Volume 29 Number 7

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BioPharmThe Science & Business of Biopharmaceuticals

EDITORIAL EDITORIAL ADVISORY BOARD Editorial Director Rita Peters [email protected] BioPharm International’s Editorial Advisory Board comprises distinguished specialists involved in the biologic manufacture of therapeutic drugs, Senior Editor Agnes Shanley [email protected] diagnostics, and vaccines. Members serve as a sounding board for the Managing Editor Susan Haigney [email protected] editors and advise them on biotechnology trends, identify potential authors, and review manuscripts submitted for publication. Science Editor Randi Hernandez [email protected] Science Editor Adeline Siew, PhD [email protected] K. A. Ajit-Simh Jerold Martin Community Manager Caroline Hroncich [email protected] President, Shiba Associates Independent Consultant Art Director Dan Ward [email protected] Rory Budihandojo Hans-Peter Meyer Contributing Editors Jill Wechsler, Jim Miller, Eric Langer, Director, Quality and EHS Audit Lecturer, University of Applied Sciences Anurag Rathore, Jerold Martin, Simon Chalk, Boehringer-Ingelheim and Arts Western Switzerland, and Institute of Life Technologies. Cynthia A. Challener, PhD Edward G. Calamai Correspondent Sean Milmo (Europe, [email protected]) Managing Partner K. John Morrow Pharmaceutical Manufacturing President, Newport Biotech and Compliance Associates, LLC ADVERTISING David Radspinner Suggy S. Chrai Publisher Mike Tracey [email protected] Global Head of Sales—Bioproduction President and CEO Thermo Fisher Scientific National Sales Manager Steve Hermer [email protected] The Chrai Associates East Coast Sales Manager Scott Vail [email protected] Tom Ransohoff Leonard J. Goren Vice-President and Senior Consultant European Sales Manager Linda Hewitt [email protected] Global Leader, Human Identity BioProcess Technology Consultants C.A.S.T Data and List Information Division, GE Healthcare Ronda Hughes [email protected] Uwe Gottschalk Anurag Rathore Biotech CMC Consultant Reprints 877-652-5295 ext. 121/ [email protected] Vice-President, Faculty Member, Indian Institute of Outside US, UK, direct dial: 281-419-5725. Ext. 121 Chief Technology Officer, Pharma/Biotech Technology Lonza AG PRODUCTION Susan J. Schniepp Fiona M. Greer Fellow Production Manager Jesse Singer [email protected] Global Director, Regulatory Compliance Associates, Inc. BioPharma Services Development AUDIENCE DEVELOPMENT SGS Life Science Services Tim Schofield Senior Fellow Audience Development Rochelle Ballou [email protected] Rajesh K. Gupta MedImmune LLC Vaccinnologist and Microbiologist Paula Shadle Jean F. Huxsoll Principal Consultant, Senior Director, Quality Shadle Consulting Product Supply Biotech Bayer Healthcare Pharmaceuticals © 2016 UBM. All rights reserved. No part of this publication may be reproduced Alexander F. Sito or transmitted in any form or by any means, electronic or mechanical including President, by photocopy, recording, or information storage and retrieval without permission Denny Kraichely BioValidation in writing from the publisher. Authorization to photocopy items for internal/ Associate Director educational or personal use, or the internal/educational or personal use of Johnson & Johnson Michiel E. Ultee specific clients is granted by UBM for libraries and other users registered with the Principal Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 Stephan O. Krause Ulteemit BioConsulting fax 978-646-8700 or visit http://www.copyright.com online. For uses beyond Director of QA Technology those listed above, please direct your written request to Permission Dept. fax 440- AstraZeneca Biologics 756-5255 or email: [email protected]. Thomas J. Vanden Boom Steven S. Kuwahara VP, Biosimilars Pharmaceutical Sciences UBM Americas provides certain customer contact data (such as customers’ Pfizer names, addresses, phone numbers, and e-mail addresses) to third parties who Principal Consultant GXP BioTechnology LLC wish to promote relevant products, services, and other opportunities that may Krish Venkat be of interest to you. If you do not want UBM Americas to make your contact information available to third parties for marketing purposes, simply call toll-free Eric S. Langer Managing Partner 866-529-2922 between the hours of 7:30 a.m. and 5 p.m. CST and a customer President and Managing Partner Anven Research service representative will assist you in removing your name from UBM Life BioPlan Associates, Inc. Sciences’ lists. Outside the U.S., please phone 218-740-6477. Steven Walfish Principal Scientific Liaison BioPharm International does not verify any claims or other information Howard L. Levine appearing in any of the advertisements contained in the publication, and cannot President USP take responsibility for any losses or other damages incurred by readers in reliance BioProcess Technology Consultants of such content. Gary Walsh Herb Lutz Professor BioPharm International welcomes unsolicited articles, manuscripts, Principal Consulting Engineer Department of Chemical and photographs, illustrations, and other materials but cannot be held responsible for Merck Millipore Environmental Sciences and Materials their safekeeping or return. and Surface Science Institute To subscribe, call toll-free 888-527-7008. Outside the U.S. call 218-740-6477. University of Limerick, Ireland INTERNATIONAL BioPharm International integrates the science and business of BioPharm biopharmaceutical research, development, and manufacturing. We provide practical, Contents peer-reviewed technical solutions Volume 29 Number 7 July 2016 to enable biopharmaceutical professionals to perform their jobs more effectively. FEATURES CAPACITY ANALYTICAL TESTING CLEANROOM STANDARDS Managing Biomanufacturing Forced Degradation Studies Revised ISO Cleanroom Capacity Expectations for Biopharmaceuticals Standards Improve Air Randi Hernandez Anette Skammelsen Schmidt Cleanliness Classification Capacity for complex The author addresses critical issues to consider Jennifer Markarian therapeutics is becoming prior to performing forced degradation studies Revised versions of ISO 14644 Parts increasingly difficult to predict. 14 and provides best practice recommendations 1 and 2 introduce changes to sampling for these types of studies. 24 procedures and monitoring plans for cleanrooms and clean zones. 38 QUALITY Microbiological Testing: PEER-REVIEWED Time is of the Essence Bioprocessing Technology PACKAGING TRENDS Cynthia A. Challener Trends of RNA-Based Raw Materials Pressures to accelerate Therapeutics and Vaccines Packaging Innovations current and next-gen therapies Claire Scanlan, Priyabrata Pattnaik, for Biopharmaceutical are challenging traditional Ruta Waghmare, Elina Gousseinov, Manufacturing microbiological testing methods. 20 Mikhail Kozlov, Aaron Hammons, Ling Bei, Nandu Deorkar Youssef Benchek, and Karim Pirani Recent trends in raw materials packaging This article reviews the current dynamics may impact manufacturing, quality, in the RNA therapeutics/vaccines market. 30 and cost of biopharmaceuticals. 40

