantageous acid sequences alone where material for testing may be molecule selection, we determined chemical structure limited . In this study, of antibodies using homology and information for a library The historical Kp database generated Kp database generated . The historical for antibodies using Quantitative screens can be leveraged from these create (QSPR) analysis to Relationship Structure-Property of new binding behavior model to predict the an in silico

Poster Abstracts Research, 32, 3975-3985 . Quigley, A . & Williams, D . the reliability of identifying antibody fragments with R . (2015) . The Second Virial Coefficient as a Predictor excellent biophysical properties . We are also developing of Protein Aggregation Propensity: A Self-Interaction a second method for screening the biophysical properties Chromatography Study . European Journal of Pharmaceutics of hundreds to thousands of mAb candidates obtained via and Biopharmaceutics, 96, 282-290 . immunization or related methods . Our approach is to use gold nanoparticles coated with anti-human antibodies to capture mAbs of interest and then assess the colloidal 6. High-throughput Methods for stability of the resulting immunoconjugates to evaluate Selecting and Optimizing Highly mAb self-association . Surprisingly, we find that our high- Developable Monoclonal Antibodies throughput mAb self-interaction measurements are not only correlated with conventional biophysical properties *Peter Tessier, Rensselaer Polytechnic Institute, such as antibody solubility but also with non-conventional United States ones such as non-specific interactions with non-adsorptive chromatography columns . We expect that these and related A key challenge in developing monoclonal antibodies high-throughput methods will improve the selection and (mAbs) as therapeutics is their variable and difficult- optimization of antibodies that are well suited for the to-predict biophysical properties (solubility, viscosity, extreme environments (high concentration, extremes in pH aggregation, polyspecificity) that collectively determine and temperature, exposure to various types of surfaces) their developability . Thus, methods for assessing encountered during antibody purification, formulation developability at the earliest stages of antibody discovery and delivery . are critical for reducing problems that may occur later during antibody development . We are establishing several high-throughput methods for assessing the biophysical 7. Molecular Modelling to Predict properties of antibody libraries to guide the selection of Chemical Stability highly stable and well-behaved mAb candidates . First, we are developing methods for assessing the biophysical *Lydia Beasley, Genentech, United States properties of large antibody libraries (millions to hundreds Amy Hilderbrand, Genentech, United States of millions) that are displayed on the surface of yeast . Nisana Andersen, Genentech, United States Yeast surface display is increasingly being used to identify Bill Galush, Genentech, United States high-affinity antibodies, and its compatibility with flow Vikas Sharma, Genentech, United States cytometry provides exciting opportunities for unusually Tom Patapoff, Genentech, United States high-throughput analysis . We find that selection of high- affinity antibody fragments using yeast surface display Early assessment of the stability and manufacturability often leads to variants with defects in antibody stability of multiple potential molecule candidates can be crucial and specificity . To overcome this challenge, we have to the selection of lead molecules that (1) have a high identified conformational probes specific for well-folded probability of technical success later in development, and antibodies that enable co-selection of antibodies with (2) enable the use of platform processes that accelerate high affinity and stability, and which greatly increase development . Molecular dynamics (MD) simulations Poster Abstracts

Recovery of Biological Products XVII | 59

We . We

United States United States Novo Nordisk, Denmark United States Hanne Sophie Karkov, Downstream Technology, Downstream Technology, Hanne Sophie Karkov, Rensselaer Polytechnic Institute , James A. Woo, Rensselaer Polytechnic Institute , Steven M. Cramer, Our lab recently has reported on the rational design Our lab recently has reported on the rational and of homologous series of antibody Fab variants as demonstrated that these libraries can serve in multimodal powerful tools to investigate selectivity experimental chromatographic systems . In this poster, experiments with data obtained from linear salt gradient robust quantitative these libraries are employed to develop for the structure activity relationship (QSAR) models prediction of Fab retention in several chromatographic systems . These models are the first reported to date for the a-priori prediction of retention behavior of large, complex proteins such as Fabs . Model development is enabled by extended single-molecule methods to the investigation of to the investigation single-molecule methods extended also are phenomena . We protein adsorption competitive multi- studying macroscopic novel methods of developing of constant competitor under conditions protein adsorption on agarose ion-exchange concentration, including of controlled ligand density chromatographic columns from each of these approaches, as will present results protein/adsorbent systems . applied to the same Libraries: Fab 9. QSAR For Models to Tools Predictive Powerful Development Process Facilitate and Identify Important Surface Properties Institute, *Julie R. Robinson , Rensselaer Polytechnic imaging to the study of protein adsorption, and now have adsorption, and now the study of protein imaging to

60 | Recovery of Biological Products XVII Lydia Kisley, Rice University, United States Rice University, Kisley, Lydia United States Sagar Dhamane, University of Houston, United States Katerina Kourentzi, University of Houston, United States Christy Landes, Rice University, Ujwal Patil, University of Houston, United States Ujwal Patil, University of Houston, United Competitive adsorption of proteins from multicomponent mixtures is the basis of chromatographic separations, but has not been extensively studied in mechanistic recently introduced the use of single-molecule detail . We *Richard Willson, U of Houston, United States *Richard Willson, U of Houston, United 8. Competitive Ion-Exchange From of Proteins: Adsorption Columns to Single Molecules molecule, fully atomistic molecular dynamics simulations molecule, fully atomistic molecular dynamics . Strong predictive of the antibody Fv-region were executed MD outputs relationships were found between certain and MET/TRP oxidation risk . Prediction of additional will physiochemical properties using MD outputs be discussed . that careful analysis of MD trajectories can predict the that careful analysis and tryptophan (TRP) oxidation risk of methionine (MET) potential to impact efficacy and shelf events that have the data . Experimental oxidation life of antibody therapeutics set to establish the relationship was used as a training and MET/TRP oxidation . For each between MD analyses of physiochemical properties by generating estimates of by generating estimates properties of physiochemical of each molecule and tracking thousands of conformations atom over time . This atomic-resolution the position of each be used to make detailed chemical and simulation data can demonstrate of each molecule . We structural calculations provide the opportunity to predict the chemical stability the chemical stability opportunity to predict provide the to the processes candidates even before of molecule have begun . MD sample of the molecule generate a perform in-depth analyses offer the ability to simulations

Poster Abstracts a new class of descriptors that quantified properties in optimisation tool . We have refined the simplex method to localized patches on the Fab surface . In addition to being cope with the discrete or gridded nature of input variables well suited for predicting Fab variants not included in model in biomanufacturing . For instance, pH can rarely be generation, these QSAR models were also successful in controlled to better than +/- 0 1,. which is its least count; predicting deamidated species and different isotypes . QSAR similarly, salt levels can rarely be controlled to better than models also were developed to predict the subtle selectivity +/- 5 mM or even +/- 10 mM . In this presentation, we changes resulting from the use of arginine and guanidine as extend the use of the refined simplex to make the following mobile phase modifiers with these Fab libraries in the Capto claims: i) That the refined simplex method is better suited MMC and Nuvia cPrime multimodal systems . Significantly, to, and more efficient than, typical DoE approaches to early- the descriptors selected in these models provided insight phase process development (up to Phase IIa) of individual into important properties of the proteins that determine unit operations in the downstream processing train for the nature of the change in retention in the presence of macromolecular therapeutics; and ii) That the refined these modifiers and corroborate existing theories about the simplex method can also be used effectively to design mechanisms of interaction . This work demonstrates how process sequences within a bioprocess . Experimental computational and experimental tools can be combined results from the Merck bioprocess development group are into a multi-step framework that can facilitate process used to support the first claim . It is shown that, for a variety development of recombinant biopharmaceuticals . These of polishing chromatography and protein refolding steps, the results also suggest that the selected descriptors can refined simplex method outperforms quadratic and higher- provide important insights into key interaction regions order regressions to experimental data . The refined simplex on the protein surface which can further inform future method is model-independent, and therefore captures any fundamental studies . Finally, this work sets the stage for realistic experimental trend, no matter how complex, in the the development of in-house QSAR models for a range of same way (without altering the method) . When more than complex new classes of biological products with important one optimum exists (as can occur for multimodal resins), implications for rapid bioprocessing development . the refined simplex finds the global optimum very often; the DoE methods routinely failed to identify the existence of multiple optima and often failed to find effective conditions . 10. Refined Simplex as Superior When combined with its robustness and ability to cope with Process Development Method for missing or even incorrect data, the refined simplex method Bioprocess Steps And Sequences is found to be superior to experimental designs in finding effective (and usually near-optimal) operating conditions *Ajoy Velayudhan, UCL, London simply and rapidly . Experimental work in our laboratory Spyridon Konstantinidis, UCL, London shows that the development of a two-column polishing David Roush, Merck, Sharp and Dohme, Inc., United States train can be achieved in a single set of experiments driven John P Welsh, Merck, Sharp and Dohme, Inc., USA by the refined simplex method . Results for a ternary protein mixture were generated in high-throughput format using Work done in our group has demonstrated that the classical a Tecan liquid handler and RoboColumns . With a natural simplex method, which has widely been used for numerical choice of pooling from the first column, the operating optimisation, can in fact be effective as an experimental Poster Abstracts

Recovery of Biological Products XVII | 61 . .

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This cascade of beneficial correlations of beneficial correlations . This cascade United States chains, and a common light chain . Co-expression of two homodimeric heavy chains leads to the formation of two is facilitated by a IgG contaminants, the removal of which of one of the heavy dipeptide substitution in the Fc portion . Thereby the Protein chains that ablates Fc Protein A binding perform both bulk A step of the purification process must impurities, a task capture and high resolution of these mAb not designed for current commercially available resins are Protein A resin, This talk details development of a novel VH-region binding which combines the a base stable, non ligand with a base bead exhibiting excellent mass transfer properties to allow high capacity single step capture and resolution of bispecific antibodies with high yields . During in a continuous process polishing with the MCSGP process with the MCSGP process polishing in a continuous batch tradeoff of classical the purity-yield can overcome chromatography higher results in continuous manufacturing in integrated of improved product quality productivities and yields Chromatographic in Serendipity 12. Development of Design: How for Purification a Custom Resin Human Bispecific of Fully Antibodies Advance Led to an Processing in Continuous Regeneron Pharmaceuticals, *Andrew Tustian, there is strong interest In the biopharmaceutical industry, antibodies that in the production of bispecific monoclonal or epitopes can simultaneously bind two distinct targets and efficacy to achieve novel mechanisms of action based upon a standard bispecific technology, Regeneron’s heavy IgG, consists of a heterodimer of two different Additionally, it is shown how it is shown is improved . Additionally, polishing steps . Typical . Typical . Hence the performance of subsequent 62 | Recovery of Biological Products XVII Fabian Steinebach, ETH Zurich, Switzerland Daniel Karst, ETH Zurich, Switzerland ETH Zurich, Switzerland Thomas Villiger, to classical batch processing . The lower residence time in the bioreactor and the higher loading in the multi-column capture step lead to post-Protein A pools with more uniform product quality in the continuous mode and connected to a two column in the continuous mode and connected for product continuous protein A chromatographic unit the capture . Different steady states are examined and and control is use of simulation models for process design the illustrated . In such an integrated continuous process, different compared protein quality of the capture product is the Biopharmaceutical Industry not only for the classical the Biopharmaceutical Industry not only preferred reasons which make continuous operation of over the batch one, but also for recent initiatives of discuss here a series the regulatory agencies . We with CHO cells for experiments where a perfusion reactor has been operated the production of a monoclonal antibody *Massimo Morbidelli, ETH Zurich, Switzerland considered by Continuous manufacturing is currently being for DSP steps as well as sequences of such steps . for DSP steps as well Uniformity Product Improving 11. Continuous by Integrated Biomanufacturing optimum when applied to this simultaneous design of a simultaneous design applied to this optimum when often failed to fit the experimental two-column train, and . inefficient operating conditions trends, thereby suggesting that the refined simplex method is These results suggest effective operating conditions rapidly well-suited to finding variables for both columns were optimised simultaneously both columns were variables for able simplex was found that the refined Again, it was rapidly operating conditions to find effective failed to find the global designs usually experimental

Poster Abstracts late-stage testing, it was realized that the high capacity at Operation is at low pressure (<20 psi), enabling the use of lower residence times (> 60 g/L), steep mAb breakthrough a fully disposable flow path . This eliminates the need for curve, and base stability of this new resin made it ideal for column packing / validation, greatly facilitating operation in a much broader application: continuous capture by periodic facilities designed for single use manufacturing . Contacting counter-current chromatography . in the individual steps is performed in a countercurrent fashion, with the flowing slurry moving from stage (module) 1 to stage 2 while the permeate moves in a countercurrent 13. Continuous Countercurrent direction . This significantly increases the overall throughput Tangential Chromatography while improving product yield and purification . All of the for Continuous Purification of chromatographic operations are performed simultaneously, Monoclonal Antibody Products with a fraction of the resin being used for binding while the rest of the resin is in the washing, elution, or regeneration *Andrew Zydney, Penn State University, United States steps . Recent experimental studies have demonstrated Oleg Shinkazh, Chromatan, Inc., United States that CCTC can be successfully used for initial capture of a Boris Napadensky, Chromatan, Inc., United States monoclonal antibody product from clarified CHO cell cell Amit Dutta, Chromatan, Inc., United States culture fluid produced in a fed batch bioreactor . Host cell protein removal and antibody yields and purities were One of the main challenges in the development of similar to that obtained with conventional batch columns, integrated continuous processes for the production of but CCTC provided a several-fold greater productivity high value therapeutic proteins is the replacement of (g purified mAb per liter resin per hr) . In contrast to traditional batch column chromatography, which tends multicolumn systems, CCTC provides true steady-state to dominate current downstream processing, with an operation, with the product concentration remaining effective continuous capture technology . Continuous constant over a multi-hour run . Current efforts have Countercurrent Tangential Chromatography (CCTC) has been been focused on the optimization of the CCTC system to designed to provide truly continuous product capture and increase productivity while reducing buffer requirements . purification using a column-free system that overcomes This includes novel strategies for buffer recycling, the many limitations of traditional column chromatography . use of small particle size resins to reduce mass transfer All operations in CCTC are conducted on a moving slurry limitations, and the optimization of the countercurrent that is continuously pumped through a cascade of static staging . Mathematical models for CCTC performance have mixers (for mixing and residence time) and hollow fiber been developed and provide a framework for system design membrane modules (for separation of the fluid phase from and process development efforts . In addition, experimental the resin particles) . For example, host cell proteins and studies are being extended to couple the CCTC system other impurities are removed in the permeate collected directly to a perfusion bioreactor, providing truly continuous through the hollow fiber membrane in the washing step, production of highly purified monoclonal antibody products . while the bound product is retained by the membrane . Poster Abstracts

Recovery of Biological Products XVII | 63 removal from supernatant when used in a flow-through removal from supernatant when used in have also examined mode with little loss of IgG yield . We of the HCPs the molecular weight and characteristics to guide ligand that can be removed with existing resins identification . Several peptide ligands have been identified cell culture without that can bind to HCPs from mammalian binding to human IgG . Preliminary results will be presented these ligands on the removal that can be achieved with . immobilized on chromatographic supports without retaining the target product at a given set of solvent at a given the target product without retaining . and conductivity) (buffer pH, composition, conditions and molecular weights the wide range of Because of to be ligand is not likely of HCPs, a single concentrations achieve complete HCP removal, thus effective enough to of HCP binding ligands needs to a “polyclonal mixture” used on any type of be developed . These ligands can be single-use membranes would support, but disposable, or . HCP-capture membranes be ideal for this application immediately after the cell columns can be implemented steps and can also be used in removal and clarification operation the . In truly continuous final polishing steps flow through these devices with the process stream would concentration technologies implementation of continuous . In cell culture, such as single-pass tangential flow filtration ten times lower the HCP concentration is approximately and as a result than that of the typical biological product, smaller than HCP capture devices will be considerably to process much product capture columns and will be able . Additionally, larger volume of process fluid in less time would save implementation in a disposable format have costs . We considerable time, space and manpower and HCP found that commercially available cell removal 0 .4 logs of HCP removal resins can result in approximately

, North Carolina State University, University, , North Carolina State BTEC 64 | Recovery of Biological Products XVII United States United States United States United States United States Amith Naik, North Carolina State University, Islam, Tuhidul Stefano Menegatti, North Carolina State University, North Carolina State University, Stefano Menegatti, Carolina State University, Ashton Lavoie, North capture the whole spectrum of HCPs in a specific cell line capture the whole spectrum of HCPs in which are time consuming, and exhibit large footprints which are time consuming, and exhibit waste disposal for buffer storage, buffer preparation and novel, management . Our work explores the development of removal robust, inexpensive, ligands for the continuous streams . of HCPs and other impurities from process small ligands that Combinatorial libraries are screened for dominant, even in so-called “continuous processes” that dominant, even in so-called “continuous stages of link together three or more columns in different operation . Bind-and-elute steps capture and concentrate cell protein (HCP), product while significantly reducing host however, DNA, and other impurities, These processes, steps, require binding, wash, elution, and regeneration by developing novel steps for impurity removal that result by developing novel steps for impurity removal based on in the complete elimination of unit operations the classical “bind-and-elute” mode . Flow-through mode as a polishing capture of impurities is widely accepted mode using step, but product capture via bind-and-elute is still high capacity affinity or ion exchange media Truly continuous purification of biologics can be achieved Truly *Ruben Carbonell, North Carolina State University, State University, North Carolina *Ruben Carbonell, 14. Truly Continuous Downstream Downstream Continuous Truly 14. Biologics of Processing

Poster Abstracts 15. Optimisation and Integration 16. Model-based Conversion of a of a Continuous Post Capture Single-column Batch Process to Purification Process 3- and 4-column Periodic Counter- *David Gruber, MedImmune, United Kingdom Current Chromatography Muazzam Khan, MedImmune, United Kingdom *Tobias Hahn, Karlsruhe Institute of Technology, Germany Lucy Evans, MedImmune, United Kingdom Gang Wang, Karlsruhe Institute of Technology, Germany Richard Turner, Medimmune, United Kingdom Fabian Görlich, Karlsruhe Institute of Technology, Germany Juergen Hubbuch, Karlsruhe Institute of Technology, As the Biotechnology industry matures and begins Germany focusing on strategies for increasing productivity and reducing cost there has been a move towards continuous As fully integrated continuous processing is being adopted processing . The implementation of perfusion cell culture has by the biopharmaceutical industry, the individual batch-wise increased productivity once again shifting the bottleneck processes have to be replaced by continuous equivalents . onto downstream . The use of periodic counter current Typically, chromatography steps are employed to purify the chromatography as a capture step is gaining popularity, target component from both weaker and stronger binding however this is largely due to the on/off nature of a impurities . While conventional simulated moving bed Protein A affinity step for processing antibody products . (SMB) systems can only purify binary mixtures, periodic Polishing the post capture product is a more complex counter-current chromatography (PCCC) systems are able operation as product must be separated from more closely to cope with ternary mixtures . PCCC has been successfully related impurities such as aggregates . Bind and elute employed as first capture step and design charts have been chromatography steps are typically utilised in order to developed for such cases with favourable isotherm by Carta separate aggregates, thus allowing for the development of and Perez-Almodovar . However, intermediate purification robust separations . However these operations are limited steps based on ion exchange or hydrophobic interaction by the binding capacities achieved (<80g/L) . Additionally the chromatography do not possess a favourable isotherm and use of salts as counter ions for elution limits the conditions are much more sensitive to small changes in the process available for downstream operations without the need parameters . Because of the many degrees of freedoms of for dilution or UF/DF . Here we describe the development PCCC set-ups, such as flow rates, buffer concentrations, of flow through polish steps as well as a strategy for cycle duration, etc . model-based process development their integration designed to increase productivity of is the method of choice to identify the design space and the overall purification process . A high throughput optimal conditions . In the presented ion exchange case screening methodology is used to determine the optimal study using SP Sepharose FF, the target component was an conditions for the polishing unit operations . Using these intermediate binding IgG . The model was calibrated using conditions enables the implementation of integrated high three gradient elutions in linear mode and one in non-linear productivity polishing steps that can be used in continuous mode . The optimal process conditions for single-column manufacturing processes . batch, 3-column (3C) and 4-column (4C) PCCC processing Poster Abstracts

Recovery of Biological Products XVII | 65 UCL’s Decisional Tools Decisional Tools . UCL’s Sterility is also demonstrated to be . Sterility is also demonstrated Feasibility of implementing such sterilized affinity . Feasibility of implementing such sterilized Drivers for Smart Decision-making Drivers for Smart Decision-making Development and in Bioprocess Fit Facility *Suzanne Farid, UCL, United Kingdom is critical for Understanding cost and performance drivers effective decision-making across the biopharmaceutical process development pathway tools team have developed advanced decision-support that effectively integrate concepts from bioprocess economics, dynamic simulation, risk analysis, combinatorial optimisation and advanced multivariate analysis to physical properties of the irradiated column, and pressure column, and of the irradiated physical properties the also demonstrate . We are not changed specifications media and capture maintenance of column, long-lasting irradiation . Long-lasting and sterility upon performance, and resin properties is particularly stability of column, as the columns chromatography, important in a continuous period of time, sometimes for up will be used for extended one purification campaign . Operating to two months during column at normal pressure and the sterilized, irradiated period of time showed no change flow over an extended in column integrity column during extended use at maintained inside the performance . A method of preserving operating conditions for maintenance of purification of affinity resins allows minimal loss of performance upon gamma irradiation with capacity is demonstrated . resins in prolonged continuous purification a capture The study presented here establishes that continuous chromatography solution for closed system in a downstream process can be achieved, and implemented purification process . Manufacturability Cost and 18. We demonstrate that demonstrate . We a non-irradiated column with that of However, . However, . Here we To date there . To However, applying . However, 66 | Recovery of Biological Products XVII Dana Pentia, Repligen Corporation, United States Dana Pentia, Repligen Corporation, United States James Rusche, Repligen Corporation, United This study describes the feasibility of sterilizing a pre- This study describes the feasibility of sterilizing packed disposable column packed with a capture resin by gamma radiation . This involves comparative evaluation of performance of a column sterilized by gamma irradiation only by using membrane chromatography step of is no suitable solution for a sterilized capture monoclonal antibodies from the bioreactor chromatography introduce a novel sterilized, pre-packed that can be solution for downstream capturing step scale . used in a continuous processing at manufacturing For continuous production of recombinant proteins at For continuous production of recombinant of the process manufacturing scale, sterility at every step available is vital . Upstream processing can integrate many filters, etc . alternatives for sterile systems: bioreactors, can be obtained For downstream purification part, sterility Chromatography for Continuous Chromatography Processing Downstream Repligen Corp, United States *James Peyser, optimization problem is highly non-linear optimization problem fronts allowed to identify different MVDA to the Pareto the most important ones being clusters of process variables, while overloading the column . related to the conditions Column Sterilized 17. were increased by 100 % and 280 % compared to the batch 280 % compared to by 100 % and were increased (MVDA) was employed process . Multi-variate data analysis factors of influence on the three to identify the main to explain the whole sub-objectives . MVDA was unable design space, underlining that the variation in the PCCC were determined with a multi-objective optimization optimization with a multi-objective were determined rate . The results yield, purity and production regarding yield and purity values for showed comparable 4C-PCCC 3C-PCCC and rates of the simulated the production

Poster Abstracts address such challenges . This presentation will show conditions in terms of their ease of manufacture in the final practical applications of such models to enable “smart UF/DF step . The indices were used to identify the optimal bioprocessing” for industrially-relevant problems related to formulation conditions that minimize the potential for both facility fit, implementation of novel technologies such as viscosity and aggregation issues during UF/DF . integrated continuous bioprocessing and manufacturability assessment for monoclonal antibodies (mAbs) . The first case study focuses on facility fit challenges in legacy 19. Acoustic Wave Separation – facilities exposed to higher titres as well as batch-to- A Scalable Disruptive Technology batch variability in cell culture titres, step yields and for Continuous Clarification of Fed chromatography eluate volumes . Insights from the Batch Cell Culture Prior to Capture systematic use of advanced multivariate analysis techniques will be presented that illustrate critical combinations of Chromatography factors that lead to undesirable mass loss levels and reveal *Peter Levison, Pall Life Sciences, United Kingdom the root causes of bottlenecks . A comparison of different Ron Farkash, Pall Life Sciences, USA debottlenecking solutions will be presented in term of their Mike Collins, Pall Life Sciences, USA impact on mass output, cost of goods and processing time, as well as consideration of extra capital investment and With advances in fed batch cell culture leading to higher space requirements . The second case study focuses on cell densities and higher product titers there is a drive facility fit challenges in multiproduct facilities catering for to improve the efficiency and speed of the cell harvest both low and high concentration formulations . The work and clarification stage to generate Harvested Cell explores the capability of a particular TFF system to reach Culture Fluid (HCCF) for capture chromatography and high concentration product formulations . Multiobjective subsequent downstream processing . This is further driven optimisation is used to find the optimal final UF/DF design by the evolution of continuous processes where there for different target product concentrations with both is a preference for a continuous feed of HCCF available maximum annual product output and minimum cost of goods for direct load to the continuous multicolumn capture (COG) . The third case study explores the economic feasibility chromatography step . Existing cell culture clarification of different configurations of continuous chromatography using either centrifugation or depth filtration are typically for clinical and commercial manufacture . It will address operated in batch mode and require bulk storage of feed questions such as: How does the feasibility of continuous or HCCF during the process . In the present work we report chromatography combined with pre-packed disposable on a novel disruptive and scalable single-use technology columns change across different feed characteristics, for cell culture clarification based on an acoustophoretic resin properties, development phases and commercial separation . Acoustic Wave Separation (AWS) technology production scales? What is the optimal design of integrated involves the use of low frequency acoustic forces to continuous downstream process configurations? Finally, the generate a 3 dimensional standing wave across a flow fourth case study presents a novel framework for deriving channel . Cell culture from a fed batch bioreactor enters the a set of manufacturability indices related to viscosity and flow channel and as the cells pass through the 3D standing thermostability to rank high-concentration mAb formulation wave they are trapped by the acoustic forces . The trapped Poster Abstracts

Recovery of Biological Products XVII | 67

. The performance of . The stationary phase provides both Benjamin Roman, MilliporeSigma, USA Benjamin Roman, MilliporeSigma, Genentech, USA Debola Banerjee, Roche Data on HCP, DNA, and viral clearance in industrially DNA, and viral clearance in industrially Data on HCP, under several relevant feed streams will be discussed feeds, pH, different conditions (flow rates, conductivity, this media etc .) . A brief overview of the economics around will be presented as well . 20. Performance of a Novel, Truly Truly of a Novel, 20. Performance Single-Use Adsorber Technology Purification in mAb United States *John Boyle, MilliporeSigma, has been a very significant focus from In recent years there manufacturers on developing and both suppliers and drug technologies . Drivers for the shift implementing single-use can depend on production scale, to single-use technologies base, and other factors . Single-use existing installed capital potential to reduce costs (both capital technologies have the costs and COGs) . Single-use chromatography media can rare (but costly) improve bioburden control and eliminate packed bed integrity issues . This presentation will show the version of a truly performance of a Quaternary Ammonium single-use chromatography media . The new technology is support material based on an entirely new stationary phase – a novel synthetic fiber high capacity and rapid mass transfer several benchmark this technology is presented here versus adsorbers) . technologies (bead-based resin and membrane We have not have . We This yields a partially clarified HCCF which can a partially clarified . This yields 68 | Recovery of Biological Products XVII well as perfusion applications that are gaining momentum well as perfusion applications that are in the biotech space . technology enables the continuous clarification of cell technology enables the continuous clarification single-use operation . culture from bed batch bioreactors in a well at cell The technology has been shown to perform is well positioned densities of up to 100 million cell/mL so higher cell to meet the clarification demands of emerging development as density fed batch processes currently in and depth filter costs, but also significant reductions in and depth filter costs, but also significant for Injection (WFI) used in depth the volume of Water wash following media conditioning and post-harvest buffer costs and clarification as well as reduced waste disposal significant set-up time savings . The economic benefits of approach will be discussed in more detail . AWS the AWS This enables the AWS applicable for 2000L bioreactors . This enables the AWS manufacture . The technology to be positioned for clinical continuous mode partially clarified HCCF is polished in a 3-5x less using depth filtration but typically requires depth filtration depth filter area than used for a traditional of footprint process . This offers economic benefits in terms the ability to clarify CHO cell culture at cell densities of the ability to clarify in a continuous manner at flow 30 – 100 million cells/mL, shown the . Furthermore we have rates of up to 3 .6 L/h and using prototype systems technology to be scalable clarification flow rates of 50 L/h that have demonstrated parallel enables the technology to be when configured in seen any demonstrable adverse effects on the quality of effects on the quality adverse seen any demonstrable clarification . viability following AWS the HCCF or the cell culture the continuous clarification of fed batch report We line expressing a humanised IgG1 of a CHO-S based cell (PD) scale we demonstrate MAb . At process development cells migrate to the nodes and clump till such time as their clump till such time to the nodes and cells migrate settle out of the suspension decreases and they buoyancy by gravity depth filter using a small area be polished

Poster Abstracts provides this critical capability, and we will describe in 21. Custom Affinity Chromatography detail how it is used as the pipeline front-end to discover – a Novel Platform for Rapid and characterize DNA aptamers offering high affinity and Development of Affinity Media specificity for a target biologic . Methods for chemically stabilizing aptamer ligands will also be described, along Displaying DNA Aptamer with specific end-group chemistries encoded within or Based Ligands appended to the aptamers to enable orientation-specific Aaron Ang, University of British Columbia, Canada ligand immobilization to Bio-Rad UNOsphere Epoxide or UNOsphere diol media . Panels of scaled-down candidate Eric Ouellet, University of British Columbia, Canada columns constructed using this pipeline may then be Mark Snyder, Bio-Rad Laboratories, United States screened using standard liquid-handling robotics, and we *Charles Haynes, University of British Columbia, Canada will describe how this may be done if time permits . Time-to-market pressures for recombinant protein therapeutics generally demand rapid design of product 22. An Integrated, High Throughput purification protocols . This need for speed challenges and Rational Design Platform the time-consuming strategies generally employed to optimize the economics and performance of a purification for the Development of Affinity train, including selection of a stationary phase for product Peptide Resins for Biological capture . The problem is especially acute for biologic orphan Product Purification drugs, for which production per annum is often a few kg to a few tens of kg, and for which proven downstream Divya Chandra, Rensselaer Polytechnic Institute, processing platforms are generally not available . This United States rapidly growing products class could benefit from creation Steven Timmick, Rensselaer Polytechnic Institute, of an integrated set of products and decision-making tools United States that enable one to efficiently define an affinity capture Chaz Goodwine, Rensselaer Polytechnic Institute, chromatography step that preserves yield and product United States integrity while limiting further downstream processing Nicholas Vecchiarello, Rensselaer Polytechnic Institute, demands, and thus cost . We will describe our progress United States towards development of a new pipeline for efficient *Pankaj Karande, Rensselaer Polytechnic Institute, creation of robust affinity chromatography media, based United States on DNA aptamers as ligands, that have been custom Steven Cramer, RPI, United States designed to achieve specific performance metrics when While many strategies have been employed for the applied to a particular product and feedstock . Central to discovery and design of affinity peptides, in a broad this concept is the creation of a method to rapidly select sense most fall under the categories of either brute force candidate affinity ligands for product capture from a highly screening of large libraries or targeted, rational design diverse starting aptamer library comprised of more than and evaluation of a small number of candidates . Here

1014 unique sequences . Our novel Hi-Fi SELEX protocol Poster Abstracts we present an integrated platform process for the rapid

Recovery of Biological Products XVII | 69

Jose Martinez, CSL Behring Australia, Australia Sweden Alstine, Royal Institute of Technology, James Van Australia Joseph Bertolini, CSL Behring Australia, manipulation of pH and ethanol concentration at subzero manipulation of pH and ethanol concentration and temperatures . The requirement for low temperatures of this large amounts of ethanol is a major disadvantage fractionation shows procedure . Polyacrylic acid (PAA) fractionation potential as an alternative to cold ethanol the precipitation of plasma proteins, especially given that . Previous steps can be conducted at room temperature specific (PAA) studies have shown that polyacrylic acid and albumin- precipitation of fibrinogen-, immunoglobulin- of 7, concentrations rich fractions was achieved at PAA respectively with yields of greater than 12 and 20% w/w, 80% . The current study explored whether established downstream processing methods used in the plasma fractionation industry could be applied to albumin and approach for the rapid identification and development and development for the rapid identification approach of range of new classes peptide resins for a of affinity important implications products, which has biological future downstream and streamlining for simplifying . process development Precipitation Based 23. Polyacid to Cold- as an Alternative of Plasma ethanol Fractionation Alignment with Existing Proteins: Methods Processing Downstream Behring Australia, Australia *Karl McCann, CSL biotherapeutic Plasma is the source of many important coagulation products such as albumin, immunoglobulins, factors and inhibitors . The fractionation of plasma is still for initial predominantly based on cold-ethanol precipitation of proteins through processing, which involves precipitation This platform represents a novel represents a novel process . This platform of this platform . Results . Batch results are then used In this platform, two approaches are . In this platform, two 70 | Recovery of Biological Products XVII for further optimization with regards to ligand density, ligand density, for further optimization with regards to process conditions, and resin regenerability from the development of affinity peptide resins for two molecules, human Growth Hormone and Interferon Alpha 2b, will be discussed to illustrate the successful application isotherm space allows for the rapid, orthogonal assessment isotherm space allows for the rapid, orthogonal metrics of affinity, of peptide resin performance for the key and selectivity elutability, process until to inform peptide refinement in an iterative column-scale optimum candidates are identified . Finally, peptide resin experiments are performed to select a single improve binding capacity and selectivity as well as to improve binding capacity and selectivity such as the facilitate elution under a desired condition, peptide . Top addition of histidines for low pH elution are synthesized candidates from these screening efforts for purification on chromatographic resin and evaluated performance in batch . Strategic sampling of the competitive libraries are then performed to generate leads that span libraries are then performed to generate protein . Top a range of binding affinities for the target characterization candidates are selected for validation and under various of binding via fluorescence polarization bioprocess-relevant conditions . Lead sequences are then maturation to docked in silico and subjected to affinity first used to generate a library of peptide candidates . first used to generate display is employed to screen High throughput phage known while simultaneously, large peptide libraries, utilized to rationally generate peptide binding partners are mapping and loop stitching . High candidates from epitope screenings of these designed peptide throughput microarray high throughput screening approaches geared towards geared towards screening approaches high throughput which meet criteria necessary for identifying peptides these purification . Specifically, efficient downstream elutability, selectivity, capacity, affinity, criteria include high and regenerability development of molecule-specific, affinity peptide resins affinity peptide of molecule-specific, development products . Our platform of biological for the purification of peptide design combination utilizes a synergistic novel, brute force) along with (rational as well as strategies

Poster Abstracts IgG precipitates derived from PAA fractionation . of the Quality by Digital Design paradigm to bioprocessing Downstream processing of the IgG-rich precipitate industry . We address such fundamental challenges by using a combination of caprylic acid precipitation and means of our “Rheo-Chip” technology* . It consists of a anion exchange (MacroPrep HQ) polishing successfully series of rigid and transparent microfluidic devices equipped depleted the major impurity proteins of IgA, IgM and α2- with replaceable pressure sensors and with a series of macroglobulin, resulting in a final product purity over 99% . flow actuation devices (syringe and pressure pumps, piezo The albumin-rich precipitate could be further processed stacks) with which every possible fundamental flow (steady using either cold-ethanol precipitation or a two-step ion and oscillatory shear, extensional, flow with a stagnation exchange (DEAE and CM Sepharose-FF) polishing method point) can be imposed to the tested fluid . The available data to generate final product with a purity of 98 .6% or 99 .6%, include steady shear and extensional viscosities measured respectively . The successful demonstration that the albumin over a range of deformation rates (102-105 s-1) typical of and IgG intermediates derived from PAA fractionation bioprocessing operations, the mechanical spectroscopy could be purified using established downstream processing measured up to frequencies as high as 100 Hz, and micro- methods suggests that PAA fractionation of plasma could injection forces studied by means of a novel microfluidic be a viable alternative to current cold-ethanol fractionation “syringe-on-chip” device . Moreover, by coupling microfluidic methods . PAA fractionation has the potential to reduce chips having a functionalized inner surface (hydrophobic or complexity, captial investment and cost of goods related to hydrophilic) with fluorescence measurements, we are able the manufacture of plasma derived biotherapeutic proteins . to understand the effects of different interfaces as well as of specific flow conditions on protein aggregation . It is envisaged that this research will lead to improved design 24. A “Rheo-Chip” Platform for QbD of bioprocess operations (vial filling, syringe injection, Approach to Bioprocessing tangential flow filtration etc .), as well as to an insightful understanding *Alfredo Lanzaro, School of Chemical Engineering, the University of Manchester, UK of the mechanisms leading to formation of protein aggregates under flow . *: The University of Manchester, Robin Curtis, School of Chemical Engineering, the University of Manchester, UK Patent Application No: PCT/GB2011/051476 Alain Pluen, School of Pharmacy, the University of Manchester, UK 25. Robust Multi-objective Future trends in treating various chronic diseases with Process Design recombinantly-produced therapeutic proteins, like human Lars Freier, Research Center Jülich, Germany serum albumin and monoclonal antibodies, require frequent *Eric von Lieres, Research Center Jülich, Germany and high dose of active protein ingredient in a small volume of liquid (c= 100 g/L or higher) . Understanding the link Separation processes are routinely designed and optimized between formulation, rheology, stability and aggregation using parallel high-throughput experiments and/or serial propensity of concentrated protein medicines is of lab experiments, depending on the available equipment paramount importance for a successful implementation and current state of knowledge . Well characterized Poster Abstracts

Recovery of Biological Products XVII | 71 . J . Op . Res . 243 (2015): Niklas Andersson, Lund University, Sweden Niklas Andersson, Lund University, Sweden Anders Holmqvist, Lund University, Sweden Anton Sellberg, Lund University, Novo Nordisk A/S, Denmark Peter Tiainen, Novo Nordisk A/S, Denmerk Arne Staby, result is an integrated chromatography column sequence result is an integrated chromatography of the target that performs straight through processing to formulation . protein, with minimal time from expression is well suited to This downstream processing technique process based on be connected to a continuous upstream introduce this technique it has to be scalable perfusion . To scale . This from desktop lab-scale to full size production and control of paper present a methodology for design through processing In straight sequences . column integrated the next column . the eluting pool is directly loaded on to The consequence is that the next step has to have proper capacity to handle the loaded pool without losses . An overall design procedure of integrated column sequences is discussed based on its performance and limitations, metamodels, IEEE Transactions on Evolutionary Computation on Evolutionary IEEE Transactions metamodels, algorithms et al .: Faster exact 421–439 . Hupkens 10 (2006): EMO improvement, expected hypervolume for computing al: . Binois et . 2015 9019, pp . 65–79, Springer, 2015, LNCS on Pareto fronts with Gaussian Quantifying uncertainty simulations, Eur process conditional 386–394 . Processing Through 26. Straight Chromatography Using Integrated Column Sequences Sweden University, *Bernt Nilsson, Lund with a Smart downstream processing can be performed which minimize sequence of integrated purification steps, hold-up time . The the number of storage tanks and reduce evolutionary optimization assisted by Gaussian random field by Gaussian random optimization assisted evolutionary 72 | Recovery of Biological Products XVII process fluctuations to the Pareto front, which provides process fluctuations to the Pareto front, important and useful information for robust process design . Rasmussen et al .: Gaussian processes for machine learning, MIT Press, 2006 . Emmerich et al .: Single- and multiobjective The method supports optimal design of both space . The method supports optimal design of and more conflicting serial and parallel experiments . Two (Hupkens, objectives are treated using the latest algorithms hypervolume 2015) for efficiently computing expected improvement . Recent methods (Binois, 2015) are also propagated from required for calculating the uncertainty data quantity, data quality, and process fluctuations . The data quality, data quantity, (Emmerich, statistical concept of expected improvement data would 2006) is used for determining which additional maximizing way, supplement current knowledge in the best prediction process performance and selectively minimizing of the parameter uncertainty in the most promising regions Gaussian process regression (Rasmussen, 2006) is applied Gaussian process regression (Rasmussen, performance for estimating the system behavior from yield, time) observed at systematically indicators (purity, with varied operating conditions (gradient, fractionation) random fluctuations (feed) . These predictions are provided the given with sound confidence estimates, reflecting models with most recent methods for uncertainty models with most recent optimization . The methods quantification in multi-objective simultaneously optimizing elution are demonstrated by strategy for separating a three- gradient and pooling to varying feed composition component system subject time . yield, and processing with respect to purity, Process optimization is . Process optimization acquired knowledge previously by conflicting design targets, such typically complicated yield, and by process variations, in as productivity and address all feed composition . We particular fluctuating advanced statistical regression these issues by combining processes can further be optimized using mechanistic using mechanistic can further be optimized processes experiments cases – serial/parallel models . In all these applied are customarily – iterative strategies and modeling on the based novel experiments/simulations for planning

Poster Abstracts together with requirements on each individual step . Design Jinkeng Asong , Keck Graduate Institute, United States of integrated column sequences in different scales is Jeffrey Salm, Pfizer, United States discussed and exemplified on lab-scale . The propagation Jon Coffman, Boehringer Ingelheim Fremont, Inc., of disturbances in connected column sequences forces an United States open-loop control strategy to be unnecessary conservative with limited performance . Three issues on the individual Low pH virus inactivation lends itself to continuous step design will be discussed . First the design procedure of processing . Continuous virus inactivation (cVI) allows a each individual step is modified to also handle disturbances smaller process footprint and a more tightly integrated which make it possible to find robust performance . downstream . It also can eliminate the need for cycle- Second, for some separation problems it is attractive to to-cycle cleaning validation, or disposable change over introduce local recycles over a step to enhance the yield between cycles in a disposable plant . In this work we of the target protein . Third, in many cases it is possible explore alternative options to incubate a stream of product to increase performance and yield by modifications of the for the desired incubation time of 30-60min . We have elution profile . A completely new case study is presented designed incubation chambers that meet the following which uses a multiple step elution gradient . The procedure criteria 1 .The incubation chambers axial dispersion is such is illustrated both using computer simulations and that the residence time distribution is sufficiently narrow, experimentally on an industrially relevant case . and empirically demonstrated . -<10ppm of tracer exits the chamber before the validated incubation time -Max residence time cannot be longer than 2hrs for more than 27. Design of a Continuous Virus 1% of the product 2 .The chambers must not generate more Inactivation System for Clinical and than a 5 psi of pressure, thus plug flow is not feasible . Commercial Scale 3 .The flow inside the chamber must be well characterized such that chambers at different flow rates can be readily *Raquel Orozco, Boehringer Ingelheim Fremont, Inc., generated and can meet conditions 1 and 2 stated above . In United States this work, we describe incubation chambers that meet these Scott Godfrey, Boehringer Ingelheim Fremont, Inc., requirements . These chambers are sufficient to support United States production for 25kg/year from a 100L perfusion bioreactor . Cameron Bardliving, Keck Graduate Institute, USA These solutions should work for a wide range of flow Lindsay Hernandez , Keck Graduate Institute, USA rates that can potentially be adopted for other continuous/ Linus Amarikwa , Keck Graduate Institute, United States periodic downstream processes . Shashidhar Hoskatti , Keck Graduate Institute, United States Poster Abstracts

Recovery of Biological Products XVII | 73 chromatography-mass spectrometry, and a battery of chromatography-mass spectrometry, evaluation of the chromatographic methods were used for oxidizing agent . ligand and base matrix compatibility with using design Bacterial challenge studies were performed and contact times of experiments to define concentrations . Furthermore, the for efficient reduction of bacterial spores lifetime effect on the media was assessed in functional and impurity studies where ligand leakage, product yield clearance were evaluated . media are available in pre-packed single use formats single use formats available in pre-packed media are high . With modern used for pilot scale predominantly might be formats, single use and improved capacity media of biopharmaceutical for future manufacturing a reality also of supplier pre-sanitized drugs . The potential for use GMP environment with large bulk media in a regulated will be discussed in conjunction on-site packed columns closed processing and aseptic with equipment enabling media slurry preparation, procedures for chromatography column handling . The chromatography column packing and and stabilized to tolerate media can also be improved which facilitates pre-sanitization and harsher conditions, into the biopharmaceutical process . sanitization integrated stabilized protein A ligands tolerating Development of alkali and up to 0 .5 M NaOH has improved the ability to clean . However, sanitize these media over the last decade a challenge . Protein resistant spore forming bacteria is still ligands that A ligand development is underway towards of NaOH, which tolerate even sporicidal concentrations . In the continuous will reach the market in the near future agents, search for new and more efficient sanitization for affinity oxidizing chemicals showed promising results . Analytical media, sensitive to harsh cleaning conditions liquid methods such as Surface plasmon resonance,

74 | Recovery of Biological Products XVII Tomas Bjorkman, GE Healthcare, Sweden Bjorkman, Tomas Sweden Elin Monie, GE Healthcare, GE Healthcare, Sweden Magnus Wetterhall, Sweden GE Healthcare, Johan Avallin, media development for sustainability at harsh sanitization media development for sustainability at agents for conditions and investigation of new sporicidal sanitization . Chromatography media suppliers can deliver using controlled products absent from microorganisms by manufacturing procedures . Pre-sanitization of media will . Pre-sanitized even further minimize the risk of bioburden Furthermore, the biopharmaceutical producers process . Furthermore, the biopharmaceutical producers aseptic must develop control strategies and implement their processes . procedures to prevent bioburden entering from suppliers This paper will focus on the commitment in delivery and will describe the continuous improvement chromatography of aseptic chromatography media, further To be successful in the accomplishment of a bioburden To and their free process, biopharmaceutical companies . Bioburden suppliers must fight this battle side by side It begins with must be attacked from different angles . routines the supplier developing products and operational that enable aseptic procedures in the biopharmaceutical Bioburden can enter into the biopharmaceutical process Bioburden can enter consumables, water, e .g . via air, through different routes . Bacteria and their by- raw materials, and operators affect the safety and potency of the products will negatively authorities . Therefore, regulatory biopharmaceutical drug producers . put increasing demands on biopharmaceutical *Anna Gronberg, GE Healthcare, Sweden GE Healthcare, *Anna Gronberg, 28. United Against the Against 28. United Threat Bioburden

Poster Abstracts were able to adsorb ~60 mg BSA/g resin . Interestingly, 29. Novel Chromatographic the resin modified with dG3 dendrons exhibited a better Adsorbents Based on Dendritic affinity than that one modified with dG5 as indicated by the Molecules for HIC affinity constant calculated from the adsorption isotherms . Dendronized resins were also tested for their performance Marco Mata-Gomez, Tecnologico de Monterrey, Mexico on separating PEGylated proteins biomolecules with Sena Yaman, Izmir Institute of Technology, Tirkey chains of PEG attached to their structure— . As expected, Jose Gonzalez-Valdez, Tecnologico de Monterrey, Mexico resins modified with dG5 exhibited a stronger hydrophobic Jesus Valencia-Gallegos, Tecnologico de Monterrey, interaction with PEGylated proteins, as these were not Mexico eluted into the gradient unless isopropanol was added into Canan Tari, Izmir Institute of Technology, Turkey the mobile phase . On the other hand, PEGylated proteins *Marco Rito-Palomares, Tecnologico de Monterrey, Mexico were eluted from the adsorbent modified with dG3 into the gradient without needing isopropanol suggesting a weaker A major goal in the biotechnological industry is the interaction . This innovative dendronized support opens a purification of bioproducts such as proteins from complex window to new generation chromatographic supports to mixtures . Since this represents up to an 80% of the total develop novel downstream processing strategies . production costs, innovation on downstream processing strategies is required . In this work, we introduce the use of dendrons branched molecules with well-defined 30. Nanofibres as a True Single-use structures for the synthesis of chromatographic adsorbents Chromatography Platform Achieved for hydrophobic interaction chromatography (HIC) . The Through High-Productivity adsorbents were synthesized by covalently attaching Product Capture the amino cores of polyester dendrons, of two different branching degrees i .e . generations 3 and 5 (dG3 and dG5, *Oliver Hardick, Puridify / UCL, United Kingdom respectively), with hydroxyl group (-OH) ends to NHS- Will Lewis, GlaxoSmithKline, United Kingdom activated chromatographic media . Dendronized resins were Daniel G. Bracewell, University College London, then functionalized by incorporation of organic carboxylic United Kingdom acid, containing a four carbon end as an hydrophobic ligand, by reaction with the available –OH groups . This resulted in Cellulosic nanofibres have been employed as an alternative two dendronized resins with clusters of hydrophobic ligands to traditional chromatographic media for industrial on the periphery . The aim of this work was to characterize product capture purification for a monoclonal antibody and evaluate the performance of the dendronized HIC platform process . The nanofibres are fabricated to provide adsorbents . UV-Vis spectra and FTIR analyses of the a combination of high flowrates and high capacities modified resins confirmed the presence of the dendrons and that enables productivity in terms of grams of product their ligands on the resins . Modification of these supports purified per litre of adsorbent, per hour to be increased resulted in an increased ligand density along the packed >10 fold with a chromatographic cycle time of <4 minutes, columns . Regarding adsorption capacity, the unmodified a 100x reduction when compared to traditional packed resin did not adsorb the protein, while modified resins bed operation . In practice, this means for an equivalent Poster Abstracts

Recovery of Biological Products XVII | 75 . This single-use enabling technology enabling technology . This single-use . Both Protein A ligands have been immobilized Patrick Gilbert, Purolite Life Sciences, UK Purolite Life Sciences, UK Caroline Tinsley, Mark Hicks, Purolite Life Sciences, UK However in clinical production, where resins are typically However in clinical production, where resins cost becomes used for only a few cycles, the high resin very significant . One way to address this would be to use production and a less expensive resin for early clinical (if) the drug switch to an alkaline stable Protein A when production . candidate is entering Phase III and regular of Protein A resins, Looking at the purification performance type of base matrix differences are mainly a function of the than the type of or immobilization chemistry used, rather Protein Protein A . In this study we have compared native with a Protein A modified for improved alkaline A (rSPA), stability to the same type of agarose matrix, using the same type functionalities and processes through a chromatographic through a chromatographic and processes functionalities the utilisation of that that enables support structure and greater throughput at a much powerful functionality productivity therefore in some processing scenarios such has clear advantages and stratified medicine supply, as early clinical supply, opportunities but offers promise localized manufacturing through providing a greater to many existing facilities strategy flexibility in processing for A Resins of Protein Design 31. Purification of MABSCost Effective Sweden *Hans Johansson, Purolite Life Sciences, has been an object The high cost of Protein A affinity resins of discussion for many years . The contribution to cost of are typically used goods in regular production, where resins accepted because for over one hundred cycles, is generally manufacturing cost . it only adds up to a small fraction of the capture purification by employing existing chromatographic existing chromatographic by employing capture purification 76 | Recovery of Biological Products XVII scale highlighting a resolution not typically associated to scale highlighting a resolution not typically convective flow adsorbents . Finally a nanofibre AEX unit was employed for HCP clearance and performed to the required 3 log reduction . This work demonstrates feasibility and scalability for industrially relevant single-use product Key metrics of host cell protein content column pressure . Key metrics of host cell protein content to a resin based and Protein A leaching were comparable feed material . CEX process benchmark run using the same removal, from nanofibre units demonstrated aggregate (using SEC- a feed of 4%, to below the limit of detection of <3minutes at pilot HPLC) with 96% recovery in a run time productivity has been demonstrated to be above 130g/L/h, productivity has been demonstrated to bed system, a 10 fold increase from our control packed rigs, without the need for multicolumn chromatography with platform with a purity equivalent to that achieved at >100 resins . The adsorbent lifetime was demonstrated or increase in cycles without significant loss in capacity demonstrated in the work presented here where Protein A demonstrated in the work presented here high-throughput nanofibre adsorbents were employed from systems at µL scale to screening scale through Tecan mAb from a CHO pilot scale purifying 30L of therapeutic of current mAb cell harvest selected to be representative platform systems at a titre of 0 .88g/L . Chromatography contrast to beads however, this surface area is immediately this surface area contrast to beads however, interaction rather than requiring accessible for biomolecule into a porous structure . With an diffusional mass transfer structure this capacity enables high open macro porous achievable with second or sub- productivity separations at low operating pressures . This is second residence times single-use operation with the goal of increasing facility goal of increasing facility operation with the single-use validation costs . and reducing plant productivity, flexibility, surface area to volume ratio in the 10’s Nanofibres exhibit a a binding capacity in the range m2/g range which yields by traditional porous bead resins . In of those expressed bioreactor harvest, a significantly smaller adsorbent smaller adsorbent harvest, a significantly bioreactor a batch through rapid be used to purify cartridge can be sized such that is a unit which can cycling . The output in over the batch resulting can be exhausted its lifetime

Poster Abstracts of chemistry, and amount of Protein A . The idea was to as ligand for affinity chromatography was investigated . design an inexpensive efficient resin for early clinical To provide “proof of principle”, M13-filamentous phages manufacturing where columns are rarely used for more than with binding affinity for human lactoferrin were selected 10 cycles and where the inherent alkaline stability of rSPA from 6-mer p3-phage display libraries and used as affinity is sufficient for efficient cleaning and sanitization using ligand and coupled onto the cryogel . Due to the physical 0 .1-0 .5 M NaOH for up to 20 cycles . The second, alkaline and structural properties (pores up to 100µm) of the stable protein A resin, is designed for regular manufacturing cryogel, phages can be used for coupling as affinity ligand . and will withstand use for several hundreds of purification Also crude samples such as milk, fermentation broth, cell cycles . The achieved dynamic binding capacities range from cultures, . . can be applied on the cryogel without any flow/ 40-50 mg hIgG/ml resin at residence times of 4-6 minutes . pressure restrictions . Proof of principle was provided by Purification data such as host cell protein content, DNA processing whole human milk on epoxy-phage-cryogels . levels, Protein A leakage, aggregate, and fragment levels Human lactoferrin was captured and recovered with a has been assayed to compare the two, agarose based, high purity (>95%) in a one-step chromatographic run . As Protein A resins . however multiple epoxy coupling sites can be occupied by a single phage, this may lead to a decrease of target binding capacity . By introducing spacers between the 32. A New Platform for Affinity epoxy-matrix and the phage ligand, indeed an increase of Chromatography Using Epoxy- binding capacity is observed . Several spacers, linear and Phage-Cryogels branched, and phages were tested . Up to a 20-fold increase in binding capacity was observed when using a phage Deckmyn Hans, KU Leuven KULAK, Belgium from a cyclic 6-mer peptide library in combination with a *Noppe Wim, KU Leuven KULAK, Belgium linear spacer at a molar ratio spacer/AGE=20 . A branched spacer combined with the same phage clone as above at a The aim of the present study is to develop a new affinity molar ratio spacer/AGE=10 resulted in a 15 fold increase of chromatographic method to by using supermacroporous target binding capacity . The recovered lactoferrin showed polyacrylamide (pAam) cryogel monoliths as again a high purity (>95%) in a one-step chromatographic chromatographic matrix in combination with peptide run . These results show that the new platform combining displaying phages as affinity ligands . 6%-pAam-epoxy epoxy-cryogels as matrix and phages as affinity ligand cryogels were produced and optimized for binding capacity . has great potential . Proof of principle was provided using Epoxy groups for coupling were introduced in the cryogel phages selected against a proteineous target, but as at varying molar ratios of Aam/Allyl Glycidyl Ether (AGE) . phages can also be selected against other materials, such Both the structural integrity of the columns and the ligand as metals, toxins, PCB’s,… . . the platform can be used for binding capacity were investigated . A decrease of the major applications, not only for purification but also for fast structural integrity was observed from a molar ratio Aam/ screening and analysis . For direct fast screening or analysis AGE<7,5 onwards . A molar ratio=10 did provide excellent cheap, robust and easy handling chromatographic matrices structural stability and with a sufficient number of epoxy should be available . In this respect, epoxy-phage cryogels groups for coupling . With higher molar ratio’s, the lower present an ideal choice as a chromatographic set-up to deal number of available epoxy-groups did lead to a lower with these demands . Poster Abstracts binding capacity . Next the use of peptide displaying phages

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Complex Technical Systems, , Germany Complex Technical Systems, Germany Technical Complex Technical Systems, Germany Complex Technical Systems, Germany Technical Systems,, Germany Complex Technical Dunja Bruder, Otto von Guericke University, Germany Otto von Guericke University, Dunja Bruder, of Complex Udo Reichl, Max Planck Institute for Dynamics Michael Pieler, Max Planck Institute for Complex Michael Pieler, Germany Sara Fenzel, Otto von Guericke University, Max Planck Institute for Dynamics of Lisa Fichtmüller, of Anja Bastian, Max Planck Institute for Dynamics Influenza is a global, highly contagious viral disease of Influenza is a global, highly contagious and various the respiratory tract occurring in humans animal species . The most effective means of controlling vaccination . seasonal influenza outbreaks is prophylactic either Current inactivated influenza vaccines contain viruses viral membrane components or whole influenza produced in embryonated chicken eggs or animal cells . As for other biological products, downstream processing . cleared from conjugates adsorbed and can be selectively the unconjugated efficiency between The separation conjugate and polysaccharide-protein polysaccharide the the relative sizes of dependent on is significantly chromatography molecules . In general, the CaptoCore700 better process performance than purification step enables of unconjugated protein and traditional TFF for clearance polysaccharide . Sulfated 34. Use of Magnetic Allows Fast Cellulose Beads and Blending of Potent Purification In Mice Influenza Vaccines Max Planck Institute for Dynamics of *Michael Wolff, results show that unconjugated polysaccharide and protein polysaccharide and that unconjugated results show 78 | Recovery of Biological Products XVII Joseph Joyce, Merck & Co., Inc., United States Joseph Joyce, Merck Sheng-Ching Wang, Merck & Co., Inc., United States Merck & Sheng-Ching Wang, Healthcare, Sweden Anders Ljunglof, GE Sweden John Daicic, GE Healthcare, & Co., Inc., United States Merck Marc Wenger, Competitive binding isotherms with polysaccharide and/or Competitive binding isotherms with polysaccharide carrier protein with polysaccharide-protein conjugates were also generated . In addition, confocal microscopy studies were performed to visualize the adsorption of low and high molecular weight polysaccharide and conjugates . The the limitation of TFF membrane sieving, the TFF process the limitation of TFF membrane sieving, unconjugated step usually results in poor clearance of CaptoCore700 for this characterize polysaccharide . To molecular application, binding isotherms for different were performed weight polysaccharides and carrier protein strength) . under various solution conditions (pH, ionic (> 2000 kD) depending on the conjugation chemistry used (> 2000 kD) depending on the conjugation for purification and are therefore potentially well-suited tangential flow filtration via CaptoCore700 . Conventionally, separate undesired (TFF) unit operation has been utilized to carrier protein unconjugated polysaccharide or unconjugated due to . However, from polysaccharide-protein conjugates core gives CaptoCore700 dual functionality, with both core gives CaptoCore700 dual functionality, . In this size- and adsorption-based separation mechanism of CaptoCore700 study we describe the novel application conjugates . media for purification of polysaccharide-protein be large molecules Polysaccharide-protein conjugates can *Michael Winters, Merck & Co., Inc., United States *Michael Winters, Merck for intermediate and polishing CaptoCore700 is designed such as viruses and virus- purification large biomolecules, with an inactive, porous like particles . This bead technology ligand-containing outer shell and a multimodal, octylamine Polysaccharide-Protein Conjugates Conjugates Polysaccharide-Protein 33. CaptoCore700 Chromatography Chromatography 33. CaptoCore700 of for Purification Media

Poster Abstracts (DSP) is of outmost importance for manufacturing chromatographic purification steps can be substituted by of potent and safe products . Within this project an isolation via MSCPs . Furthermore, it is anticipated that virus alternative to the elaborate chromatographic purification pretreatment prior to binding the virus onto MSCPs could and formulation of virus harvests was investigated . In be completely omitted, simplifying the DSP process even particular, magnetic sulfated cellulose particles (MSCPs) further . For human applications, however, the DSP needs were used to capture and isolate cell culture-derived virus to be expanded, e .g . by a nuclease treatment, to comply particles, and the virus loaded MSCPs were directly used with regulatory limits . Furthermore, prior injection the virus to immunize mice . Therefore, influenza A/PR/8/34 (H1N1) might be released from the MSCPs by increasing the ionic virus was propagated in Madin Darby Canine Kidney strength of the formulation solution to avoid, if necessary, (MDCK) suspension cells, and then clarified, inactivated, injection of nanoparticles . Overall, considering the broad concentrated and diafiltered against an adsorption buffer . range of virus species binding to sulfated cellulose Subsequently, virus particles were either bound to the matrices, MSCPs can be envisioned as a highly efficient and MSCPs via a short incubation in this buffer solution simple DSP platform technology for production of vaccines or purified by sulfated cellulose membrane adsorption and viral vectors . (SCMA) chromatography . Four groups of C57BL/6J mice were immunized twice intraperitoneally (i .p .) with (1) virus bound to MSCPs (1 µg HA/dose), (2) virus purified 35. Negative Chromatography of by SCMA (1 µg HA/dose; positive control), (3) the latter Virus-like Particle Using and MSCPs (w/o virus; contralateral i .p . injection), and (4) Adsorbent Grafted With the generally applied adjuvant solution (negative control) . Thermoresponsive Polymer Fourteen days after the booster immunization the mice were intranasally challenged by a lethal dose of the *Micky Fu Xiang Lee, Mr, Malaysia same influenza virus strain . After the challenge, the mice *Beng Ti Tey, Monash University Malaysia, Malaysia were weight daily for six days, sacrificed, and their lungs extracted for subsequent influenza virus RNA analysis . Prior Virus-like particle (VLP) is a large biomolecule with a to the immunizations and challenge, the antibody titer was valuable immunogenic property . However, the multivalent determined by a specific ELISA, which indicated that all binding site requirement and limitation in the diffusion groups except the negative control developed an immune of large biomolecules have reduced the binding capacity response against the influenza virus . The successful of most of the commercially available adsorbent to VLP immunization was confirmed by weight monitoring, and a particles . Hence, the downstream purification of VLP qPCR quantification of the pulmonary viral load . Only the encounters bottleneck, and the conventional bind and elute negative control group showed substantial weight losses type of chromatography is not suitable for VLP purification . and up to 400-fold higher viral load in the lung tissue . In negative chromatography, impurities are retained by Between the other groups no significant differences could the adsorbent whereas the VLPs are allowed to flow be determined . Hence, virus particles bound onto MSCPs through the column . As a result, the limitation in adsorption or unbound purified virus co-injected with MSCPs induced capacity of VLP can be avoided . Thermoresponsive polymer, a comparable immune response to chromatographically poly[oligo(ethylene glycol) methacrylate] (POEGMA), is purified virus in mice . Therefore, time-consuming excellent in repelling the protein from binding to the Poster Abstracts

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On the other hand, the . On the other Meanwhile, the impurities . Meanwhile, the impurities matrices today are infinitely superior to matrices today are infinitely superior to Future work is recommended to further improve . Future work is recommended United Kingdom United Kingdom United Kingdom United Kingdom hromatographic Thomas C. Willett, University of Birmingham, Thomas C. Willett, University of Birmingham, Thantawat Theeranan, University of Birmingham, Zhenyu J. Zhang, University of Birmingham, United Kingdom Eirini Theodosiou, Aston University, C on the basic their ancestors, and yet more than 60 years blueprint remains largely the same, with most continuing to perform just a single function . Against the backdrop of rising product titres, increasing size and complexity purity obtained under similar operation condition for SQ operation condition under similar purity obtained coated these two inert layer much higher than (62%) was has improved feed concentration . Using a lower adsorbents 1000 from InertLayer of flow-through HB-VLP the purity effect over SQ . Therefore, by 51 .5% but has no significant in handling higher feed concentration SQ is more capable SQ has not fully excluded 1000 . Yet, compared to InertLayer of impurities still remained in the the HB-VLP and a portion HB-VLP flow-through pool of than HB-VLP will co-flowthrough that having a size larger that reduced the purity of flowthrough together with HB-VLP HB-VLP of POEGMA without affecting the the shielding of HB-VLP from E . coli host cells . adsorption of the impurities Brush 36. Mixed Polyelectrolyte ‘Chameleon-like’ Surfaces Display Elution Binding And Protein Properties Edgbaston, Thomas, University of Birmingham, *Owen R.T. HB-VLP with 43 .7% of purity HB-VLP with . Furthermore, We have further accessed have further accessed . We . A longer chain length of POEGMA 80 | Recovery of Biological Products XVII SQ was further benchmarked against inert layer . SQ was further benchmarked against inert were prototypes for negative chromatography adsorbents . were prototypes for negative chromatography InertShell has a thicker inert shell which resulted in the thorough flow-through of the proteins while the thinner inert shell of InertLayer 1000 allowed 60% recovery of more impurities to adsorb which resulted in 86% removal more impurities to adsorb which resulted of impurities . The single step negative chromatography compared to of HB-VLP has shown better performance of previous studies of anion exchange chromatography HB-VLP 1000), which coated adsorbents (InertShell and InertLayer (LQ) restricted the penetration of impurities (from E . coli (LQ) restricted the penetration of impurities were embedded HCPs) to access the cationic ligands which beneath the POEGMA chains . On the other hand, a shorter better penetration chain length of POEGMA (SQ) allowed a of impurities through the POEGMA layer of SQ allowed the exclusion of HB-VLP from the surface on the effect of the chain length of grafted POEGMA on on the effect of the chain length of grafted of impurities . the HB-VLP exclusion and the adsorption POEGMA grafted adsorbents were fabricated, namely Two chain length) . SQ (shorter chain length) and LQ (longer The feedstock used was clarified E . coli lysate containing recombinant HB-VLP along with 79% of medium size BSA, while the small size along with 79% of medium in the column . Further increase in insulin was retained the lower critical solution temperature temperature above of the POEGMA chain, thus (LCST) led to the collapse of medium size BSA, while still increased the adsorption of HB-VLP retaining the exclusion When a feed contained When a feed onto the adsorbent . adsorbed [HB-VLP ~ 4 MDa (large), BSA ~ 66 mixture of proteins to the ~ 5 .8 kDa (small)] was loaded kDa (medium), insulin grafted adsorbents, the large packed column of POEGMA (88%) excluded from the column size HB-VLP was mostly Hence, in our work, POEGMA was grafted on the was grafted on the our work, POEGMA surface . Hence, in the large size recombinant adsorbents to repel cationized adsorbent, the binding site of VLP (HB-VLP) from hepatitis B host cell protein (HCP) the small size while allowing

Poster Abstracts of emerging bioproducts, escalating waste generation, –109 . [2] Cao, P . et al . (2015) J . Chromatogr . A, 1403: 118 and mounting competition from alternative techniques/ – 131 . [3] Hinrichs, K . et al . (2009) Langmuir 25: 10987 – formats the continued incremental improvement of existing 10991 . media represents an increasingly untenable approach to the development of new chromatographic materials for the biopharma industry . The past few years have seen 37. Development of a Novel a marked surge in research into ‘smart’ chromatography and Efficient Cell Culture adsorbents, largely driven by the realization that ‘smart Flocculation Process Using a materials’, especially ‘smart’ polymers, afford potential solutions to some of the aforementioned problems . By Stimulus Responsive Polymer to combining chromatographic supports modified with ligand- Streamline Antibody/Bispecific bearing temperature-sensitive copolymers with a novel Antibody Purification Processes purpose-built device, the Travelling Cooling Zone Reactor (TCZR), we recently demonstrated continuous thermally *Kenneth Kang, Eli Lilly and Company, United States mediated bioseparation of proteins without any alteration Richard Chen, Eli Lilly and Company, USA in mobile phase composition (i .e . under normal binding Bryan Dravis, Eli Lilly and Company, USA conditions), and also presented the first ever demonstration Dayue Chen, Eli Lilly and Company, USA of single column continuous chromatography [1,2] . Here we Michael Barry, Eli Lilly and Company, USA describe some of our work with pH responsive polymers . By tethering two oppositely charged smart polymer chains, Recent advances in mammalian cell culture processes have poly(2-vinyl pyridine) and poly(methacrylic acid), adjacent significantly increased product titers, but have also resulted to one another on the support at high grafting density, we in substantial increases in cell density and cellular debris have succeeded in generating high capacity adsorbents, as well as process and product-related impurities . As such, which in response to discrete changes in environmental pH with improvements in titer, corresponding improvements have the ‘Chameleon-like’ ability to reversibly transform between anion exchange, hydrophobic mixed mode and in downstream processing are essential . In this study cation exchange functionalities . In this presentation we we have developed an alternative harvest process that shall: (i) briefly explain the rationale behind the mixed incorporates flocculation using a novel stimulus responsive polyelectrolyte (PEL) brush concept and what’s known polymer, benzylated poly(allylamine) . As tested on multiple about pH induced changes that occur in mixed PEL layers antibodies including bispecific antibodies, this process [3]; (ii) describe three manufacturing approaches we demonstrates high process yield, improved clearance of have used to create well-characterized mixed PEL brush cells and cell debris, and efficient reduction of aggregates, modified adsorbents; (iii) illustrate the effects of pH, host cell proteins (HCP), DNA and viruses . A wide operating brush composition, polymer chain length and inter-graft window was established for this novel flocculation process spacing on the selectivity of binding of anionic, neutral and through design of experiments condition screening and basic proteins out of a four protein mix (Ovalbumin, beta- optimization . Residual levels of impurities in the Protein A Lactoglobulin, Carbonic Anhydrase, Lysozyme); and finally eluate were achieved that potentially meet requirements (iv) show the effectiveness of ‘pH shift only’ mediated of drug substance thus alleviating the burden for additional elution of bound protein species from mixed PEL adsorbents . impurity removal by subsequent chromatography steps . Poster Abstracts [1] Müller, T .K .H . et al . (2013) J . Chromatog . A, 1285: 97

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Germany Dariusch Hekmat, Technical University of Munich, Germany Dariusch Hekmat, Technical University of Munich, Technical Dirk Weuster-Botz, Preparative Packed Chromatography Chromatography Packed Preparative by Discrete Beds is Revealed Modeling Particle University of Munich, Germany *Martin Dorn, Technical using polymer- Preparative packed-bed chromatography based, compressible, porous resins is an essential separation method especially for macromolecular bioproducts . Due to a downstream processing bottleneck because of limited purification capacities, In other cases, novel ligands were developed novel ligands achieved . In other cases, when >0 .5 pH units, elution pH, on average to give milder a resulted on average pH gradient . This increase tested in a yield compared to traditional 30% increase in greater than an Fc fusion . In one case, where recombinant ligands the yield was improved from 11% to protein was tested, and antibody fragments 93% . . Capacity for antibodies combination with novel bead designs . was investigated in “steric barrier” to capacity were proven Previous claims of a binding capacity values on the to be untrue, with dynamic being achieved using various process order of 80-100 g/L each of these individually, . Taken and product designs are impressive, but combining performance improvements is an exercise in optimization . them into a single design ligands will be The design rationale chosen for various different discussed, as different applications require be prioritization . As a final point, these improvements will economy and analyzed in terms of their effect on process costs . the overall impact on antibody manufacturing 39. The Origin of the Complex of Behavior Hydrodynamic In some cases, up to 5X increases in alkali-stability were in alkali-stability up to 5X increases In some cases, 82 | Recovery of Biological Products XVII Tomas Björkman, GE Healthcare Life Sciences, Sweden Tomas GE Healthcare Life Sciences, Sweden Magnus Wetterhall, Sweden Ronnie Palmgren, GE Healthcare Life Sciences, Mats Ander, GE Healthcare Life Sciences, Sweden GE Healthcare Mats Ander, Healthcare Life Sciences, Sweden Gustav Rodrigo, GE antibody affinity ligands were investigated to evaluate their antibody affinity ligands were investigated effect on key performance characteristics for industrial purification . These modifications were made in singular and in plural due to the codependent nature of amino acid substitutions and structural changes in affinity ligands . less desirable characteristics . This case-study will describe in conjunction how high throughput screening was used, next generation with surface plasmon resonance, to develop capacity and protein ligands with improved alkali stability, strong affinity elution characteristics while retaining the for human antibodies . In total, over 400 modifications to L, Protein G, etc .) are the result of millions of years of against evolution by bacteria as a protective mechanism the needs . Today, mammalian antibody immune response faster than ever, of biological manufacturing are changing to preserve the and an accelerated evolution is required while improving the advantageous aspects of these ligands, *John Daicic, GE Healthcare Life Sciences, Sweden *John Daicic, GE Healthcare Life Sciences, Native protein affinity ligands (e .g . Protein A, Protein Technology to Meet the Needs of Meet the Needs to Technology Antibody Processes Generation Next process can be easily integrated into current mAb or bsAb into current mAb be easily integrated process can . purification platforms Evolution: 38. Accelerated AntibodyAdvancing Affinity In addition, efficient clearance of residual polymer was of residual polymer efficient clearance In addition, . of a stimulus reagent in the presence demonstrated removal during of HCP and aggregate The mechanism and efficient . This novel was also explored flocculation

Poster Abstracts strategy of combined flow and mechanical compression the chromatography equipment is often operated at its in a quantitative manner will be proposed in order to hydrodynamic limit leading to a complex, hysteretic, thus, substantially improve packing homogeneity . References: history-dependent packed bed behavior [1] . The theoretical [1] Hekmat D, Mornhinweg R, Bloch G, Sun Y, et al . understanding of the causes is still limited . Therefore, Macroscopic investigation of the transient hydrodynamic a rigorous modeling approach of the chromatography behavior of preparative packed chromatography beds . J . column on the particle scale will be presented which Chromatogr . A . 2010;1218:944-950 . [2] Dorn M, Hekmat D . takes into account the interparticle micromechanics Simulation of the dynamic packing behavior of preparative and fluid-particle interactions for the first time . A three- chromatography columns via discrete particle modeling . dimensional deterministic model was developed by applying Biotechnol . Prog . 2015 . DOI:10 .1002/btpr .2210 . Computational Fluid Dynamics (CFD) coupled with the Discrete Element Method (DEM) [2] . The column packing behavior during either flow or mechanical compression was 40. Designing Efficient Separation investigated in-silico as well as in laboratory experiments Processes of Modified Proteins by using a novel micro-chromatography column . The simulation satisfactorily reproduced the experimental observations Chromatography: PEGylated Proteins regarding the complex packing compression behavior as and Protein Aggregates well as the pressure-flow dependency . Pronounced axial *Shuchi Yamamoto, Yamaguchi University, Japan compression-relaxation hystereses were identified that Noriko Yoshimoto, Yamaguchi University, Japan differed for both compression strategies . It will be shown for the first time that the direction of the hystereses When native proteins are modified (intendedly or switched depending on the hydrodynamic conditions . unintendedly), modified forms must be separated from The individual study of flow and mechanical compression the native protein and other unwanted contaminants . A revealed distinct differences in the packing behavior which typical intended protein modification is PEGylation, where were related to wall support . The packed bed during flow PEG molecules are attached to a target protein . PEGylation compression exhibited nearly linear axial compression reaction mixtures contain various unwanted species, such leading to a most compressed region at the column outlet . as PEGylated isoforms (positional isomers), multi-PEGylated In contrast to this, mechanical compression led to an proteins (PEGmers), the native protein, non-reacted PEGs exponential compression profile with the most compressed and more . Electrostatic interaction based chromatography region at the top of the packing . By applying a novel UV- microscopy method, still existing void spaces were detected (ion-exchange chromatography, IEC) is known to be efficient in the packed bed even during high compression levels . It for PEGylated protein separations . We have analyzed was assumed that these void spaces are surrounded by PEGylated protein separations by linear gradient elution a particle force-chain network . Therefore, compaction of (LGE) IEC based on our models . PEGylated proteins were the chromatography packing is rather a result of particle much more weakly retained on the IEC columns . Weak rearrangement than particle deformation . Simulation results retention of PEGylated proteins was to be due to a steric indicate that packing anisotropy as well as the packing hindrance between the ion-exchange ligand and charges dynamics are governed by particle-wall and interparticle of PEGylated proteins . The shift of the retention volume of Poster Abstracts friction effects . A novel modeling-based systematic PEGylated proteins was well correlated with the calculated

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Hence, it becomes pertinent to solve . Hence, it becomes Technology, India Technology, Pamela Jha, DBT-ICT-CEB, India Pamela Jha, DBT-ICT-CEB, India Sandeep B. Kale, DBT-ICT-CEB, an affordable and effective pseudoaffinity adsorbent an affordable and effective pseudoaffinity by its use in the for purification of antibodies, followed process . An development of the antibody purification was, therefore, indigenous pseudobioaffinity adsorbent structure designed by studying the X-ray crystallographic of Protein A of the complex formed between B domain the potential and Fc portion of IgG . Based on this study, by molecular ligands were selected and further assessed docking studies . The ligand with maximum dockScore onto a and minimum internal energy was immobilized rigid polymethacrylate base matrix (particle size 90 µm, pore size 400 A0) . The novel pseudobioaffinity adsorbent, thus designed was named ‘ISep’ . The selectivity of ‘ISep’ was demonstrated by the purification of mAbs generated 41. ‘ISep’: A Pseudobioaffinity 41. Adsorbent the Purification for Of Antibodies Institute of Chemical DBT-ICT-CEB, *Sushmita Koley, (pAbs) and monoclonal antibodies Polyclonal antibodies class of biopharmaceuticals . (mAbs) are an important mAbs and plasma biologics The current rise of global to be up to $58 billion and $21 market is estimated billion, respectively manufacture of the underlying problems present in the antibodies . Cohn plasma fractionation for purification such as poor of polyclonal IgG suffers from limitations and difficulty in process yield due to the use of ethanol process automation . Protein A/G affinity chromatography, from various widely used for purification of mAbs suffers drawbacks, including high cost ($9000-$12000/L), (ligand instability to strong base (1M NaOH), leaching is toxic) and elution at low pH (>3 .5), which leads to IgG develop aggregates . The objective of the present work is to 84 | Recovery of Biological Products XVII Finally, in addition to linear in for protein monomer separation . Finally, gradient elution (LGE) mode, flow-through chromatography (or weak-partitioning chromatography) operation was designed and tested based on our method using LGE experimental data and the pore diffusivity correlations . monomer-dimer separation . The binding site values were monomer-dimer similar to those for conventional IEC . In order to understand salts such as the retention mechanism various different employed as an arginine chloride and sodium sulfate were additive or a gradient substance . Since no additives are attractive method needed this polyamine-based IEC is an binding site values as well as the peak salt concentrations binding site values as well as the peak examined for of protein monomer and aggregates were ligands . Among the various different types of ion-exchange anion exchange ligands tested, a salt-tolerant polyamine for the ligand showed quite high separation performance and PEGmers within ca . 4 minutes while the pressure drop was below 1 MPa . Another unintended protein modification is protein aggregation . Dimer and aggregate removal is . Since important for protein-drug separation processes and have more dimer and aggregates are more hydrophobic in IEC . The charges, their retention volumes are larger conditions and linear gradient elution experiments . Good conditions and linear between the diffusivities and correlations were established it is possible . Based on these data, the hydrodynamic radius . protein separation processes to design efficient PEGylated disk IEC was designed, which As an application, monolith isoforms can permit separation of 12 PEGylated protein Another important . Another important those for native proteins similar to . We PEGylated proteins is pore diffusion of parameter diffusivities of PEGylated proteins measured molecular and PEGs by the laminar flow as well as native proteins method . Pore diffusivities were dispersion based Taylor elution experiments at non-binding determined by isocratic . the protein molecule of PEG layer around thickness by were separated protein positional isomers PEGylated were proteins binding sites for mono-PEGylated IEC and the

Poster Abstracts against glycated hemoglobin (Hb1Ac) and pAbs from CCS on size exclusion . However, recent results point to the and human plasma, respectively, on a preparative scale . It fact, that virus particle retention can also be impacted was revealed that the elution of antibodies occurs at pH by adsorptive interactions between virus particles and 7 .0 for ‘ISep’, unlike low pH elution required by Protein A . membranes . Furthermore, the impact of flow rate and The integrity and purity of the antibody were confirmed flow interruptions on virus retention has been observed by SDS-PAGE and size exclusion chromatography (SEC) . and possible mechanisms discussed . The goal of this The centrifugal membranes of different porosity (30, 50, investigation is to gain a holistic picture of the underlying 100kDa) were screened for desalting and it was found that mechanisms for virus particle retention in size exclusion 50kDa performed best . The optimized protocol for ‘ISep’ membranes as a function of membrane structure and chromatography gave the purity of >95% (mAbs and pAbs) . membrane surface properties, as well as flow- and solution In a similar study, the conventional adsorbent, Protein A conditions . Therefore, parvo virus retentive membranes was used to purify IgG1 and the results were comparable . with different pore sizes and different levels of surface Moreover, ‘ISep’ is found to be stable in 0 .5M NaOH, the hydrophilization were generated in order to systematically most common SIP agent . The inhouse adsorbent designing change the level of size exclusion and adsorptive action process of ‘ISep’ is simple, robust, reproducible, economical on particle retention . The respective membranes were and can be scaled up . The critical parameters for indigenous characterized with regards to their permeability, non- purification process for both pAbs and mAbs using ‘ISep’ specific protein adsorption, surface zeta potential and have been optimized and product characterization in terms pore size distribution by using liquid-liquid displacement of quality, safety and efficacy is underway . Hence, ‘ISep’ porometry (LLDP) . Furthermore, an experimental space of promises to serve as an alternative to the conventional solution conditions regarding pH and salt concentration platforms for the purification of pAbs and mAbs . was chosen, that impacts the adsorptive interaction between virus model particles and membrane surface . Bacteriophages (like PP7) were chosen as model particles, 42. Mechanistic Study of Virus due to the established correlation to parvo virus retentive Particle Retention by Size Exclusion behavior and the related ease of handling . Filtration Membranes: The Interplay of Size runs to determine the respective levels of retention Exclusion and Adsorptive Action were complemented by flow stop conditions and a set of permeate fractions for titer determination was taken Under Different Flow Regimes at different process stages . This poster will introduce to Peter Kosiol, Sartorius Stedim Biotech , Germany the generated set of data and will draw first conclusions . Catharina Kahrs, Clausthal University of Technology, Based on the DOE character of the experimental space and Germany respective analysis, interactions between different factors Bjoern Hansmann, Sartorius Stedim Biotech , Germany can be determined . The results enable to differentiate *Volkmar Thom, Sartorius Stedim Biotech, Germany size exclusion from adsorptive action with respect to virus particle retention within the given space of material, Parvo virus removal by filter membranes is regarded as a process and solution conditions, significantly sharpening robust and efficient unit operation . The retention of virus our understanding of retention mechanisms in parvo Poster Abstracts particles by virus filter membranes is predominantly based virus filtration .

Recovery of Biological Products XVII | 85 . [1] B Cunha et al, J Membr Sci . Overall, this work shows that TFF is a scalable . Overall, this work shows that TFF is a scalable a hurdle that needs to be circumvented by manufacturers, a hurdle that needs to be circumvented in the cell therapy in order for these devices to be applied industry of hMSC . and efficient methodology for the concentration workflow of It can be incorporated in the biomanufacturing to other stem cell-based therapies, having also applicability for cell types (e .g . human pluripotent stem cells) relevant the cell therapy industry . 2015 2015 478, 117-129 . [2] B Cunha et al, J Biotechnol 213, 97-108 . processing the large expansion volumes (one to hundreds of volumes (one to hundreds the large expansion processing scale-up therapies, its and allogeneic liters) for autologous and quality needs characteristics on hMSC’s and impact (0 .25 to 2 was to scale-up The aim of this work assessment TFF-based concentration process [1,2] L) already established assessed (load, shear rate and based on process parameters we have compared the permeate flux) . More specifically, fiber and flat sheet cassette devices performance of hollow . The impact of all methodologies to concentrate hMSC identity viability, cell morphology, on cells’ quality (i .e . yield was evaluated . The most and potency) and recovery were validated with hMSC derived promising conditions adipose tissue . Results show that from bone marrow and concentrated up to a factor of hMSC could be successfully potency and high cell 50, while maintaining their identity, sheet cassette viability using both hollow fiber and flat that flat devices . Nonetheless, it was possible to observe 10% of cells at sheet cassettes allowed to recover more both processing the end of the concentration process in fluid dynamics (more scales (0 .25 and 2 L) . The device’s material of the turbulent), coupled to the nature of the such results . membrane (more hydrophilic) could explain unlike hollow fiber devices, pre-sterilization is However, 5 L), for TFF to be applied for lower scale (0 .5 L), for TFF studies at

. Despite previous

86 | Recovery of Biological Products XVII e Tecnológica, Oeiras, Portugal e Tecnológica, Experimental Oeiras, Portugal e Tecnológica, Oeiras, Portugal e Tecnológica, e Tecnológica, Oeiras, Portugal e Tecnológica, Oeiras, Portugal e Tecnológica, Oeiras, Portugal e Tecnológica, e Tecnológica, Oeiras, Portugal, Portugal e Tecnológica, Manuel J. T. Carrondo, iBET, Instituto de Biologia Carrondo, iBET, Manuel J. T. Instituto de Biologia Experimental Margarida Serra, iBET, Instituto de Biologia Experimental Paula M. Alves, iBET, Tiago Aguiar, iBET, Instituto de Biologia Experimental Instituto iBET, Aguiar, Tiago Instituto de Biologia Experimental Sofia Carvalho, iBET, Experimental Instituto de Biologia Marta M. Silva, , iBET, Bárbara Cunha, iBET, Instituto de Biologia Experimental Instituto de Biologia Experimental Bárbara Cunha, iBET, well established technology that has been arising as an well established technology that has been attractive solution for cell therapy DSP culture volume, in order to guarantee the required cell culture volume, in order to guarantee the be applied in numbers to be delivered to the clinic . To are required, and therapies low volumes for administration workflow should therefore downstream processing (DSP) compromising comprise a concentration step without filtration (TFF) is a flow cells’ characteristics . Tangential *Cristina Peixoto, IBET, Portugal *Cristina Peixoto, IBET, (hMSC) are Currently human mesenchymal stem cells microcarrier-based expanded using planar technologies or of liters of stirred culture systems from one to hundreds of Human Mesenchymal Stem Cells: Stem Mesenchymal of Human betweenComparison Hollow Fiber Cassette Devices and Flat Sheet 43. Scale-up of Filtration of Filtration 43. Scale-up the Concentration for Methodologies

Poster Abstracts progenitors, osteoblast-like derivatives and fibroblasts 44. Progress in Label-free using atomic force microscopy and data processing Techniques for Scalable Downstream algorithms to characterize the stiffness of cell populations . Processing of Cellular Therapies Undifferentiated hESC exhibited CEMs three-fold lower than those of differentiated cells, information we exploited *Nik Willoughby, Heriot-Watt University, United Kingdom to develop a label-free separation device based on the Helen Bridle, Heriot-Watt University, United Kingdom principles of tangential flow filtration (TFF) . With this device Mike MacDonald, University of Dundee, United Kingdom we segregated hESC mixed with fibroblasts and hESC- mesenchymal progenitors induced to undergo osteogenic There are currently whole cell therapeutic treatments in differentiation . The device is easily scaleable, permitted a development for a wide-range of “mass market” diseases . high throughput of cells, and achieved up to 50% removal of It is commercially inconceivable to consider an autologous specific cell types per single pass . The level of enrichment model for indications affecting large populations; soft, pluripotent hESC in the permeate was found to therefore a shift towards an allogeneic model is desirable . rise with increasing stiffness of the differentiating cells, There is currently much debate regarding the regulatory suggesting CEM can serve as a major discriminator . • Cell- landscape for cellular therapies, however we believe that cell separation using inertial focusing devices to separate the regulatory bodies are certain to require as stringent on a combined size/shape/density basis . Specifically we a demonstration of purity as is currently demanded for will present data regarding segregation of nucleated and protein therapeutics . This will be especially the case for enucleated cells at the latter stages of manufacture of allogeneic therapies . Consequently, there is an urgent red blood cells (RBCs) using a custom-designed inertial need to address the challenges associated with large- focusing device that can be stacked to enable scale-out scale downstream processing of cell therapies This work to high throughputs . • Cell-cell separation in optical and will present results from within our group investigating acoustic energy landscapes where segregation of cells is several novel techniques to separate cells of different achieved via focusing within the landscapes . Demonstration phenotypes and stage of differentiation based on physical of 1-D cell separation using optical landscapes has been and mechanical properties rather than the more currently previously demonstrated however the work here combines used “gold standard” of biological surface markers . this with ultrasonic acoustic focusing and translates Specifically the research presented here will demonstrate into 2-D and 3-D landscapes to illustrate the scale-up promising results and progress made over the last two potential of the technique . Our results demonstrate the years in the areas of: • Cell-cell separation on the basis of principle of a scalable, label-free, solution for separation elastic modulus using modified tangential flow filtration . of heterogeneous cell populations deriving from human Here, we identify differences in cell stiffness, expressed as pluripotent stem cells through a number of approaches . cell elastic modulus (CEM), for hESC versus mesenchymal Poster Abstracts

Recovery of Biological Products XVII | 87 No one can yet predict if a platform . No one can yet predict Maria Gencoglu, Michigan Tech, United States Tech, Maria Gencoglu, Michigan United States Tech, Ashish Saksule, Michigan flocculation in viral particles . This is likely due to the high hydrophobicity of viral particles . The high hydrophobicity of water makes viral particles flocculate in the presence the structuring osmolytes, as compared to stabilizing have demonstrated structure, as occurs in proteins . We (PPV), and an that a non-enveloped virus, porcine parvovirus be flocculated and enveloped virus, Sindbis virus (SINV) can of osmolytes . removed with a micropore filter by a variety particles and not The osmolytes were preferential to virus demonstrated a diafiltration have now model proteins . We process using mannitol as a common flocculant for both virus types . Infectious recoveries of >60% for both 46. Moving Towards Platform Virus Virus Platform Towards 46. Moving Purification Particle States United Michigan Tech, *Caryn Heldt, has evolved into a standard While antibody production viral particle purification technology, platform purification is still in its infancy for such a wide array of products technology can be used size, pI, and surface charge that have such a large lab is to explore methods distribution . One key goal of our to become a platform technology for that have the potential the viral particles . Last Recovery meeting, we presented on (OFT) to purify potential of osmolyte flocculation technology particles . Here, both enveloped and non-enveloped virus demonstrates we will present a purification process that providing a purification for both types of viral particles, purification . potential platform process for viral particle stabilize intracellular Osmolytes are natural compounds that . This is done by proteins against environmental stresses structure around a the ability of osmolytes to control water have capitalized on this osmolyte trait to induce protein . We

. The collaborative analysis from the 88 | Recovery of Biological Products XVII International Co., United States International Co., United Eileen Wilson & Olga Galperina, GSK, UK Novartis, Switzerland Konstantin Zoeller, Mike Clark, AbbVie, United States Mike Clark, AbbVie, United States Shengjiang Liu, Bayer, United States John Pieracci, Biogen, BMS, United States Thomas R. Gervais, United States Xinfang Li, ImmunoGen, David Roush, Merck, United States John Mattila, Regeneron, United States John Mattila, Regeneron, . Included the design and interpretation of future studies is an abundance of data that would have been difficult but collectively will help support to generate individually, modular viral clearance claims . . The extensive viral inactivation and nine for viral filtration the industry with a data set presented in this paper provides in addition reference point for establishing robust processes (ex . ASTM Std . to other protocols available in the literature E2888-12 for low pH inactivation) . In addition, it identifies set and instructs points of weakness in the existing data that are known to have robust and effective retrovirus that are known to have robust and effective clearance capability Clearance Viral members of BioPhorum Development Group common virus reduction steps considers two Team Working viral inactivation in biopharmaceutical processes: low pH and viral filtration . Analysis included eight parameters for Considerable resources are spent within the Considerable resources are spent within clearance biopharmaceutical industry to perform viral used unit operations studies which are conducted for widely Viral Filtration Data from Multiple Data from Filtration Viral Collaboration Company Pharmaceuticals Takeda *Norbert Schuelke, 45. Retrospective Evaluation of Evaluation 45. Retrospective and Inactivation Viral Low pH

Poster Abstracts In the presented case study, I will show how this strategy viruses was achieved with diafiltration using a micropore was applied at Janssen for fast development of a process membrane . When an ultrafiltration membrane is used, for inactivated polio vaccine production from high cell >90% of infectious virus recovery can be obtained . There density/high titer PER .C6® based cell culture harvests is a great protein reduction due to this operation, providing for which the use of existing polio virus purification purification of the virus particles, while maintaining technologies would be unsuitable . This was followed by infectivity and activity . OFT has a high potential to become optimization of bottleneck steps using statistical DoE and the key step in a platform virus purification technology small scale experiments and finally process verification at that could revolutionize the production of viral particle pilot scale . therapeutics . Mannitol is commonly used in antibody formulation and is currently on the FDA inactive ingredient list for approved drugs for both powder and intravenous 48. Mitigating Impurity Interactions applications, reducing the need for stringent removal from with Protein A and Monoclonal the final product . Higher purity with a low cost, platform process would increase the safety and decrease process Antibodies During Chromatography development time and production costs for future viral with PEGylated Ligands products . *Justin Weinberg, Carnegie Mellon University, United States 47. Cost-effective Manufacturing Shaojie Zhang, University of Virginia, United States of Viral Vaccines: Systematic Giorgio Carta, University of Virginia, United States Development of Inactivated Polio Todd Przybycien, Carnegie Mellon University, United States Vaccine Production Process Protein A (PA) chromatography is a highly selective capture and separations technique in use throughout the *Beckley Kungah Nfor, Janssen Vaccines, The Netherlands biopharmaceutical industry for the downstream purification The production of safe and efficacious vaccines at low cost of monoclonal antibodies (mAbs) . While PA is often is essential to guarantee world-wide vaccine availability responsible for removing ≥98% of mAb process impurities, and affordability, especially in developing countries . Our the remaining small, but significant amounts of host cell proprietary PER .C6® cell line and PIN platform technologies proteins (HCPs), DNA, and product aggregates necessitate have enabled the expression and production of viral multiple subsequent polishing steps downstream . In this vaccines at very high titers in cell cultures, thereby shifting work, we aim to decrease the post-PA chromatography the challenges and opportunities for further cost reduction burden on mAb downstream processes by discouraging to the downstream processing (DSP) . Our DSP development impurity interactions with PA media via chemical strategy to deal with these challenges involves the use of modification of the PA ligands with polyethylene glycol our established purification platform, which was designed (PEGylation) . Here, the main hypothesis is that PEG will for treating high cell density/high titer cell culture harvests . impede non-specific binding of contaminant species to Poster Abstracts

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United Kingdom United Kingdom Martina Micheletti, University College London, Martina Micheletti, College London, United Kingdom University Gary J Lye, using a novel scale-down or microscale bioprocessing using a novel scale-down or microscale process analysis tool we created [1] which enabled linked g . in situ DNA of the impact of upstream operation, e . recovery hydrolysis during homogenisation, on primary operation via crossflow microfiltration . The novel device and a 100-fold requires 10-fold smaller process volume available lab- decrease in membrane area compared to scale crossflow microfiltration module . This approach of recovery of a employing cell engineering to improve primary modification biological product complements biomolecular 49. Scale-down Tools to Assess to Tools 49. Scale-down by Engineering of Cell the Impact of Staphylococcal Co-expression E.Nuclease in to Improve coli Fragments of Fab Primary Recovery Microfiltration via Crossflow University College London, *Andrea CME Rayat, is a key parameter that impacts the recovery and Viscosity materials . The the overall manufacturability of biological resulted in almost a release of DNA during homogenisation have shown . We 2-fold increase in viscosity of E .coli cells reduced by over that the homogenate viscosity could be . coli strain via 3-fold through cell engineering of the E with antibody co-expression of Staphylococcus nuclease and compared the crossflow evaluated Fab fragments . We microfiltration performance of E . coli cell homogenate (Control) against the cell-engineered E .coli homogenate In a follow up study, we . In a follow up study, 90 | Recovery of Biological Products XVII . make the case for commercial consideration a higher percentage of static binding capacity over the a higher percentage of static binding capacity resin . same digestion period compared to unmodified potential benefits, we offers multiple While PEGylated PA operations and confront the tradeoffs of simplified polishing dynamic binding increased resin lifetime with reduced IgG simulation to capacity and increased resin costs via process fluid (CHO HCCF) . Here, we examine the relationships modification used between the size and extent of the PEG HCP eluate content, and on the resin on product recovery, we aggregate eluate content . In a third and final study, attack resists proteolytic demonstrate that PEGylated PA by retaining against chymotrypsin and host cell proteases results not only suggest that the PEGylated resins will have results not only suggest that the PEGylated also result in a lower level of but anti-fouling behavior, source of impurity product- associated impurities, a major chromatography carryover in PA via separation test the selectivity of the PEGylated resins cell culture of mAbs from Chinese hamster ovary harvest PEGylated PA is effective at reducing the interaction of the is effective at reducing the interaction PEGylated PA that are bound to the affinity ligand; contaminant with mAbs reduction is dependent on the size of the magnitude of the that PEGylation we reveal Additionally, the PEG chain used . of contaminant aggregates that bind reduces the amount of the resin particles . These promising to the outer surface PA resins using confocal laser scanning microscopy laser scanning microscopy using confocal resins PA with both sequential (CLSM) . Experiments were conducted of mAb and contaminant and and simultaneous loading both 5 and 20 kDa PEG chains . on resins modified with the CLSM images, we demonstrate that Through analysis of the ligand and to bound mAbs via steric hindrance as well via steric hindrance and to bound mAbs the ligand . attack and fouling ligand from proteolytic as shield the mAb and a interactions of a we explore the In one study, unmodified and PEGylated contaminant on model protein

Poster Abstracts for enhancing manufacturability . Very few articles in the operations such as homogenisation followed by crossflow literature reflect the inherent complexity in the crossflow microfiltration . This has ultimately informed larger scale microfiltration of biopharmaceutical feedstocks such as crossflow microfiltration operation and the necessary Fabs . Most focus on membrane fouling and flux decay design improvement of the cell engineered-strain . [1] Rayat, studies, not on the effect of process parameters on steady A .C .M .E ., Lye, G .J ., Micheletti, M . 2014 . A novel microscale state flux or product transmission which are important in crossflow device for the rapid evaluation of microfiltration bioprocessing and modelling studies . We demonstrated the processes . Journal of Membrane Science Vol . 452, 284–293 utility of the novel microscale crossflow filtration device in manufacturability assessment of the E . coli homogenates . This was mainly due to the wider range of experimental 50. Utilizing Mass Spectrometry and data it enabled us to generate which allowed for a more Mechanistic Modeling to Support in-depth analysis of crossflow microfiltration that described Development of a Cation Exchange not only flux behaviour, but including transmission of the Chromatography Capture Step key product: antibody fragments, as well as transmission of impurities as reflected by the total protein and DNA . Our *Stephen Hunt, KBI Biopharma, United States findings show that at 22kPa transmembrane pressure (TMP), Mark Sleevi, KBI Biopharma, United States viscosity reduction did not impact steady state permeate Garo Dombourian, KBI Biopharma, United States flux (12 LMH) but has improved %Fab transmission from Lindsey Irish, KBI Biopharma, United States 70% for the Control to 100% Fab transmission for the Robert Todd, KBI Biopharma, United States cell-engineered homogenate . At this TMP, total protein transmission from the Control is significantly lower Although the ability to develop mechanistic models for (45% vs 80%) . In contrast, DNA transmission is higher protein-based chromatography has been demonstrated, for the Control (45% vs 10%) . Furthermore, we have the industrial application to process development remains demonstrated that flux and Fab transmission was found limited . One of the challenges of mechanistic modeling to be independent of TMP at the high cell concentration for industrial applications is the complexity of the feed (90 gDCW L-1) used in the study . However, total protein streams and therefore, the amount of analytical data and DNA transmissions were not TMP-independent . required to estimate model parameters . In this case study, Results at the higher TMP (75 kPa) illustrated a decrease a combination of UPLC and mass spectrometry data are in %protein transmissions for both homogenates while used to estimate binding isotherm parameters for a complex for %DNA transmissions, only the Control homogenate set of host related impurities and product related variants showed lower transmission (20%) with no significant eluting during a cation-exchange capture step for an difference to the cell-engineered E .coli homogenate . Such industrially relevant recombinant protein produced in E . coli . rapid determination of key process information depicted the The challenges encountered in developing the model and interaction of the linked process sequences of upstream the insights gained by applying the model during process operation, including cell engineering, and primary recovery development will be discussed . Poster Abstracts

Recovery of Biological Products XVII | 91 . Osberghaus, A ., Lars Sejergaard, Novo Nordisk A/S, Denmark Denmark Ernst Broberg Hansen, Novo Nordisk A/S, 52. Process Validation (Stage 1) (Stage 1) Validation 52. Process Sampling: Using Latin Hypercube Case Study Chromatography *Thomas Hansen, Novo Nordisk A/S, Denmark the goal According to the process validation guideline for routine of stage 1 is to design a process suitable commercial manufacturing that can consistently deliver a product that meets its quality attributes . Based on this statement one should validate likely combinations of the UV absorption measurements are the large concentration are the large concentration measurements UV absorption and contaminants . It is between product differences measured at variable UV absorption spectra shown how with PLS . In combination overcome this issue pathlengths allows the selective quantification modelling, the approach the co-eluting contaminants even of both product and conditions . In summary, in overloaded chromatographic UV absorption spectra has a great PLS modelling with control in chromatography and potential for process from batch to continuous might simplify the transition pathlengths can processing . Measurements at variable by increasing the range of detectable support this method ., Briskot, T concentrations . [1] Brestrich, N inline quantification Hubbuch, J . (2014) . A tool for selective using spectral of co-eluting proteins in chromatography . Biotechnology analysis and partial least squares regression ., and Bioengineering 111:1365-1373 . [2] Brestrich, N . Advances in inline Sanden, A ., Kraft, A ., Hubbuch, J . (2015) quantification of co-eluting proteins in chromatography: application Process-data-based model calibration and towards real-life separation issues . Biotechnology and Bioengineering 112: 1406-1416 . A difficulty regarding species . A difficulty of all co-eluting quantification

In chromatography, the . In chromatography, 92 | Recovery of Biological Products XVII Germany Germany Matthias Ruedt, Karlsruhe Institute of Technology, Institute of Technology, Matthias Ruedt, Karlsruhe Germany of Technology, Karlsruhe Institute Laura Rolinger, Institute of Technology, Juergen Hubbuch, Karlsruhe and HCP concentrations in the feed . In a second part of the presentation, advances in PLS modelling for pooling control will be discussed . In contrast to the load control, where only the product is monitored, pooling decisions require the In a first part of this presentation, an approach conditions . In a first part of this presentation, an approach tool can be applied for will be presented how the PAT load control in capture steps . A PLS model is calibrated effluent . The to selectively quantify mAb in the column for load calibrated PLS model is subsequently applied varying mAb control in Protein A chromatography under variability and in the worst case to batch failure . In order to variability and in the worst case to batch tool based on inline UV absorption tackle this issue, a PAT (PLS) modelling spectra and Partial Least Square Regression has been published recently [1-2] . The tool was successfully and real- used for selective inline protein quantification under diluted time pooling decisions in polishing steps have been discussed increasingly decision in load volume in capture steps and the pooling . Due polishing steps are important process parameters inline protein to a lack of analytical tools for selective adapted quantification, both parameters are usually between batches . This may lead to higher lot-to-lot Since the publication of the PAT guidance by the FDA in of the PAT Since the publication of real-time monitoring of critical 2004, the implementation the real-time control of important quality attributes and production process parameters in biopharmaceutical to Polishing to Polishing , Germany Institute of Technology *Nina Brestrich, Karlsruhe 51. Application of PAT in in PAT of Application 51. Capture From Chromatography:

Poster Abstracts process parameters, i .e . the variation occurring on a routine with the number of parameters . As a consequence, the basis, and focus on robustness rather than “worst case” risk assessment has to end up with a suitable low number conditions . In this work we present a practical example of of relevant parameters (4-6), and this can compromise an alternative approach to the classic DoE methodology the credibility of the risk assessment exercise . Since used for process validation . The alternative approach interactions in DoE are studied as a combination of end is called Latin Hypercube Sampling (LHS) . LHS can be points, one for each parameter range, the likelihood of each considered an approximate Monte Carlo simulation and combination occurring in an actual production scenario delivers a good approximation of the probability function decreases exponentially with the number of parameters even with a limited number of experiments . A big advantage (assuming normal distributed ranges) . Thus, a substantial of the LHS approach is that one can choose the number of number of experiments performed within the DoE study will experiments independent of the number of parameters to be cover conditions which will practically never occur during investigated . Based on the distribution for each parameter production . According to the process validation guideline (e .g . normal or uniform) the LHS approach will come up with the goal of stage 1 is to design a process suitable for a statistically relevant design leading to output data within routine commercial manufacturing that can consistently the “routine manufacturing” range . An example of an actual deliver a product that meets its quality attributes . Based on validation study on a chromatography step is presented this statement one should investigate likely combinations including experimental design and data interpretation in of the process parameters, i .e . the variation occurring detail . on a routine basis, and focus on robustness rather than “worst case” conditions . In this work we present an alternative to the DoE approach for process validation 53. Process Validation (Stage 1) called Latin Hypercube Sampling (LHS) . LHS is an extension Using Latin Hypercube Sampling of the classic Latin square concept to more than three dimensions (forming a hypercube) and can be considered an *Lars Sejergaard, Novo Nordisk A/S, Denmark approximate Monte Carlo simulation . Using an algorithm, Thomas Hansen, Novo Nordisk A/S, Denmark the parameter values are distributed in the hyperspace Ernst Broberg Hansen, Novo Nordisk A/S, Denmark according to the stated probability distribution and even with a limited number of experiments we get a fairly good To validate and document a robust process design, approximation to this distribution . In the LHS approach laboratory studies are often required . The widely accepted one chooses the number of experiments independent of approach is to investigate the variation of the process the number of parameters . Based on the distribution for parameters in a classic DoE set up . Since a process each parameter (e .g . normal or uniform) the LHS approach step often has 10 – 20 input parameters it is practically will come up with a statistically relevant design leading impossible to include all parameters . Therefore a pre- to output data within the “routine manufacturing” range . selection of the relevant process parameters is done Thus, the LHS approach addresses the issues raised for using a risk based approach (e g. . FMEA type risk DoE . In this presentation we discuss the advantages of the assessment) . An inherent drawback of the DoE approach LHS approach compared to DoE based on both theoretical is that the number of experiments increases exponentially considerations and practical examples . Poster Abstracts

Recovery of Biological Products XVII | 93 Taiwan Taiwan Taiwan Taiwan Taiwan Ai-Ning Irene Lin, Development Center for Biotechnology, for Biotechnology, Ai-Ning Irene Lin, Development Center Hui-Chun Li, Development Center for Biotechnology, Pan, Development Center for Biotechnology, Yi-Hsuan Wei-Kuang Chi, Development Center for Biotechnology, Chi, Development Center for Biotechnology, Wei-Kuang *San-Cher Chen, Development Center for Biotechnology, Biotechnology, *San-Cher Chen, Development Center for affected by the Quality of therapeutic antibodies are deeply basal and choice of host cells, expression vector systems, feed media, fed-batch and perfusion process, downstream processes and bulk formulations . Appropriate analytical methods and bioassays are required to characterize the biosimilarity for antibodies but is being further developed is being further developed for antibodies but biosimilarity . In this for novel antibodies development tool as a process of antibody the structures PCA is used to predict study, different expressed from compare antibodies aggregates, and monitor antibody structural cell culture conditions, the downstream process and different changes caused by analysis by buffer conditions . The aggregate structure certain correlations with HDX-MS Protein Array found for different processing results . The antibody analysis the conformational changes conditions demonstrated including pH and along the process . The buffer conditions significant impact on antibody salt concentration showed well with aggregate level and structure, which correlate analysis by . Higher order structure Thermal Shift results to provide in- Protein Conformational Array is very useful . depth product and process understanding Antibody 55. Therapeutic Process During Characterization Development

94 | Recovery of Biological Products XVII United States Mukeshkumar Mayani, Bristol-Myers Squibb, Mukeshkumar Mayani, Bristol-Myers Squibb, United States Angela Lewandowski, Squibb, United States Zhengjian Li, Bristol-Myers Yuanli Song, Bristol-Myers Squibb, United States Song, Bristol-Myers Squibb, Yuanli United States Bristol-Myers Squibb, Elizabeth Schutsky, or Luminex®-based method uses a panel of 34 antibodies or Luminex®-based method uses a panel to detect epitopes that cover the whole antibody and thus indicate the antibody 3-D structure . It is simple to perform with high throughput and can be a complementary technique to HDX-MS . It was initially used to determine but are not suitable for large proteins . Mass Spectrometry Spectrometry and Hydrogen/Deuterium Exchange Mass protein (HDX-MS) are increasingly utilized to determine expertise and sequence and conformation but need special have low throughput . Protein Conformational Array (PCA) HOS . This ELISA is a new technique to determine protein determination is challenging for large proteins like determination is challenging for large proteins levels antibodies . Different biophysical tools have different of resolution and throughput . Light Scattering for protein structure provide size and Circular Dichroism for secondary low resolution structural information . X-ray Crystallography high resolution and Nuclear Magnetic Resonance have process understanding in biopharmaceutical development . process understanding in biopharmaceutical of biologics are The clinical effect and biological functions by processing, closely related to HOS which can be affected formulation and storage conditions . Therefore, HOS is relationships important in determining structure-function biologics . HOS and in guiding process development for Higher order structure (HOS) is critical for product and Higher order structure Array for In-depth Product and and Product In-depth for Array Understanding Process States Bristol-Myers Squibb, United *Deqiang Yu, 54. Antibody Higher Order Structure Structure 54. Antibody Order Higher Conformational by Protein Analysis

Poster Abstracts structure of both protein moieties and glycans attached . In to increase understanding of ADC conjugation processes, this study analytical data will be presented to show that mechanisms for product aggregation, and formation of even for the same antibody, different cell clones will exhibit minor Drug-to-Antibody Ratio (DAR) species . The study different aggregate levels and glycan compositions (Go, included 6 process parameters and allowed for detection G1, G2 etc .) . Also different processes using batch culture, of interactions across multiple reaction steps . Key outputs fed-batch culture and concentrated fed-batch culture will of the study demonstrated the overall robustness of the generate different glycan distributions . A fast way to conjugation process for delivery of consistent product determine glycan structure distribution (G0, G1, G2) using quality, but also provided specific observations, such as a LC/MS/MS were developed for the screening of culture correlation between formation of minor DAR species and conditions and media formulation designs leading to desired aggregates which was not previously known . The study quality attributes such as CDC and ADCC . Comparison of showed that these product quality outputs are driven by qualities of monoclonal antibody produced by shake flask similar parameters, such as temperature, conjugation cultures, 2 – 5 liter and 20 liter bioreactor culture show time, pH, and drug/Ab ratio, which may suggest formation that glycan profiles are quite different between batch through common mechanisms . In addition, conjugation culture and fed-batch culture, wheras that for concentrated reaction time and temperature were identified as process fed-batch and batch cultures are quite similar, the glycan parameters contributing to vHMWS formation . The process distributions are less affected during scale-up from 5 liter to design study enabled identification of key conjugation- 20 liter . specific parameters for robust control of product quality, delivering consistent manufacture for ADCs . 56. High Resolution Experimental Design to Study Antibody-Drug 57. Use of DOE and Bottom-Up Conjugation Process Robustness Mass Spectrometry Methodologies to Enable Rapid Process *Ben Sackett, Genentech, United States Thanmaya Peram, Genentech, United States Characterization and Optimization Jayme Franklin, Genentech, United States of a Complex Glycoprotein Jeff Gorrell, Genentech, United States Downstream Process

Antibody-drug conjugates (ADCs) are produced after *John Cundy, Pfizer, USA antibody intermediate (AI) purification through a series Emily Barr, Pfizer, USA of chemical reactions . To deliver ADCs of consistent Paul Brown, Pfizer, USA product quality and robust manufacturing, an increase in Brian X. Chen, Pfizer, USA process understanding of conjugation-specific parameters Keith Johnson, Pfizer, USA is required . In conjugation processes, kinetic parameters Joseph P. Martin, Pfizer, USA spanning multiple reaction steps can confound the John Plihal, Pfizer, USA identification of parameters impacting Critical Quality Thomas W. Powell, Pfizer, USA Attributes (CQA) . A high resolution study was designed Nicholas Russell, Pfizer, USA Poster Abstracts

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Dayana Soler, CIGB, Cuba Dayana Soler, CIGB, Cuba Laura Varas, Cuba Maelys Miyares, CIGB, Zárate, CIGB, Cuba Yasser Cuba Aniurka Panfet, CIGB, Cuba Alain Alfonso, CIGB, Cuba Milaid Granadillo, CIGB, CIGB, Cuba Isis Torrens, development to obtain an active pharmaceutical ingredient development to obtain an active pharmaceutical based on the HPV, for a cancer vaccine candidate against implementation of QbD . It helped us to build the knowledge non- critical space and get information about critical, also encompasses attributes and process parameters; and ranges as well as the design space and normal operating that unacceptable areas where the CIGB 550-E7 it is known product is produced . Finally the SuperPro Designer Software consider the was used as a computer tool in order to parameters such economic impact of some selected process the recovery purity and lifetime of matrices on as recovery, of CIGB 550-E7 downstream process . This methodology was found very convenient to identify where cost reduction can be achieved at early stage of the development project before the final stage of clinical trials will be conducted . 58. QbD and Process Simulation Simulation and Process 58. QbD for CIGB 550-E7, an Active for Ingredient Pharmaceutical HPV a New Vaccine Cuba *Miladys Limonta, CIGB, E7 HPV16 the comprising protein a fusion is CIGB 550-E7 The immunostimulatory antigen fused to a cell penetrating and terminal peptide which corresponds to the carboxy region of LALF 32-51 . Previously we demonstrate that response CIGB 550-E7 induces a potent antitumoral could become a against E7 expressing tumors, therefore of HPV 16 promising vaccine candidate for the treatment related malignancies . This work describes the process We have identified proteases . We 96 | Recovery of Biological Products XVII required dataset to support manufacturing, but also required dataset to support manufacturing, improvement provided key process understanding enabling and control . in overall process robustness, product quality, analytical control strategy process samples, and other host cell proteins in mid-stream modifications whose removal we can assure by appropriate the to key steps . DOE experimentation in conjunction with of an optimized mass spectrometry allowed for definition operating space . Our approach not only delivered the (DOE) and heightened in-process analytical methodologies (DOE) and heightened rapid process characterization and as appropriate to enable unit operations to achieve our goals . optimization for key spectrometry was used on key bottom-up mass Specifically, and studies which enabled an enhanced in-process samples with the established understanding not achievable Given the aggressive pace of project aggressive pace of scale . Given the to 13,000-L fit experimentation and process progression, rapid facility required to enable an understanding characterization was required and a basis-of-design prior of facility modifications design of experiment this end, we applied to start-up . To A complex recombinant glycoprotein expressed in CHO expressed in recombinant glycoprotein A complex clinical . To-date in late-phase development is currently . at 1000-L scale had been performed manufacture a process upscale forecast necessitated Commercial

Poster Abstracts a typical monoclonal antibody using a cell with 0 .1 mm cell 59. Approaches Enabling High length is demonstrated as well as examples from some unit Protein Concentration UV Sensing operations mentioned above . Requirements and possible in Bioprocess Applications limitations are discussed as well . *Hanno Ehring, GE Healthcare, Sweden 60. Purification of Recombinant Increased titers in cell culture, increased capacity of Polyclonal Antibody (rpAb) Mixtures: chromatography resins and Ultrafiltration/Diafiltration Impact of Polishing Modality on operations with concentrations beyond 200 g/l in combination with stronger PAT requirements all put new Aggregate Selectivity and mAb demands on UV/Vis absorption sensing in bioprocess Component Ratios applications . Currently, results obtained often show *Timothy Pabst, MedImmune, United States elution peaks that are saturated or non-linear so that no information about fine structure or sample concentration Frank Bartnik, MedImmune, United States can be extracted, which is not acceptable from a PAT Hongji Liu, MedImmune, United States perspective . To extend the dynamic range a wavelength Jihong Wang, MedImmune, United States where the protein extinction coefficient is lower is often Xiangyang Wang, MedImmune, United States used . For quantification, off-line sampling is utilized to Alan Hunter, MedImmune, United States estimate the final product yield . However these methods are cumbersome and not robust and the dynamic range is Recombinant polyclonal antibodies (rpAbs) represent a often not sufficient . In this study devices, concepts and novel class of biopharmaceuticals that enable targeting results are presented that can overcome the limitations of multiple antigens . To reduce cost, it is anticipated that above . By combining different wavelengths and pathlengths rpAbs may be manufactured in a single batch in which including dual pathlength flow cells with very short the individual component monoclonal antibodies (mAbs) pathlength down to 0 .1 mm many different options are are co-expressed in the same bioreactor and purified together . For mAbs, multimeric species are often the most possible that can address multiple needs . The cells can challenging product related impurity to remove . Separation be adapted to flow ranges from laboratory to large scale of multimers in mAb processes is frequently achieved using production equipment . Unit operations that currently cation exchange chromatography (CEX), where monomer see limitations regarding in-line sensing of high protein purity of the final antibody preparation can exceed 99% . concentrations include sample loading, check for impurities For therapeutic IVIg preparations derived from human in elution peaks, UV-based product pooling criteria, plasma, the level of multimers is often higher, likely due to quantification of total product concentration, product the diverse nature of the material (i .e ., different isoelectric concentration in ultrafiltration, straight-through processing points and IgG subclasses) . As a result of this diversity, it and dynamic control in continuous chromatography is difficult to remove multimers without simultaneously applications . Data will be presented showing the usability separating monomers that differ based on characteristics of the concepts in different situations . Linearity of such as charge . Similar to mAbs, it is desirable to control measurement up to 200 AU/cm corresponding to 150 g/l of multimeric species at low levels for rpAbs . Dissimilar to Poster Abstracts

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R-based  Hammad Shaikh, Novo Nordisk A/S, Demark Gong, Novo Nordisk Research Centre China, China Wei . One of the major system for therapeutic protein production in cytoplasm . expression formats is soluble expression from the cell, host Upon recovering soluble target protein released as well . cell proteins and chromosome DNA are most likely be If the target protein is acidic, capture will . As over 50% carried out using an anion exchange resin they all of E .coli host cell proteins are acidic in nature, binding sites during compete with the target protein for the DNA and endotoxin, which are both capture . Moreover, heavy negatively released during the recovery steps, are charged entities that bind aggressively and compete for the binding capacity with the target protein . Consequently, the binding capacity for the target protein is decreased dramatically and the CIP of the used anion exchange resin speciation of IgG2, IgG3, nonfucosylated and bisected and bisected of IgG2, IgG3, nonfucosylated speciation evaluated for elution peaks were the glycans . Additionally, Killer cell degranulation of both Natural their potentiation phagocytosis as well as Monocyte (ADCC surrogate) revealing FcgRIIIa-based in in vitro cellular bioassays significantly increased effector chromatography most the use of Fc function . This work demonstrates plasma IgG and directly chromatography to reformulate and illustrates the potential its impact effector function to develop new therapeutics from use of this technology plasma IgG . Mix Mode 62. Use Cationic (CMMC)Chromatography to Purify E.coli from Bi-functional Protein Cell Lysate China, China *Honghao LI, Novo Nordisk Research Centre expression E .coli is one of the most wildly employed using high-throughput techniques reveal receptor-specific reveal receptor-specific techniques using high-throughput

. . Conceptual design Rs) on immune cells, which in  98 | Recovery of Biological Products XVII Rs as chromatographic affinity ligands, we were able MIT, United States MIT, United States  Alison Mahan, Ragon Institute of MGH, Harvard, Alison Mahan, Ragon Institute of MGH, Ragon Institute of MGH, Harvard, MIT, Galit Alter, United States Margaret Ackerman, Dartmouth College, Ty Thach, Dartmouth College, United States Thach, Dartmouth Ty . Leveraging cellular cytotoxicity (ADCC) and phagocytosis Fc to enrich in high affinity subclasses and glycoforms using human plasma derived IgG as a load material . Biophysical characterization of of both subclass and glycosylation Human plasma is used as a manufacturing feedstock for Human plasma is used as a manufacturing both immunity a number of licensed IgG therapeutics in and infectious disease . Part of the mechanism of action engagement of for some of these therapies relies on the Fc gamma receptors (Fc turn elicit effector functions such as antibody-dependent Receptors as Affinity-based as Affinity-based Receptors Ligands Chromatography *Austin Boesch, Zepteon, Inc., United States ratios are discussed for each modality ratios are discussed strategies, and data will be of rpAb processes, purification presented . of IgG Subclasses Separation 61. Fc Gamma Using and Glycoforms In this work we examine . In this work not be appropriate as CEX may to be used in the manufacture of purification options hydrophobic interaction, rpAb therapeutics, including multi-modal chromatography hydroxyapatite, and on component mAb yield and impact Aggregate selectivity, mAbs, rpAb purification adds the additional constraint the additional constraint purification adds mAbs, rpAb mAbs individual component ratios of the that the relative challenging . For such a and controlled be well understood such used for mAbs traditional approaches separation,

Poster Abstracts also often remains a challenge . To improve this situation, testing purification cases show very nice selectivity and basic tags are designed and fused with the target protein, binding capacity, which indicates that the idea is worth to so that the pI of fusion protein can be increased for CIEX be further explored . In future it could be possible to become column . There have been successful cases reported . Further a generic protein production platform . study indicates that the basic tag can form a distinct motif from target protein so that the fusion protein behaves as a ‘bi-functional’ protein with two pI domains instead of 63. Demonstration of a mAb one average pI . With this character, the fusion protein Chromatography Platform Process can be captured by CIEX at pH base on pI of fusion tag Operating in a Continuous and but not average pI of fusion protein . With conventional Integrated Mode: Flexibility for cation exchanged chromatography purification at acidic condition(for example at pH 5-7), the removal of acidic HCP, Facility Fit and Process Economics endotoxin and DNA is enhanced, but the selectivity to the Optimization basic host cell proteins is not as efficient as that to acidic Xhorxhi Gjoka, Pall Life Sciences, United States host cell proteins As the pKa of basic residues on the tag Rene Gantier, Pall Life Sciences, United States of the bi-functional molecule are much higher than 7, they *Mark Schofield, Pall Life Sciences, United States can easily be protonated at neutral or even higher pH . So the positively charged tags can interact with the cationic The biopharmaceutical industry is adopting continuous groups on the resin . As CMMC can tolerate relative higher processing to address the challenge of process concentration of salt, which can enhance the hydrophobic intensification for monoclonal antibodies (mAbs) . interaction between the target protein (if the target protein Continuous multi-column chromatography (MCC) is a key exhibits medium or higher hydrophobicity) and hydrophobic facilitative technology for the adoption of continuous group on the resin . The actual binding strength between downstream processing . The major focus so far has been on the CMMC resin and the bi-functional protein is much the implementation of MCC for the Protein A capture step, higher than the simple sum up of independent cationic and leading to significant reduction in cost through minimization hydrophobic interaction strength . As a result, the selectivity of Protein A resin volume . In production scenarios, where to the bi-functional molecule is much higher than to the resin cost is amortized over many cycles, additional benefits basic host cell proteins, while the acidic host cell proteins, including facility utilization, buffer consumption and DNA and endotoxin are in flowthrough . The relatively higher overall process cost can be achieved through the operation conductivity would not be an issue as it would be when of a fully integrated and continuous chromatography anion exchange chromatography was employed . Overall, purification process . In this study we establish a complete the absorption shows not only very high selectivity but mAb chromatography purification platform, operating in a also high binding capacity and salt tolerance comparing continuous and integrated mode, with optimized process with that of conventional chromatography techniques to productivity, yield of recovery and purity . The process, normal proteins . The absorbed bi-functional proteins can comprising a Protein A capture step followed by low pH be eluted out by relative higher pH and/or combination of viral inactivation and two polishing steps, anion exchange proper concentration of salt, which compare with most of

(AEX) and mixed-mode chromatography, is performed with Poster Abstracts the affinity elution condition(low pH), is much milder . The

Recovery of Biological Products XVII | 99 Cassia Andrade, MedImmune, United States States Matthew Aspelund, MedImmune, United MedImmune, United States Ronald Schoner, MedImmune, United States Alan Hunter, Bispecific Antibodies Using a Three Bispecific Antibodies Using a Three Process Column mAb-like Platform Keen Chung, MedImmune, United States *Wai (BisAbs) are a Full length IgG-like bispecific antibodies novel class of therapeutics that allow for the binding of multiple targets or two distinct epitopes of the same target, Fab’s with hinge cysteines can be assembled into a Fab’2, be assembled into cysteines can with hinge Fab’s forming a heterodimer preference for but no structural very which can lead to exists, different Fab’s between two leaving process using a yields . An improved low process during an of two Fab’s group to promote heterodimerization been developed . The formation of assembly reaction has in the assembly of two distinct Fab’s a Fab’2 bispecific by heterodimer poses several unique order to form a Fab-Fab from combining the two challenges . The assembly mixture in order to remove unique assembly must be purified Fab’s homodimers, free light chain and heavy byproducts such as Fab’, leaving group, and others . chain, adducts, unreacted including host cell process-related impurities Typical molecular weight species must also proteins, DNA and high be be removed . Stability of the process intermediates must investigated . Multiple novel unit operations were developed separation . High in order to achieve effective assembly and rapidly develop a throughput screening was leveraged to . Non-standard significantly off-platform purification process in order to analytical methods were also implemented . support the purification process development for Removal Aggregate 65. Improved Two . Two immunogenicity risk and may pose an heterogeneity

.

To perform the four unit operations, perform the four . To 100 | Recovery of Biological Products XVII a new format, a bispecific Fab’2 . Different approaches can be taken to generate a Fab’2 bispecific . Enzymatic cleavage of a full-length bispecific can generate a bispecifc the cleavage site can introduce product Fab’2 . However, Recent advances in biologic drug development have led to Recent advances in biologic drug development target diseases in creation of new formats of biologics to biologic new ways . A large and rapidly growing area of new . A diverse formats is bispecific and multispecific antibodies for manufacturing set of bispecific formats and approaches them now exists . This talk will cover the development of 64. Assembly and Purification of 64. Assembly a Bispecific Fab’2 *Glen Giese, Genentech, United States equipment requiring a total bench length of less than 20 equipment requiring a total bench length in feet . This represents a significant improvement to a traditional process time and facility usage compared may normally be batch process where the 4 unit operations using more than 1l operated for at least 2 consecutive days of total chromatography media . operation of this continuous and integrated platform operation of this continuous at 1mg/ content of a 25L bioreactor, allows processing the 8 hours by deploying a total volume in under ml mAb titer, media of less than 200 ml, leading of chromatographic to 4g of purified mAb per hour to throughputs of up using process development This operation is conducted connected to the second system operating the two polishing operating the two to the second system connected inactivation step integrated in steps with low pH viral the process, by combining between . The simplification of high productivity and throughput, unit operations, enables in footprint and process cost . The and further reduction minimal buffer adjustments enabling unit operations are unit operations adjustments enabling minimal buffer directly integrated Capture are employed: MCC systems only two BioSMB® is a first system which A is performed onto on Protein

Poster Abstracts potentially allowing mechanisms of action that may not be achievable with mAbs or mAb combinations . However, 66. A New Fab-Fusion Protein a common trend observed in many BisAb processes is Therapeutic a higher level of aggregates (10-20%) at the end of cell *Michiel Ultee, Ulteemit BioConsulting, LLC, United States culture compared with mAbs . The increased aggregate burden at the start of purification exceeds the capabilities Emily Schirmer, Catalent Pharma Solutions, United States of a conventional mAb platform purification process, Dustin Armstrong, Valerion Therapeutics, Inc, USA typically employing cation exchange chromatography We describe the design, development and manufacture (CEX) for aggregate removal . In addition, the high of a unique antibody-fusion protein consisting of a Fab- aggregate levels have been implicated in poor virus linked enzyme rather than the more typical Fc-linked fusion removal on anion exchange flowthrough unit operations . protein . VAL-1221, a preclinical product candidate for Alternative chromatography options (e .g . multimodal, glycogen-storage diseases, contains a humanized anti- hydroxyapatite, HIC) usually provide better aggregate dsDNA Fab genetically linked to the acid alpha glucosidase clearance compared to CEX, but they each present enzyme (GAA) . The Fab portion of this construct was distinct manufacturing challenges . Various methods of derived from a murine antibody previously demonstrated introducing improved aggregate removal capability into a as the Mab or single-chain fragment to possess cell- commercial three column mAb-like platform purification penetrating properties . These were dependent on its process were evaluated . This included the use of i) pH DNA-binding activity and membrane expression of a gradient elution in the Protein A capture step; ii) calcium nucleoside transporter (ENT2), highly elevated in human phosphate precipitation after harvest and/ or low pH skeletal muscle . The VAL-1221 fusion protein enables viral inactivation; iii) High Performance Tangential Flow increased muscle delivery via its dual modes of both Fab- Filtration (HP-TFF) . In all cases, the process modifications mediated and M6P-targeted (GAA) cellular uptake . This novel construct was initially produced in transient CHO showed sufficient aggregate clearance to enable use of cultures, followed by a stable CHO cell line based on the CEX polishing . The different process options were evaluated GPEx expression system . To substitute for the Fc-directed based on scalability, robustness, and platformability . High Protein-A capture step found in most antibody and Fc- throughput development data as well complete process protein downstream processes, we compared alternative scale demonstration run data are provided . Results indicate affinity supports focused on the Fab, either on its CH1 or that product quality and process performance obtained light-chain region . We selected a Protein L support rather from precipitation and Protein A pH gradient processes are than anti-CH1, since the fusion of the GAA reduced capacity similar to that of a conventional mAb platform . of the anti-CH1 support for the Fab-GAA . A series of ion- exchange and hydrophobic-interaction chromatography polishing steps followed to remove both host-cell proteins (HCP) and excess light chain . Purification challenges included the sensitivity of the GAA to pH levels below pH 3 .5 and above pH 7 .0, a tendency for HCP to track with the fusion protein on some steps, and high levels of free and dimeric light chain in the Protein L eluate . The final process

overcame these challenges to result in production of clinical Poster Abstracts material at the 250L scale .

Recovery of Biological Products XVII | 101 . There is no approved reversal agent for direct . There is no approved reversal agent for Stuart Builder, Strategic BioDevelopment, United States Strategic Stuart Builder, Kent Iverson, Consultant, Un Portola Pharmaceuticals, United States Kentaro Takeda, States Genmin Lu, Portola Pharmaceuticals, United States Portola Pharmaceuticals, United Pam Conley, surgery Factor Xa inhibitors . Andexanet alfa was developed as a and indirect fast acting, universal antidote for both direct Factor Xa inhibitors . It is a recombinant protein, derived activity from Factor Xa, with no pro- or anti-coagulant anticoagulation in Phase 3 trials demonstrated reversal of healthy volunteers (Siegel et al ., NEJM 2015) . FDA older, approval granted breakthrough status and accelerated for Andexanet alfa . The company thus maintained an process for established 1st generation manufacturing meet all the expected demand, earliest BLA submission . To a 2nd generation manufacturing process was developed and scaled-up to 10,000L . This process is based on a novel affinity capture step, utilizing a naturally occurring protein biochemical studies and the precautions taken to perform taken to studies and the precautions biochemical to final drug of the ADC’s and purifications conjugations drug . are being used as as cytotoxic agents substance 68. of a Second- Development for Andexanet Process Generation for Protein Alfa, a Recombinant the Reversal by of Anticoagulation Xa Inhibitors Factor Pharmaceuticals, United States *Mark Karbarz, Portola patients will be It is estimated that by 2020, 30 million prevention of treated with a Factor Xa inhibitor for the atrial fibrillation, DVT or PE . Of these patients, an estimated due to a life- 500,000 will be admitted to the hospital emergency threatening bleed, or because they require characterization of the ADC’s with physicochemical/ with of the ADC’s characterization .

. The mechanism of action consists 102 | Recovery of Biological Products XVII Netherlands Netherlands Netherlands Netherlands Patrick Beusker, Synthon Biopharmaceuticals BV, BV, Synthon Biopharmaceuticals Patrick Beusker, Netherlands BV, Synthon Biopharmaceuticals Henri Spijker, Biopharmaceuticals BV, Ruud Coumans, Synthon Guy de Roo, Synthon Biopharmaceuticals BV, Netherlands Biopharmaceuticals BV, Guy de Roo, Synthon Netherlands Biopharmaceuticals BV, Bram Kamps, Synthon Biopharmaceuticals BV, Synthon Ruud Verstegen, . As second disease and anaplastic large-cell lymphoma (Kadcyla®) from Roche received FDA product T-DM1 approval as improved treatment for HER2-breast cancer on the development will show the current progress We of purification processes for Antibody Drug Conjugates, by cleaving the linker and the monoclonal antibody from by cleaving the linker and the monoclonal to block cellular the ADC so that the cytotoxic agent is able processes followed by cell death . In the past few years the (Adcetris®) from first new ADC variant brentuximab vedotin as a new treatment Seattle Genetics received FDA approval for U .S . patients with a pair of rare lymphomas—Hodgkin’s are new biotherapeutic medicines consisting of a drug are new biotherapeutic medicines consisting linker and a (chemotherapeutic agent), a (non-)cleavable monoclonal antibody on the cells by of the recognition of a specific receptor is internalized monoclonal antibodies, the complete ADC in the cell into the cell and the cytotoxic agent is released In the past years biopharmaceutical companies have In the past years biopharmaceutical companies as a follow- searched for new biological entities (NBE’s) . One of up of the very successful monoclonal antibodies are the Antibody the most promising new lines of NBE’s Drug Conjugates . Antibody Drug Conjugates (ADC’s) Therapeutic Molecules Therapeutic Biopharmaceuticals BV, *Michel Eppink, Synthon 67. Antibody Drug Conjugates: Antibody Drug Conjugates: 67. of Protein Platform A New

Poster Abstracts ligand from soybean . The entire process is simpler, more the target protein and host cell protein impurities . Several robust, and increases overall yield by 4-fold, with half of members of the heat shock protein family were identified the improvement coming from downstream operations . A via mass spectrometry as persistent impurities throughout case study will be presented detailing the issues that arose the purification process . However, a set of optimal during development and the decisions that were made . precipitation conditions were identified that, when used in combination with the appropriate depth filter, could significantly reduce the abundance of these impurities . 69. Purification of a 900 kDa The combination of traditional precipitation techniques and Trimeric Glycoprotein: Marrying a novel affinity-based capture step were instrumental in the Old and the New enabling the purification of a 900 kDa protein . *Anne Kantardjieff, Alexion Pharmaceuticals, United States Abe Friedman, Alexion Pharmaceuticals, United States 70. Purification of Secretory Immunoglobulins A process was developed to purify a 900 kDa heterotrimeric extracellular matrix (ECM) protein with a high propensity *Rainer Hahn, BOKU Vienna, Austria for polymerization . Low titers from cell culture, the Hannah Engelmaier, BOKU Vienna, Austria presence of closely-related contaminating species, and Gottfried Himmler, Angothera GesmbH, Austria close interactions between the target protein and several Alexander Matlschweiger, BOKU Vienna, Austria members of the heat shock protein family, posed significant challenges during development . A novel affinity-based Immunoglobulin A (IgA) is the most abundant antibody capture step was developed which exploits the naturally class in the human body . In its secretory form (SIgA) it is occurring high affinity binding between the target protein the main effector of the mucosal immune system . With its and another ECM protein . An Fc-tagged version of both the high therapeutic potential it is a promising candidate for full length and binding domain of the binding partner were prophylaxis or therapy against various diseases associated generated and used as an affinity ligand in combination with the respiratory, gastrointestinal or urogenital tract . with Protein A . Elution conditions were identified that So far, medical application of SIgA has been hampered by allowed for elution of the target protein, while keeping the difficulties in producing and purifying large quantities . We Fc-tagged affinity ligand bound to Protein A . In addition, have studied the isolation of SIg from animal whey, which a refolding step was incorporated following elution of the is a comparably rich source with SIg representing 4-5% of target protein and re-use of the ligand was demonstrated total protein . Besides SIgA, whey also contains considerable out to 6 cycles . To increase binding capacity, the unit amounts of secretory immunoglobulin M (SIgM) . In this operation was optimized using a Protein A monolith, leading work, the purification characteristics of these antibodies to an increase in resin load of more than ten-fold . The was studied on the basis of standard unit operations for development of an affinity capture step was instrumental in protein purification, including membrane separations, achieving purity targets . Low pH precipitation was utilized precipitation and various chromatographic methods . as a key step for host cell protein removal . Conditions were Difficulties in the present study aroused from the high identified that provided significant separation between abundance of accompanying whey proteins . Due to the large Poster Abstracts

Recovery of Biological Products XVII | 103 . In our . For baculovirus . The presented study highlights many of the . The presented study Sofia B. Carvalho, IBET, Portugal Sofia B. Carvalho, IBET, Portugal Ricardo J. S. Silva, IBET, Portugal Mafalda G. Moleirinho, IBET, Portugal Paula M. Alves, IBET, Portugal Carrondo, IBET, Manuel J.T. Portugal Cristina Peixoto, IBET, *Alex Xenopoulos, Merck Millipore, United States *Alex Xenopoulos, Merck Millipore, United particles (VLP) have become prime vaccine Virus-like immunogenicity and candidates because of their versatility, safety profile . The diversity of surface epitopes contributes, to a variability in downstream purification, that however, could ultimately affect manufacturability Under these conditions the residual IgG did not conditions the residual column . Under these mg/mL . at capacities of 20 could be captured bind and SIg could SIgA and SIgM a linear salt gradient, By applying process step . The overall during the elution be separated low at 15% when all steps were yield for SIg was rather achieving maximal purity operated at conditions be a too serious problem since whey case, this would not raw material available in practically is an extremely cheap results of this work can be unlimited amounts . However, may go beyond the isolation of SIg of general interest and from whey associated with purification of critical steps and bottlenecks potential provides furthermore and molecule complex a such processing options . Envisioning solutions and alternative SIgA or SIgM, yield purification of recombinant monoclonal has certainly to be increased . This may be achieved by i) selectivity better ii) and material starting the of purity higher more homogenous of the respective unit operations due to characteristics of a monoclonal antibody for Process Platform Filtration 71. Like Particles Purification of Virus sodium chloride and directly loaded onto a POROS 50 HQ onto a POROS and directly loaded sodium chloride . As has been Polyethylen glycol at . Polyethylen glycol at -lactoglobulin, which remained in -lactoglobulin, which  104 | Recovery of Biological Products XVII -lactalbumin and SIg was collected in the flow through . This step utilized the low effective pore diffusion coefficient of SIg in the small-pore medium, an effect that was further enhanced when operated under conditions where the impurities were bound . The flow through was then supplemented with was hindered by a multi-component adsorption effect of SIg was hindered by a multi-component adsorption a capacity of only being displaced by oligomers leading to could 3 mg/mL . High purity and good process performance exchange steps . be obtained by a combination of two anion operating conditions In the first step on Q-Sepharose FF, were captured and were chosen, at which albumin and IgG A general problem of processing large molecules needed . A general problem of processing large accessibility with chromatography is the restricted pore resulting in slow diffusion and/or low capacity monoliths have the potential to overcome shown previously, diameter and this problem due to their large channel case the application convective transport but in the present fractional precipitation and the dissolved precipitate was fractional precipitation and the dissolved in a purity of ~ 75% . then subjected to diafiltration resulting obtain high purity of > 95% several chromatographic To methods were investigated . Chromatography with a core efficient . In this bead technology (Capto core 700) was not 500 kDa would be case, a different exclusion limit of 300 or However, fractional precipitation at 3% was effective to fractional precipitation However, of large molecular weight fraction remove around 50 % of . Furthermore, concentration containing Ig oligomers  was significantly reduced . In the residual supernatant, ultrafiltration was followed by an optimized process, to achieve high purities of 60-70%, whereas the membrane whereas the high purities of 60-70%, to achieve factor cut-off was a less dominating was very efficient in precipitating concentrations of 6-7% selectivity was poor with respect SIg out of solution but IgA dimers and albumin . to removal of IgG, non-secretory molecular weight of SIg with 400 and 900 kDa, respectively, 400 and 900 kDa, respectively, weight of SIg with molecular was the obvious ultra/diafiltration low titer, and also the . Low transmembrane the purification sequence first step in parameter import operating found to be most pressure was

Poster Abstracts expression systems in particular, the similarity between provides the yields to meet growing therapy demands . residual baculovirus and VLP particles causes significant Key to this however, is to consider the impact on product problems . For that purpose, we have undertaken an effort quality and process performance . The cell culture media to develop platform processes for purification of VLPs . In plays a critical role in providing the primary source of the one approach described here, we focus on size exclusion raw materials that eventually end up in the drug itself . as the key mechanism of separation, with the ultimate Many of the amino acids making up the primary structure goal of an all filtration purification process . The first step of the therapeutic protein are derived directly from the was to evaluate a legacy purification that was not robust culture medium . Therefore determining the correct feeding or efficient and replace the ion exchange chromatography strategy that meets all the process requirements, including step with size exclusion chromatography . Performance of the impact on downstream and product stability are central the SEC step will be described and the shortcomings of such to meeting the demands for the requirements for consistent a method for a scaled up, GMP process will be discussed . product quality profiles with little product variance . In this The proposed all-filtration process will then be described, study, we describe the impact of changing the upstream employing either normal or tangential flow filtration for the feeding regime and varying the concentrations of the feed clarification stage, followed by multiple ultrafiltration steps to the bioreactor on the subsequent downstream process to achieve the needed concentration and diafiltration purity steps and the effect on the product stability profiles . The specifications . Efforts to clear nucleic acid without the use feeding regime is shown to directly impact key product of an endonuclease digestion step will also be described . quality attributes, such as the level of monomer and To show the potential for a universal, platform process, charged species and therefore the yield achieved in the two insect cell systems producing two different VLPs were downstream process steps . Having then been processed to studies and preliminary results will be presented . the drug substance the stability was evaluated to determine if following purification the stability profiles were affected by the differences in the feeding regimes . 72. Assessment of the Impact of CHO Cell Culture Feeding Strategy on IgG Key Quality Attributes Throughout 73. Theoretical Analysis of the the Subsequent Downstream Process Benefits of Multi-step Loading Steps and the Stability for Affinity Chromatography Based on the Shrinking-Core *Nicola Roberts, UCB, United Kingdom Breakthrough Model Laura Gimenez, UCB Pharma, Belgium Mariangela Spitali, UCB Pharma, United Kingdom *Brian Bowes, Eli Lilly and Company, United States Andrew Yates, UCB, UK As cell cultures titers have increased in recent years, Stefanos Grammatikos, UCB Pharma, Belgium so has the scrutiny placed on the productivity of the Development of an efficient, commercially viable process to purification unit operations . For Fc-containing proteins, protein A chromatography is widely employed as an affinity

produce a therapeutic antibody involves many challenges, Poster Abstracts among them being finding the cell culture medium which capture step due to the purity it can deliver, often with

Recovery of Biological Products XVII | 105 . Jason Reck, University of Virginia, USA Jason Reck, University of Virginia, States Pabst, MedImmune, United Timothy MedImmune, USA Alan Hunter, MedImmune, USA Xiangyang Wang, The removal of soluble dimers from monoclonal antibodies The removal of soluble dimers from monoclonal challenge . (mAbs) is a significant downstream processing mass, monomer- Despite the large difference in molecular because of low dimer resolution is often difficult, in part mass transfer selectivity and in part because of slow resins has Although protein adsorption in cation exchange model systems, been studied extensively for a variety of about the behavior of less is known, fundamentally, mixtures with respect to their actual mAb monomer-dimer loadings . In competitive binding properties at high protein and two-component this work, we have examined the single binding behavior on the cation exchange resin Nuvia HR-S (BioRad Laboratories) of a purified mAb monomer and of which was isolated from a process feedstock its dimer, taking the loading productivity as DBC/(load time + fixed as DBC/(load time loading productivity taking the is optimal approach reveals that the time) . This approach a time to be approximately residence for each increased steps, the one (e .g ., with three of the previous fixed multiple be 3, 3×1 .5 = 4 .5, and 3×1 .52 = 6 .75 residence times might it is possible to make clear minutes) . With this approach, of the potential productivity gains and fair comparisons number of loading segments, including afforded by a certain a continuous flow ramp, allowing one to the limiting case of process simplicity or other priorities . seek a balance with Binding of Antibody Competitive 74. Monomer-Dimer on CEX Mixtures and Equilibrium, Kinetics, Resins: Analysis by Frontal Separation United States *Giorgio Carta, University of Virginia, order to assume minimal yield loss during loading when loss during loading minimal yield order to assume

 1%) in ≤ and  = -1 and  ), where qmax is the is obtained, providing   ∙[(RT)]^  106 | Recovery of Biological Products XVII are fitted values . With the appropriate functional form for the DBC vs . residence time, one can seek to maximize productivity for one or more loadings steps, seeking to find how to select those steps . Here, the approach was to take a low threshold for the breakthrough percentage ( equilibrium capacity of the column and RT is the residence equilibrium capacity of the column and many practical time; reasonable data fit is achieved in and cases . In the limiting case of a constant pattern negligible external mass transfer resistance, a physically meaningful value of where some context for the more general case may require more time and/or expertise than is available may require more time and/or expertise to development scientists . In this work, the goal was to to determine use the shrinking-core breakthrough model between the expected functional form of the relationship the DBC and the residence time . This approach reveals a form of DBC=q_max (1- time(s) where capacity is high, but the productivity would time(s) where capacity is high, but the productivity . Examples to be low if used for the entire loading phase loading segments date of successful implementation of 2+ driven . In have been somewhat anecdotal or empirically been applied, the cases where theoretical analyses have holistically and they have not necessarily been applied optimization of the single loading flow rate, it has been optimization of the single greater improvement can be achieved recognized that much at increasing residence times . The via segmented loading loading can be executed at a premise is that the initial while the where the DBC may be low, short residence time be executed at increased residence additional step(s) can The product mass loading is often based mass loading is often . The product flow limitations capacity (DBC) at a given residence on the dynamic binding safety factor; the result could time, with an additional the 10% DBC, for example . While be loading to 80% of may be possible through some productivity improvements As such, protein . As such, protein development minimal molecule-specific choice for productivity is a logical A chromatography been A columns have protein efforts . Typically, optimization based on pressure/ single flow rates, perhaps loaded at

Poster Abstracts using size-exclusion chromatography . The CEX resin used in this work contains relatively large pores allowing both 75. Nanofiltration Optimisation monomer and dimer to diffuse freely within the particles . Using Micro-titre Plates Single and two-component adsorption behaviors were studied both through macroscopic and microscopic (CLSM) *Alison Tang, MedImmune, United Kingdom means using fluorescently labeled forms of each protein . Richard Turner, Medimmune, United Kingdom The selectivity was found to vary substantially with ionic strength, selectivity being lowest when conditions A robust method to remove potential viral contaminants is favor the strongest binding . The adsorption rates are a requirement for purification processes producing products strongly influenced by pore diffusion, although a kinetic from animal cell culture . A common technique and central to resistance to the displacement of monomer by the dimer Medimmune’s viral clearance strategy is Nanofiltration . This also exists, especially at low ionic strengths when protein approach is typically very effective and generally agnostic binding is strongest . A model is developed to describe the to any product to product variation i .e . if the protein is competitive binding process accounting for both diffusion smaller than the pore size then the protein will pass through and kinetic effects, providing a tool to optimize column the filter . Success of the step is measured in terms of the design and explain how process parameters such as feed quantity of product to pass through an acceptable amount of composition and salt concentration affect resolution . The membrane in a manageable period of time . Whilst still the model is also used to examine the feasibility of separating exception rather than the rule it is becoming increasingly monomer-dimer mixtures by fontal analysis . In such a common that proteins such as antibody or antibody related process, the mixture is continuously fed to the column for proteins do not easily pass through nanofilter using conditions where selectivity favors binding of the dimer, standard nanofilter optimisation strategy . MedImmune has yielding essentially pure monomer at the column outlet developed, in conjunction with Merck Millipore, a multi- until breakthrough of the dimer occurs . Following dimer well filter plate with an integral nanofilter membrane that breakthrough under appropriate conditions, the column has the potential, when used with the Tecan automation contains little bound monomer, resulting in a purification with high monomer recovery . Dimer species and any platform, to help simultaneously evaluate multiple multimer present as trace components in the feed mixture parameters of nanofiltration, and determine optimal can be recovered quantitatively with a high-salt strip . conditions rapidly with milligram quantities of product . Compared to other chromatographic modes, frontal analysis In addition this technology can generate a ‘fingerprint’ maximizes the protein load, providing a very high utilization of filtration performance as a function of operating of the column binding capacity and yields the purified conditions that provides a clear operating window for monomer in the same buffer in which it was fed to the specific molecules . The standard approach to nanofiltration column . For such a process to be successful, resins (such as optimisation, the advantages of scaled-down nanofiltration Nuvia HR-S) that permit rapid diffusion as a result of larger device and data to support scale-up of the unit operation pores and smaller particle size are needed to achieve the using the multi-well filter plates will be discussed . desired chromatographic efficiency . Poster Abstracts

Recovery of Biological Products XVII | 107 . .

. Although We show that there is a morphological change in the . We University College London, Gordon Street, London WC1H University College London, 0AH, United Kingdom New Delhi, India IIT Delhi, Hauz Khas, United Kingdom Kent, CT2 7NJ, Canterbury, United Kingdom Kent, CT2 7NJ, Canterbury, IIT Delhi, Hauz Khas, New Delhi, India Gordon Street, Engineering, University College London, London WC1H 0AH, United Kingdom Katherine Lintern, Department of Biochemical Engineering, of Biochemical Engineering, Katherine Lintern, Department of Chemical Engineering, Mili Pathak, Department of Biosciences, University of Kent, Kevin Howland, School of Biosciences, University of Kent, C. Mark Smales, School of Chemical Engineering, Anurag Rathore, Department to understand their role in this ageing process, and any to understand their role in this ageing process, is a significant risk to product quality they might represent, metrological challenge . Here we combine scanning electron mass spectrometry microscopy and liquid chromatography protein carry- to observe and map this phenomenon of over cycle number surface appearance of resin with increasing cohort present the composition of the protein Significantly, number on the resin is shown to shift with cycle cell proteins an expected increase in the number of host (HCPs) identifiable was observed with increasing cycle the functional class of predominant HCPs was also number, seen to change . This was all observed in the context of total HCP in the eluate remaining constant with cycle number 77. Analysis of Residual Host Host of Residual Analysis 77. A Protein During Cell Protein Lifetime Chromatography *Daniel G. Bracewell, Department of Biochemical chromatography Capacity reduction in protein A affinity manufacture with extended use during therapeutic antibody is well documented . These changes present a challenge in cycle numbers . demonstrating process robustness at high previous cycles Identification of proteins remaining from . The combination of the knowledge 108 | Recovery of Biological Products XVII Ernst Broberg Hansen, Novo Nordisk A/S, Denmark Ernst Broberg Hansen, Nordisk A/S, Denmark Thomas Hansen, Novo increased significantly of chemistry and the use of mathematical modelling has proven to be efficient . Combining the tools resulted in a better process understanding and a better process . Finally, at the end of the reaction a final diafiltration step at the end of the reaction a final diafiltration Finally, purification prepared the reaction mixture for the following reaction step . Using this set-up of for the conjugation 50 allowed us to increase the yield from approximately % to above 75 % . The side chain and the reducing agent the purity was consumption were halved . Additionally, and the reducing agent . The conjugation reaction was set it possible to up in a cross flow filtration system making for obtaining increase the concentration of the protein reagents and unwanted faster reactions . Additionally, after the side products could be removed by diafiltration alkylation step . first reaction step preparing for a better . In order to chain and reducing agent used in the process reaction steps were optimize the process the kinetics of the The model was used described using a mathematical model . resulted in for describing a set-up of the reaction which consumption increased yield, higher purity and decreased the side chain of expensive starting materials such as This introduces new challenges both regarding cost (e .g . This introduces new demand for purification . This side chain) and further step consisting of reduction study looks at a conjugation into a free cysteine followed by of a protected cysteine were product an alkylation step . The main challenges the amount of side degradation during reduction as well as *Charlotte S Hunneche, Novo Nordisk A/S, Denmark S Hunneche, Novo *Charlotte has become an increasingly important Protein conjugation production of modern pharmaceuticals . process step in the 76. Optimization of a Conjugation of a Conjugation Optimization 76. Modelling Mathematical Step Using

Poster Abstracts Low cycle number resin contained greater proportions of A significant improvement to the productivity and process residual mAb than did resin used for more cycles . A greater robustness for manufacture of a legacy Fc-fusion protein proportion of cytoskeletal components and proteins known with a complex and tightly-specified range of quality to play a role in protein synthesis and metabolism were attributes was achieved through development of a second- associated with resin from a higher number of cycles . These generation manufacturing process . The primary challenge progressive changes raise the question as to what initiates to the downstream process is the diverse population of this fouling process, three possible causes are suggested product glycoforms generated in the cell culture step in the literature; histones, conformational changes to mAb, that require consistent control to ensure drug substance and proteases . To investigate this feedstreams varying comparability, efficacy and patient safety . This work in these components have been studied . This data is outlines two case studies where mechanistic modeling, interpreted to give an insight into the dominant mechanism DoE and QbD principles were incorporated into the of resin fouling in the chromatographic process, and characterization of the manufacturing process . The first highlights those HCPs which are present at elevated levels example demonstrates how mechanistic modeling, with as the resin ages and as a consequence may represent a the fundamental parameters measured using conventional risk to process robustness . This suggests that screening and high throughput screening (HTS) methods, and a DoE feedstocks for critical HCPs which are associated with these approach were integrated to establish the scale-down mechanisms so they can be moderated upstream may help model of an anion exchange chromatography (AEX) step . to improve resin performance at extended cycle numbers . Due probably to wall effects, small diameter AEX columns were unable to achieve the bed compression specified for manufacturing scale columns, resulting in practically 78. Implementation of Mechanistic higher protein loading per resin particle volume when the Modeling, DoE and QbD Principles column loading was specified according to packed bed into Process Characterization for an volume . A correction to the column loading based only on Fc-Fusion Protein bed compression appeared inadequate to ensure equivalent column performance and to establish a representative *Xuankuo Xu, Bristol-Myers Squibb, United States scale-down model . To assess the impact of bed compression Chao Huang, Bristol-Myers Squibb, United States within the design space, a DoE framework was applied to a Zhiqiang Chen, Bristol-Myers Squibb, United States mechanistic column model describing glycoform separation Steven Traylor , Bristol-Myers Squibb, United States in AEX using a minimal set of fundamental experimental Na Zhang, Bristol-Myers Squibb, United States measurements, which allowed for efficient identification Amanda Lewis, Bristol-Myers Squibb, United States of a representative operating condition for the scale-down Nicholas Abu-Absi, Bristol-Myers Squibb, United States model . The second example focuses on characterizing two polishing steps (AEX-HIC) in a single custom multivariate Michael Borys, Bristol-Myers Squibb, United States DoE study using QbD principles to achieve improved and Zhengjian Li, Bristol-Myers Squibb, United States Poster Abstracts

Recovery of Biological Products XVII | 109 . . . The results show that the . This is impossible in batch the adsorption/desorption of UV-detectable histidine that the adsorption/desorption of UV-detectable in each well and, allows to quantify the adsorber amount thus, the static binding capacity is highly equivalent column volume for a batch experiment difference in resin dependent and that the protein-specific as source of error static and dynamic binding still remains targeted model While HTS is faced with these challenges, Experimental calibration in column mode using Optimal with highest Design allows to determine the parameters of experiments . statistical quality using the least number Hence, column experiments become on a par with typical DoE campaigns in terms of sample volume, time and labor must be high, and the parameters must be representative must be representative and the parameters must be high, these discusses whether . This contribution and transferable and if it is more efficient can be met by HTS challenges reliable . Quality: A column experiments than conventional slurry in the 96-well plates must distribution of adsorber errors must be minimized and be guaranteed, pipetting in the 96-well plates must be UV measurement variations of errors can be taken into account . Even if these sources fitting is not reliable per se . For model eliminated, isotherm adsorber Capto phenyl, we obtained proteins on the HIC confidence intervals compared to much larger parameter Under weak binding column mode . Representativeness: of diffusion parameters can be as conditions the influence parameters themselves . Muca important as the isotherm et al . (J Chromatogr A, 2009) measured an exponential broadening such influence of salt concentration on peak must be determined that four different diffusion parameters mixed-mode for each involved protein to model HIC and chromatography accurately For low salt concentrations, e .g . mode . Transferability: is the main issue . when using IEX, parameter transferability cope with this, we developed a method to determine To mode, based on the ionic capacities in column and batch for model-based process development, the data quality the data quality process development, for model-based

The knowledge gained in . The knowledge gained 110 | Recovery of Biological Products XVII Germany Tobias Hahn, Karlsruhe Institute of Technology, Germany Hahn, Karlsruhe Institute of Technology, Tobias Germany Karlsruhe Institute of Technology, Gang Wang, Technology, Juergen Hubbuch, Karlsruhe Institute of model-based process development, only few experiments model-based process development, only of a DoE campaign are of relevance, those that provide information for model calibration . In order to use batch data High-throughput screening (HTS) in batch format is often High-throughput screening (HTS) in batch (DoE) used in combination with Design-of-Experiments of the process and promises to characterize a large part short time . For design space with low sample volume in Equal Time Germany *Thiemo Huuk, Karlsruhe Institute of Technology, 79. No Use for High-Throughput Use for High-Throughput No 79. Batch Isotherms in Modelling? Yield Column Experiments Why Information in Accurate More The extensive process understanding would specifications . The extensive process if the two polishing steps were not be easily achievable characterized independently was finally used to define the process characterization the drug substance manufacturing control strategy for protein . process of this fusion . With variables for HIC of AEX as the input output results models developed, an easily- the predictive statistical was proposed, where the PPs of the implemented strategy according to the quality of the AEX step could be adjusted achieve the target drug substance AEX input material to robust control of product glycoform distribution . Process of product glycoform robust control based on risk assessment (PPs) were selected parameters space the optimal design knowledge to explore and process the operations using two successive unit across these

Poster Abstracts Participant List

Jeff Allen Hanne Bak Lydia Beasley PFENEX INC REGENERON PHARMACEUTICALS GENENTECH 10790 Roselle 777 Old Saw Mill River Road 1 DNA Way San Diego, CA 92121 Tarrytown, NY 10591 South San Francisco, CA 94080 United States United States United States jallen@pfenex .com hanne .bak@regeneron .com lydiab@gene .com Ashraf Amanullah Gregory Barker Christopher Belisle ATYR PHARMA BRISTOL-MYERS SQUIBB BIO-RAD LABORATORIES 3545 John Hopkins Court, Suite 250 519 Route 173 West 3210 Meadow View Ln San Diego, CA 92121 Bloomsbury, NJ 8804 Walnut Creek, CA 94598 United States United States United States aamanullah@atyrpharma com. gregory .barker@bms .com christopher_belisle@bio-rad .com Dorothee Ambrosius Kristopher Barnthouse Jean Bender BOEHRINGER INGELHEIM JANSSEN / J&J MEDIMMUNE Biberach 200 Great Valley Parkway One Medimmune Way Germany Malvern, PA 19355 Gaithersburg, MD 20878 dorothee .ambrosius@ United States United States boehringer-ingelheim .com kbarntho@its .jnj .com benderj@medimmune .com Patrick Asplund Ronald Bates Joseph Bertolini COOK PHARMICA, LLC BMS CSL BEHRING 1300 South Patterson Drive 6000 Thompson Road 189-209 Camp Rd Bloomington, IN 47402-0970 East Syracuse, NY 13057 Broadmeadows, Victoria 3047 United States United States Australia patrick .asplund@cookpharmica .com ronald .bates@bms .com joe .bertolini@cslbehring .com .au Morrey Atkinson Pascal Baumann Bharat Bhut BRISTOL-MYERS SQUIBB KARLSRUHE INSTITUTE OF MERCK & CO . 311 Pennington Rocky-Hill Rd TECHNOLOGY (KIT) 2000 Galloping Hill Rd Hopewell, NJ 8534 Engler-Bunte-Ring 3 Kenilworth, NJ 7033 United States Karlsruhe, Baden-Wuerttemberg 76131 United States morrey .atkinson@bms .com Germany bharat .bhut@merck .com pascal .baumann@kit .edu Engin Ayturk Marc Bisschops PALL CORPORATION Kevin Beam PALL LIFE SCIENCES 20 Walkup Drive SEATTLE GENETICS Nijverheidsweg 1 Westborough, MA 1581 21823 30th Drive SE Medemblik, 1671 GC

United States Bothell, WA 98021 Netherlands Participant List engin_ayturk@pall .com United States marc_bisschops@pall .com kbeam@seagen .com Austin Boesch ZEPTEON, INC . 42 Chauncy St . STE 10A Boston, MA 2111 United States austin .boesch@zepteon .com

Recovery of Biological Products XVII | 111 .com

.com boehringer-ingelheim AMGEN 360 Binney St Cambridge, MA 2142 United States mcoufal@amgen .com Canada chickosky@natrixseparations .com Keen Chung Wai MEDIMMUNE One MedImmune Way Gaithersburg, MD 20878 United States chungw@medimmune .com Jonathan Coffman BOEHRINGER INGELHEIM Fremont United States jonathan .coffman@ Charles Cooney MIT MIT 56-469B Cambridge, MA 2139 United States ccooney@mit .edu Myra Coufal PFIZER INC Parkway West 700 Chesterfield MO 63017 Cheterfield, United States shuang .chen@pfizer Qi Chen GENENTECH, INC 1 DNA Way CA 94080 South San Francisco, United States qi@gene .com Jie Chen SHIRE 300 Shire Way Lexington, MA 2421 United States jchen@dyax .com John Chickosky INC . SEPARATIONS NATRIX 5295 John Davis Drive Burlington, Ontario L7L 6A8 Shuang Chen Shuang

.com . .michelle@gene .com FOR BIOTECHNOLOGY 102 Engineers Way 22904 Charlottesville, VA United States gc@virginia .edu San-Cher Chen DEVELOPMENT CENTER 101, Lane 169, Kangning St ., Xizhi Dist 22180 City, New Taipei Taiwan scchena@mail .dcb .org .tw United States jordan .byrd@regeneron .com Ruben Carbonell UNIVERSITY NORTH CAROLINA STATE 850 Oval Drive Raleigh, NC 27695 United States ruben@ncsu .edu Giorgio Carta UNIVERSITY OF VIRGINIA s .burton@prometic .com Michelle Butler GENENTECH, INC . 1 DNA Way South San Francisco, CA 94002 United States butler Jordan Byrd REGENERON PHARMACEUTICALS INC 81 Columbia Turnpike NY 12144 Rensselaer, Stuart Builder BIODEVELOPMENT STRATEGIC Dr 2827 Wemberly Belmont, CA 94992 United States .com sbuilder@ix .netcom Steve Burton PROMETIC BIOSCIENCES LTD Horizon Park, Barton Road Cambridge, CB23 7AJ United Kingdom Deirdre Buckley Deirdre ELI LILLY Dunderrow Cork, Ireland buckley_deirdre_a@lilly .com 112 | Recovery of Biological Products XVII TECHNOLOGY Kenilworth, NJ 7033 United States mark_brower@merck .com Denmark eha@novonordisk .com Mark Brower MERCK & CO ., INC . 2000 Galloping Hill Road nina .brestrich@kit .edu Ernst Broberg Hansen NOVO NORDISK A/S Smørmosevej 17 Bagsværd, 2880 Nina Brestrich KARLSRUHE INSTITUTE OF 7 Fritz-Haber-Weg 76131 Karlsruhe, Baden Wuertemberg Germany UCL Gordon Street London, WC1H 0AH United Kingdom d .bracewell@ucl .ac .uk ROCHE DIAGNISTICS GMBH Nonnenwald 2 Penzberg, Bavaria 82377 Germany reinhard .braaz@roche .com Daniel Bracewell 80 Ashby Road Bedford, MA 1730 United States john .boyle@emdmillipore .com Reinhard Braaz Indianapolis, IN 46285 United States bowes_brian_d@lilly John Boyle MILLIPORE SIGMA United States gbolton@amgen .com Brian Bowes AND COMPANY ELI LILLY Lilly Corporate Center Glen Bolton AMGEN St 360 Binney MA 2141 Cambridge,

Participant List Steven Cramer Evi Dimitriadou Michel Eppink RPI LONZA BIOLOGICS SYNTHON BIOPHARMACEUTICALS BV 3211 CBIS, 110 8th St . 224 Bath Road Microweg Troy, NY 12180 Slough, Berkshire SL1 4DX Nijmegen, Nederland 6503 GN United States United Kingdom Netherlands crames@rpi .edu evi .dimitriadou@lonza .com michel eppink@synthon. .com John Cundy Florian Dismer John Erickson PFIZER NOVO NORDISK A/S GSK 700 Chesterfield Parkway West Novo Nordisk Park 709 Swedeland Rd Chesterfield, MO 63017 Maalov, DK-2760 King of Prussia, PA 19406 United States Denmark United States john .cundy@pfizer .com fld@novonordisk .com john .c .erickson@gsk .com Bárbara Cunha Mandar Dixit Derek Ettie IBET / ITQB-NOVA SARTORIUS STEDIM BIOTECH GEA Apartado 12 5 Orville Dr . . 17 Overidge Ln Oeiras Bohemia, NY 11716 Wilton, CT 6897 Portugal United States United States bcunha@itqb .unl .pt mandar .dixit@sartorius-stedim .com derek .ettie@gea .com John Curling Martin Dorn Rob Fahrner JCC AB TECHNISCHE UNIVERSITÄT PFIZER Swedenborgsgatan 5 MÜNCHEN 700 Chesterfield Pkwy W Uppsala, 753 34 Boltzmannstr . 15 Chesterfield, Missouri, MO 63017 Sweden Garching, Bavaria 85748 United States john@consultcurling .se Germany robert .fahrner@pfizer .com martin .dorn@tum .de John Daicic Suzanne Farid GE HEALTHCARE LIFE SCIENCES Chris Dowd UCL Björkgatan 30 GENENTECH Gordon Street Uppsala, 75184 1 DNA Way London, WC1H 0AH Sweden South San Francisco, CA 94080 United Kingdom john .daicic@ge com. United States s .farid@ucl .ac .uk cjdowd@gene .com Andy Davies Conan Fee PROMETIC BIOSCIENCES Andre Dumetz UNIVERSITY OF CANTERBURY Horizon Park GSK Ilam Road Comberton, Cambridgeshire CB23 7AJ 709 Swedeland Road Christchurch, 8041 United Kingdom King of Prussia, PA New Zealand a .davies@prometic .com United States conan .fee@canterbury .ac .nz andre .c .dumetz@gsk .com Nuria de Mas Gisela Ferreira BRISTOL-MYERS SQUIBB Michael Egholm MEDIMMUNE 38 Jackson Rd PALL CORPORATION 1 MedImmune Way Devens, MA 1434 20 Walkup Drive Gaithersburg, MD 20877 United States Westborough, MA 1581 United States nuria .demas@bms .com United States FerreiraG@medimmune .com Participant List michael_egholm@pall .com Kumar Dhanasekharan Stefan Fischer-Frühholz COOK PHARMICA Hanno Ehring SARTORIUS STEDIM BIOTECH GMBH 1300 S Patterson Dr . GE HEALTHCARE August-Spindler-Str . 11 Bloomington, IN 47402 Björkgatan 30 Goettingen, 37079 United States Uppsala Germany kumar .dhanasekharan@ Sweden stefan fischerf@sartorius-stedim. .com cookpharmica .com Hanno .Ehring@ge .com

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.cmu .edu .com .Hahn@boku .ac .at OF TECHNOLOGY Thomas Hansen NOVO NORDISK A/S Smørmosevej 17-19 Bagsværd, DK-2880 Denmark tmhs@novonordisk .com United States qingu@andrew Xuejun Gu LILLY Lilly Research Lab Indianapolis, IN United States sherrygu@lilly Rainer Hahn BOKU VIENNA Muthgasse 18 1190 Vienna, Austria Rainer Hahn Tobias KARLSRUHE INSTITUTE 3 Engler-Bunte-Ring Karlsruhe, BW 76135 Germany tobias .hahn@kit .edu GE HEALTHCARE 30 Bjorkgatan Uppsala, 75184 Sweden com anna .gronberg@ge . Robert Gronke BIOGEN 225 Broadway Cambridge, MA 2142 United States .com rob .gronke@biogen David Gruber MEDIMMUNE Milstein Building Cambridge, CB21 6GH United Kingdom gruberd@medimmune .com Qin Gu CARNEGIE MELLON UNIVERSITY Doherty Hall, 5000 Forbes Avenue 15213 Pittsburgh, PA Anna Gronberg .com . 1 .com .goehmann@gea .com .goetz@lilly Munchensteinerstrasse 38 Basel, 4002 Switzerland uwe .gottschalk@lonza .com Jan Griesbach ROCHE Nonnenwald 2 Penzberg, 82377 Germany jan .griesbach@roche .com Germany ruediger Elizabeth Goodrich MILLIPORESIGMA 900 Middlesex Turnpike Billerica, MA 1821 United States Elizabeth .goodrich@emdmillipore .com Uwe Gottschalk LONZA godawatr@alxn .com Goetz Victor AND COMPANY ELI LILLY 33 ImClone Drive Branchburg, NJ 8876 United States victor Rüdiger Göhmann GEA Werner-Habig-Str Oelde, NRW 59302 Ranga Godavarti PFIZER One Burtt Road MA 1810 Andover, United States ranga .godavarti@pfizer Rahul Godawat ALEXION PHARM 100 College St New Haven, CT 6501 United States Glen Giese GENENTECH 1 DNA Way Francisco, CA 94080 South San United States ggiese@gene .com

.com 114 | Recovery of Biological Products XVII AT AUSTIN AT .com boehringer-ingelheim Research Triangle, NC Research Triangle, United States sanchayita .ghose@biogen .com United States gg@che .utexas .edu Sanchayita Ghose BIOGEN Research Triangle George Georgiou THE UNIVERSITY OF TEXAS 200 E . Dean Keeton St ., C0400 Austin, TX 78712 Rene Gantier LIFE SCIENCES PALL Drive 20 Walkup MA 1581 Westborough, United States rene_gantier@pall .com Stuart Gallant ANTHERA PHARMACEUTICALS 55 Hill Road, Suite 706 Belmont, MA 2478 United States sgallant@outlook .com AVITIDE, INC AVITIDE, PO Box 1774 Grantham, NH 3753 United States dfreymey@gmail .com Fremont, California, 94555 Fremont, California, United States nuno .fontes@ Dan Freymeyer United States jace@lilly Nuno Fontes BOEHRINGER INGELHEIM 6701 Kaiser Drive Jace Fogle ELI LILLY Co . DC 35353 Eli Lilly and IN 46285 Indianapolis,

Participant List Oliver Hardick Naokatsu Hirotomi Günter Jagschies PURIDIFY / UCL ASAHI KASEI BIOPROCESS AMERICA GE HEALTHCARE LIFE SCIENCES Stevenage Bioscience Catalyst 1855 Elmdale Avenue Björkgatan 30 London, SG1 2FX Glenview, IL 60026 Uppsala, 75184 United Kingdom United States Sweden oliver@puridify .com naokatsu .hirotomi@ak-bio .com guenter .jagschies@ge .com Roger Hart Regina Holzhauser Canping Jiang AMGEN, INC . TOSOH BIOSCIENCE GMBH BIOGEN 360 Binney Street Im Leuschnerpark 4 250 Binney Street Cambridge, MA 02142-1097 Griesheim, 64347 Cambridge, MA 2142 United States Germany United States rhart@amgen .com regina .holzhauser@tosoh .com canping jiang@biogen. .com Charles Haynes Juergen Hubbuch Mi Jin UNIVERSITY OF BRITISH COLUMBIA KARLSRUHE INSTITUTE TEVA PHARMACEUTICALS Vancouver OF TECHNOLOGY 145 Brandywine Pkwy Canada Engler-Bunte-Ring 3 West Chester, PA charles haynes@ubc. ca. Karlsruhe, 76131 United States Germany mi .jin@tevapharm .com Xuemei He Juergen Hubbuch@kit. .edu BIO-RAD LABORATORIES Hans Johansson 6000 James Watson Drive Charlotte S Hunneche PUROLITE LIFE SCIENCES Hercules, CA 94547 NOVO NORDISK A/S Vårdkasvägen 7 United States Hagedornsvej 1 Uppsala, 75655 Xuemei_He@biorad .com Gentofte, 2820 Sweden Denmark hans .johansson@purolite .com Milton Hearn chun@novonordisk .com MONASH UNIVERSITY David Kahn Building 23 Stephen Hunt MEDIMMUNE Clayton, Victoria 3800 KBI BIOPHARMA 1 MedImmune Way Australia 2500 Central Ave Gaithersburg, MD 20878 milton .hearn@monash edu. Boulder, CO 80503 United States United States kahnd@medimmune .com Sarah Hedberg shunt@kbibiopharma com. IMPERIAL COLLEGE LONDON Anne Kantardjieff Department of Chemical Engineering Thiemo Huuk ALEXION PHARMACEUTICALS South Kensington Campus KARLSRUHE INSTITUTE 352 Knotter Drive London, SW7 2BY OF TECHNOLOGY (KIT) Cheshire, CT United Kingdom Engler-Bunte-Ring 3 United States s .hedberg13@ic .ac uk. Karlsruhe, 76131 kantardjieffa@alxn .com Germany Caryn Heldt thiemo .huuk@kit .edu Pankaj Karande MICHIGAN TECH RENSSELAER POLYTECHNIC 1400 Townsend Dr . Michael Inglis INSTITUTE Houghton, MI 49931 PFIZER BIOTECH 110 8th St . United States Grange Castle, Grange Castle Bus . Pk . Troy, NY 12180 heldt@mtu .edu Dublin United States Participant List Ireland karanp@rpi .edu Pim Hermans michael .inglis@pfizer .com THERMO FISHER SCIENTIFIC Mark Karbarz J .H Oortweg. 21 PORTOLA PHARMACEUTICALS Leiden, 2333 CH 270 East Grand Avenue Netherlands South San Francisco, CA pim .hermans@thermofisher .com United States mkarbarz@portola .com

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CENTRE CHINA CIGB 31 e/ 158 y 190 Ave La Habana Cuba miladys .limonta@cigb .edu .cu United Kingdom peter_levison@pall .com Paul Lewus AMGEN, INC 11804 N . Beasly Rd Longmont, 80504 United States plewus@amgen .com Honghao LI NOVO NORDISK RESEARCH Building #2, 20 Life Science Park Road Beijing 102206 China hhli@novonordisk .com Zhengjian Li BRISTOL-MYERS SQUIBB Devens, MA 1434 United States zhengjian .li@bms .com Miladys Limonta ROCHE DIAGNOSTICS GMBH ROCHE DIAGNOSTICS 2 Nonnenwald Bavaria 82377 Penzberg, Germany .com thorsten .lemm@roche Abraham Lenhoff UNIVERSITY OF DELAWARE 150 Academy Street Newark, DE 19716 United States lenhoff@udel .edu Philip Lester GENENTECH 1 DNA Way South San Francisco, CA 94080 United States lesterp@gene .com Peter Levison LIFE SCIENCES PALL 5 Harbourgate Business Park Portsmouth, PO6 4BQ Thorsten Lemm Thorsten .ac .uk . Lafayette, IN 47906 Sunway City, Selangor 47500 Sunway City, Malaysia mickylfx@gmail .com Steven Lehrer CIPLA BIOTEC 1771 Ringling Blvd PH 203 Sarasota, Florida 34236-6872 United States steven .lehrer@cipla .com alfredo .lanzaro@manchester Michael Laska MODERNA THERAPEUTICS 5th Floor Cambridge, MA 2139 United States michael_laska@live .com Micky Fu Xiang Lee MONASH UNIVERSITY Jalan Lagoon Selatan Asif Ladiwala GENENTECH 1 DNA Way South San Francisco, CA 94080 United States ladiwala .asif@gene .com Alfredo Lanzaro THE UNIVERSITY OF MANCHESTER 131 Princess Street Greater Manchester M1 Manchester, 7DN United Kingdom Karol Lacki N-ONE Sweden k .lacki@icloud .com Michael Ladisch PURDUE UNIVERSITY LORRE W United States ladisch@purdue .edu Beckley Kungah Nfor Beckley VACCINES JANSSEN 4-6 Archimedesweg CN Leiden, 2333 Netherlands bkungahn@its .jnj .com .

116 | Recovery of Biological Products XVII .brian@gene .com PHARMA BIOTECH PENZBERG CHEMICAL TECHNOLOGY APPLIED SCIENCES PORTOLA PHARMACEUTICALS INC . 270 E Grand Ave South San Francisco, CA 94080 United States pkumar@portola .com Nonnenwald 2 Penzberg, Bavaria 82377 Germany wolfgang .kuhne@roche .com Pawan Kumar United States mikhail .kozlov@emdmillipore .com Kuhne Wolfgang ROCHE DIAGNOSTICS GMBH, India sushsparky@gmail .com Mikhail Kozlov MILLIPORESIGMA 80 Ashby Rd . Bedford, MA 1730 Sushmita Koley OF INSTITUTE DBT-ICT-CEB, N . P Institute of Chemical Technology, Marg, Matunga(E) Mumbai, Maharashtra 400019 ROCHE DIAGNOSTICS GMBH 5b Nantesbucher Weg Iffeldorf Germany annika .kleinjans@roche .com Karlstrasse 11 Biberach, 88400 Germany .de kiefer@hochschule-bc Annika Kleinjans United States kelley Hans Kiefer OF BIBERACH UNIVERSITY Brian Kelley GENENTECH 1 DNA Way Francisco, CA 94080 South San

Participant List Naichi Liu Christian Manzke John Moscariello BIOMARIN SARTORIUS STEDIM BIOTECH GMBH CMC BIOLOGICS 105 Digital Drive August-Spindler-Strasse 11 22021 20th Ave SE Novato, CA 94949 Goettingen, 37120 Bothell, WA 98133 United States Germany United States Naichi .liu@bmrn .com christian .manzke@ jmoscariello@cmcbio .com sartorius-stedim .com Lotta Ljungqvist Morten Munk GE HEALTHCARE LIFE SCIENCES Bruno Marques NNE PHARMAPLAN Björkgatan 30 GLAXOSMITHKLINE Nybrovej 80 Uppsala 709 Swedeland Road GENTOFTE, 2820 Sweden King of Prussia, PA 19406 Denmark lotta .ljungqvist@ge com. United States mbmn@nnepharmaplan .com bruno .f .marques@gsk .com Tim Lowery Christopher Murray JSR MICRO Tiago Matos INSTITUTE FOR HEALTH METRICS 1280 North Mathilda Avenue NOVO NORDISK AND EVALUATION Sunnyvale, CA 94089 Novo Nordisk Park 2301 5th Ave . United States Malov Seattle, WA 98121 tlowery@jsrmicro .com Denmark United States tgmm@novonordisk .com cjlm@uw .edu Mats Lundgren GE HEALTHCARE LIFE SCIENCES Karl McCann Jill Myers Bjorkgatan 30 CSL BEHRING AUSTRALIA FORTRESS BIOTECH Uppsala, 75184 189 – 209 Camp Road 95 Sawyer Rd . Sweden Broadmeadows, Victoria 3047 Waltham, MA 2453 matslundgren@ge .com Australia United States karl .mccann@cslbehring com. .au jmyers@fortressbiotech .com Herb Lutz MILLIPORE SIGMA Amit Mehta Amith Naik 6220 Pacific Ave . GENENTECH NORTH CAROLINA STATE UNIVERSITY Playa del rey, CA 90293 1 DNA Way 1791 Varsity Drive United States South San Francisco, CA 94080 RALEIGH, NC 27695 herb lutz@emdmillipore. com. United States United States mehta@gene .com adnaik2@ncsu .edu Chris Major PUROLITE Lee Mermelstein Alpana Naresh Unit D PORTOLA PHARMACEUTICALS JSR LIFE SCIENCES Wales 270 East Grand Avenue 1280 N Mathilda Ave United Kingdom South San Francisco, CA 94080 Sunnyvale, CA 94089 chris major@purolite. .com United States United States lmermelstein@portola .com anaresh@jsrmicro .com Jan Makela GE HEALTHCARE Atul Mohindra Darren N. Nesbeth Little Chalfont LONZA BIOLOGICS UNIVERSITY COLLEGE LONDON Amersham, Buckinghamshire HP7 9LL 224 Bath Road Bernard Katz Building, Gordon Street United Kingdom Slough, Berkshire SL1 4DX London, WC1H 0AH jan makela@ge. .com United Kingdom United Kingdom Participant List atul mohinDr. .a@lonza .com d .nesbeth@ucl .ac uk. Gunnar Malmquist GE HEALTHCARE Massimo Morbidelli Sara Nilsson Bjorkgatan 30 ETH ZURICH GSK Uppsala, SE-751 84 Vladimir-Prelog-Weg 1 / HCI F131 Medicines Research Centre (5G104) Sweden Zurich Stevenage, Hertfordshire SG1 2NY gunnar .malmquist@ge com. Switzerland United Kingdom nadine widmer@chem. .ethz .ch sara .x .nilsson@gsk .com

Recovery of Biological Products XVII | 117 .com .cmu .edu 3M 400 Research Parkway Meriden, CT 6450 United States lraman@mmm .com United States jennifer_pollard@merck .com Przybycien Todd CARNEGIE MELLON UNIVERSITY 5000 Forbes Avenue 15213 Pittsburgh, PA United States todd@andrew Maen Qadan LILLY Lilly Corporate Center Indianapolis, IN 46285 United States qadan_maen@lilly Natraj Ram ABBVIE INC . 100 Research Drive MA 1581 Worcester, United States natraj .ram@abbvie .com LP Raman MILLIPORESIGMA Road 80 Ashby 1730 Bedford, MA United States .com michael .phillips@emdmillipore John Pieracci BIOGEN CORPORATION 125 Broadway Cambridge, MA 1890 United States .com john .pieracci@biogen Nuno Pinto MERCK & CO ., INC . Room K15-B412C Kenilworth, NJ 7033 United States nuno .pinto@merck .com Jennifer Pollard MERCK & CO ., INC . 2000 Galloping Hill Rd Kenilworth, NJ 7033 Michael Phillips Michael .com .parra@thermofisher COMPANY REPLIGEN CORPORATION 41 Seyon St MA 02453 Waltham, United States dpentia@repligen .com James Peyser REPLIGEN CORP 41 Seyon Street MA 2453 Waltham, United States jpeyser@repligen .com Cumberland, RI 2864 United States shelly Cristina Peixoto IBET Apartado 12 Oeiras, 2781-901 Oeiras Portugal peixoto@ibet .pt Dana Pentia United States david .n .paolella@gsk .com Siddharth Parimal BIOGEN 5000 Davis Drive Durham, NC 27709 United States siddharth .parimal@biogen .com Shelly Parra THERMO FISHER SCIENTIFIC 27 Windsong Road Lars Pampel PHARMA NOVARTIS WKL-693 .1 .139 .14 Basel, 4002 Switzerland .com lars .pampel@novartis David Paolella GLAXOSMITHKLINE 709 Swedeland Rd King of Prussia, 19406 Olga Paley PHARMACEUTICAL TAKEDA 35 Landsdowne MA 2139 Cambridge, United States com olga .paley@takeda .

118 | Recovery of Biological Products XVII com FREMONT, INC . FREMONT, GMBH & CO . KG .com boehringer-ingelheim MEDIMMUNE 1 MedImmune Way Gaithersburg, MD 20872 United States pabstt@medimmune .com Julianalaan 67 Delft, Zuid Holland 2628 BC Netherlands m .ottens@tudelft .nl Pabst Timothy United States . raquel .orozco@boehringer-ingelheim Marcel Ottens DELFT UNIVERSITY OF TECHNOLOGY brian .omara@bms .com Raquel Orozco BOEHRINGER INGELHEIM 6701 Kaiser Drive Fremont, CA 94555 Brian O’Mara BRISTOL-MYERS SQUIBB E 1201 Eastlake Ave 98102 Seattle, WA United States Birkendorfer Straße 65 Biberach, 88397 Germany stefan .oelmeier@ King of Prussia, PA 19406 King of Prussia, PA United States .com kevin .odonnell@tosoh Stefan Oelmeier BOEHRINGER INGELHEIM PHARMA Belgium .be wim .noppe@kuleuven-kulak J. Kevin O’Donnell LLC TOSOH BIOSCIENCE Suite 100 3604 Horizon Drive Wim Noppe CAMPUS KORTRIJK KU LEUVEN 53 E . Sabbelaan Kortrijk, B-8500

Participant List Andrew Ramelmeier Frederik Rudolph Pierre Schelling PORTOLA PHARMACEUTICALS BOEHRINGER INGELHEIM PHARMA MILLIPORESIGMA 270 East Grand Avenue GMBH & CO KG Merck & Cie South San Francisco, CA 94080 Birkendorfer Strasse 65 8200 Schaffhausen, United States Biberach an der Riss, 88397 Switzerland aramelmeier@portola .com Germany pierre .schelling@merckgroup .com frederik .rudolph@boehringer- Andrea Rayat ingelheim com. Emily Schirmer UNIVERSITY COLLEGE LONDON CATALENT PHARMA SOLUTIONS Department of Biochemical Ben Sackett 726 Heartland Trail Engineering GENENTECH Madison, WI 53717 LONDON, London WC1H 0AH 1 DNA Way United States United Kingdom South San Francisco, CA 94080 emily .schirmer@catalent .com andrea rayat@ucl. .ac uk. United States sackettb@gene .com Christine Bruun Schjødt Marco Rito-Palomares NOVO NORDISK A/S TECNOLOGICO DE MONTERREY Jeffrey Salm Hagedornsvej 1 Ave Eugenio garza sada 2501 PFIZER Gentofte, 2820 Monterrey, 1 Burtt Rd Denmark Mexico Andover, 1810 chbs@novonordisk .com mrito@itesm .mx United States jeff .salm@pfizer .com Stefan Schmidt David Robbins RENTSCHLER BIOTECHNOLOGY MEDIMMUNE Mark Saltzman Erwin Rentschler Sr . 21 One MedImmune Way YALE UNIVERSITY Laupheim, BW 88471 Gaithersburg, MD 20878 55 Prospect Street Germany United States New Haven, CT 6520 stefan schmidt@rentschler. .de RobbinsD@MedImmune .com United States mark .saltzman@yale .edu Mark Schofield Nicola Roberts PALL LIFE SCIENCES UCB Nooshafarin Sanaie 20 Walkup Dr . 638 Ajax Avenue GILEAD SCIENCES Westborough, MA 1581 Slough, Berkshire SL1 4BG 5582 Renaissance Ave United States United Kingdom San Diego, CA Mark_Schofield@pall .com nicola .roberts@ucb .com United States nooshies@yahoo .com Tim Schroeder Julie Robinson ATOLL GMBH RENSSELAER POLYTECHNIC Kimo Sanderson Ettishofer Str . 10 INSTITUTE ASAHI KASEI BIOPROCESS Weingarten, 88250 110 8th Street AMERICA, INC . Germany Troy, NY 12180 1855 Elmdale Ave . tschroeder@repligen .com United States Glenview, IL 60026 robinj6@rpi .edu United States Norbert Schuelke kimo .sanderson@ak-bio .com TAKEDA PHARMACEUTICALS David Roush INTERNATIONAL CO . MERCK & CO ., INC . Lalit Saxena 35 Landsdowne Street 2015 Galloping Hill Road, Mailstop CIPLA BIOTEC PVT .LTD . Cambridge, MA 2139 K15-2-H206 L147/ B United States Participant List Kenilworth, NJ 7033 Goa, Goa 403722 norbert .schuelke@takeda .com United States India david_roush@merck .com lalit saxena@ciplabiotec. .com Lars Sejergaard NOVO NORDISK A/S Smørmosevej 17-19 Bagsværd Denmark lsej@novonordisk .com

Recovery of Biological Products XVII | 119 Shinya Sekine Joseph Shultz Daniel Strauss ASAHIKASEI AMR ICA. NOVARTIS PHARMA AG ASAHI KASEI BIOPROCESS AMERICA 525 Middlefield Road, Suite 110 Klybeckstrasse 141 1855 Elmdale Ave Menlo Park, CA 94025 Basel, 4058 Glenview, IL 60026 United States Switzerland United States sekine .sd@om asahi-kasei. .co .jp joseph .shultz@novartis .com daniel .strauss@ak-bio .com Ian Sellick Nripen Singh Alison Tang PALL LIFE SCIENCES BRISTOL MYERS SQUIBB MEDIMMUNE 20 Walkup Drive 38 Jackson Road Aaron Klug building Westborough, MA 1581 Devens, MA 1748 Cambridge, CB21 6GH United States United States United Kingdom ian_sellick@pall com. nripen .singh@bms .com tanga@medimmune .com Hammad Shaikh Marty Siwak Peter Tessier NOVONORDISK JSR LIFE SCIENCE RENSSELAER POLYTECHNIC Smørmosevej 17-19 , 6BC2 .59,1 1280 N Mathilda Ave INSTITUTE 2880, Copenhagen Bagsværd Sunnyvale, CA Center for Biotech & Interdisciplinary Denmark United States Studies hams@novonordisk .com msiwak@jsrmicro .com Troy, NY 12180 United States Rahul Sheth Mark Snyder tessier@rpi .edu BIOMARIN PHARMACEUTICAL INC . BIO-RAD LABORATORIES 105 Digital Drive 6000 James Watson Drive Volkmar Thom Novato, CA 94949 Hercules, CA 94547 SARTORIUS STEDIM BIOTECH United States United States August Spindler Str . 11 rsheth@bmrn .com mark_snyder@bio-rad .com Göttingen, 37083 Germany Eric Shierly Mariangela Spitali volkmar .thom@sartorius-stedim .com REGENERON PHARMACEUTICALS UCB PHARMA 81 Columbia Turnpike 638 Ajax Avenue Owen R.T. Thomas Rensselaer, NY 12144 Slough, UNIVERSITY OF BIRMINGHAM, United States United Kingdom EDGBASTON eric .shierly@regeneron .com Mari .Spitali@ucb .com School of Chemical Engineering, College of Engineering and Joseph Shiloach Rick St. John Physical Sciences NIDDK/NIH GENENTECH Birmingham, UK, West Midlands B15 Bldg 14 A Room 173 1 DNA Way 2TT Bethesda, Maryland 20852 South San Francisco, 94030 United Kingdom United States United States o .r .t .thomas@bham .ac .uk Josephs@niddk .nih .gov st-john .richard@gene .com Jorg Thommes Russell Shpritzer Arne Staby BIOGEN PFIZER NOVO NORDISK A/S 250 Binney Street 1 Burtt Road Smørmosevej 17 Cambridge, MA 2142 Andover, MA 1810 Bagsværd, 2880 United States United States Denmark jorg thommes@biogen. .com russell .shpritzer@pfizer .com ast@novonordisk .com

Peter Tiainen Notes Abhinav Shukla Sofie Stille NOVO NORDISK A/S KBI BIOPHARMA INC . GE HEALTHCARE LIFE SCIENCES Novo Nordisk A/S 2 Triangle Drive Björkgatan 30 Måløv, 2760 Durham, NC 27704 Uppsala, 75184 Denmark United States Sweden ptii@novonordisk .com ashukla@kbibiopharma .com sofie stille@ge. .com

120 | Recovery of Biological Products XVII Ben Tillotson James Van Alstine Suresh Vunnum AMGEN ROYAL INSTITUTE OF TECHNOLOGY AMGEN One Amgen Center Drive JMVA Biotech One Amgen Center Drive Thousand Oaks, CA 91320 Stockholm, 11254 Thousand Oaks, CA 91320 United States Sweden United States bent@amgen com. jim .vanalstine@telia com. vvunnum@amgen .com Steven Timmick Ganesh Vedantham Cornelia Walther RENSSELAER POLYTECHNIC AMGEN BOKU & BOEHRINGER- INSTITUTE Amgen Drive INGELHEIM-RCV 110 8th Street Cambridge, MA Muthgasse 18 Troy, NY 12180 United States Vienna, 1190 United States vedanthg@amgen com. Austria timmis@rpi edu. cornelia .walther@boku .ac .at Ajoy Velayudhan Nigel Titchener-Hooker UCL Sheng-Ching Wang UCL Bernard Katz Building MERCK & CO ., INC . Faculty of Engineering Sciences London WC1E 7JE 770 Sumneytown Pike London, WC1E 7JE United Kingdom West Point, PA 19486 United Kingdom a .velayudhan@ucl .ac .uk United States nigelth@ucl ac. .uk sheng-ching_wang@merck .com Louis Villain Alejandro Toro SARTORIUS STEDIM BIOTECH Chen Wang AMGEN August-Spindler Str . 11 ABBVIE PO Box 4060 Göttingen, 37097 100 Research Drive Juncos, Puerto Rico 00777-4060 Germany Worcester, MA atoro@amgen .com louis .villain@sartorius-stedim .com United States c .wang@abbvie .com Benjamin Tran Monchois Vincent GENENTECH, INC . NOVASEP PROCESS Yong Wang 1 DNA Way 81 Boulevard de la Moselle SHIRE San Francisco, CA 94080 POMPEY, 54340 200 Shire Way United States France Lexington, MA 2421 benjampt@gene com. vincent .monchois@novasep .com United States ywang@shire .com Nihal Tugçu Alexei Voloshin MERCK 3M COMPANY Veena Warikoo 2000 Galloping Hill Road 3M Center SANOFI Kenilworth, NJ 7033 St . Paul, MN 55144 31 New York ave United States United States Framingham, MA 1701 nihal_tugcu@merck .com amvoloshin@mmm .com United States veena .warikoo@sanofi .com Andrew Tustian Thomas von Hirschheydt REGENERON PHARMACEUTICALS ROCHE INNOVATION CENTER Michaela Wendeler 777 Old Saw Mill River Road PENZBERG MEDIMMUNE Tarrytown, NY 10591 Roche Diagnostics GmbH One MedImmune Way United States Penzberg, 82377 Gaithersburg, MD 20878 andrew .tustian@regeneron .com Germany United States Participant List

Notes thomas .von_hirschheydt@roche .com WendelerM@medimmune .com Michiel Ultee ULTEEMIT BIOCONSULTING, LLC Eric von Lieres Marc Wenger 800 Atkinson Circle RESEARCH CENTER JÜLICH MERCK & CO ., INC . Hillsborough, NJ 8844 Wilhelm-Johnen-Strasse 1 PO Box 4 United States Jülich, 52425 West Point, PA mike@ulteemitbio com. Germany United States e .von .lieres@fz-juelich .de marc_wenger@merck .com

Recovery of Biological Products XVII | 121 for their support and encouragement. Organization Sponsors Organization We are grateful to the ACS BIOT Division and to the Recovery Series Board are grateful to the ACS BIOT Division and to the Recovery We .psu .edu United States nzecherle@bmrn .com Min Zhu MEDIMMUNE 1 Medimmune Way Gaithersburg, MD 20878 United States zhum@medimmune .com Andrew Zydney UNIVERSITY PENN STATE 118B Fenske Lab 16802 University Park, PA United States zydney@engr BRISTOL-MYERS SQUIBB BRISTOL-MYERS Rd 38 Jackson 1434 Devens, MA United States xuankuo .xu@bms .com Shuchi Yamamoto UNIVERSITY YAMAGUCHI Tokiwadai Ube, 755-8611 Japan .ac .jp shuichi@yamaguchi-u Deqiang Yu BRISTOL-MYERS SQUIBB 38 Jackson Road Devens, MA 1434 United States deqiang .yu@bms .com Nick Zecherle BIOMARIN PHARMACEUTICAL INC 105 Digital Drive Novato, CA 94945 Xuankuo Xu Xuankuo Woodruff Ave . Woodruff DYNAMICS OF COMPLEX DYNAMICS OF COMPLEX TECHNICAL SYSTEMS 675 West Kendall Street 675 West Cambridge, MA 2142 United States mxenelis@momentapharma .com Alex Xenopoulos MERCK MILLIPORE 80 Ashby Road Bedford, MA 1730 United States Alex .Xenopoulos@EMDMillipore .com Oceanside, CA 92056 United States james .woo@gilead .com David Wood UNIVERSITY OHIO STATE 151 W Columbus, OH 43210 United States wood .750@osu .edu Michael Xenelis PHARMACEUTICALS MOMENTA Michael Wolff FOR MAX PLANCK INSTITUTE Sandtorstrasse 1 Magdeburg, Germany .mpg .de mwolff@mpi-magdeburg James Woo GILEAD SCIENCES De La Plata 4049 Avenida Richard Willson Richard U OF HOUSTON 4004 UH CHBE 77004 Houston, TX United States willson@uh .edu

.ac .uk 122 | Recovery of Biological Products XVII Physical Sciences N .A .Willoughby@hw Nik Willoughby UNIVERSITY HERIOT-WATT School of Engineering and Edinburgh, EH14 4AS United Kingdom GENENTECH 1 DNA Way South San Francisco, CA 94080 United States christrw@gene .com UCB 638 Ajax Avenue Slough United Kingdom gavin .wild@ucb .com Chris Williams Hagedornsvej 1 Gentofte, 2820 Denmark mbsc@novonordisk .com Gavin Wild Novato, CA 94949 United States cwhite@bmrn .com Matthias Wiendahl NOVO NORDISK A/S United States .com mattwestoby@gmail Catherine White INC BIOMARIN PHARMACEUTICAL 105 Digital Drive Matthew Westoby Matthew BIOGEN Drive 5000 Davis NC 27709 Chapel Hill,

Participant List Organization Sponsors

The Recovery of Biological Products Conference Series is associated with the American Chemical Society’s Division of Biochemical Technology (BIOT.) This relationship maintains the Not-for-Profit and tax-exempt status of the Conference Series and provides certain fiscal services, including insurance.

The Recovery of Biological Products Conference Series is the leading forum for discussion and development of new areas relating to separation, purification and processing of biological products. This biennial conference provides a unique venue for discussions between leaders in the field from both academia and industry of the latest developments in downstream bioprocessing.

We are grateful to the ACS BIOT Division and to the Recovery Series Board for their support and encouragement.