COLUMNS AND DEPARTMENTS

6 From the Editor 44 Troubleshooting CPhI Pharma Awards The author provides a seek nominations for review of the concepts of excellence in biopharma. design and qualification that Rita Peters apply to single-use systems. Jerold M. Martin 8 US Regulatory Beat Volume 29 Number 7 INTERNATIONAL Agency guidance and industry 49 New Technology Showcase BioJuly 2016 PharmThe Science & Business of Biopharmaceuticals standards aim to reduce lapses www.biopharminternational.com and improve quality operations. 49 Product Spotlight MANAGING Jill Wechsler BIOMANUFACTURING 49 Ad Index CAPACITY EXPECTATIONS 12 Perspectives on Outsourcing 50 Biologics News Pipeline CDMOs need to be aware that unfavorable public markets put

emerging bio/pharma R&D QUALITY PEER-REVIEWED ANALYTICAL TESTING MICROBIOLOGICAL BIOPROCESSING TECHNOLOGY FORCED DEGRADATION TESTING: TIME IS TRENDS OF RNA-BASED STUDIES FOR spending at risk in 2017. OF THE ESSENCE THERAPEUTICS AND VACCINES BIOPHARMACEUTICALS Jim Miller Cover: JurgaR/Getty Images

BioPharm International
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BioPharm International ISSN 1542-166X (print); ISSN 1939-1862 (digital) is published monthly by UBM Life Sciences 131 W. First Street, Duluth, MN 55802-2065. Subscription rates: $76 for one year in the United States and Possessions; $103 for one year in Canada and Mexico; all other countries $146 for one year. Single copies (prepaid only): $8 in the United States; $10 all other countries. Back issues, if available: $21 in the United States, $26 all other countries. Add $6.75 per order for shipping and handling. Periodicals postage paid at Duluth, MN 55806, and additional mailing offices. Postmaster Please send address changes to BioPharm International, PO Box 6128, Duluth, MN 55806-6128, USA. PUBLICATIONS MAIL AGREEMENT NO. 40612608, Return Undeliverable Canadian Addresses to: IMEX Global Solutions, P. O. Box 25542, London, ON N6C 6B2, CANADA. Canadian GST number: R-124213133RT001. Printed in U.S.A.

4 BioPharm International www.biopharminternational.com July 2016 BD™ CHO CD Medium and Feed Kit

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BD CHO CD Medium and Feed Kit, and the BD OneFeed supplement are prototypes in the BD Preview Series. BD Larger quantities are available through BD Custom Products Services. 7 Loveton Circle Sparks, MD 21152 © 2016 BD. BD, the BD Logo and BD OneFeed are trademarks of Becton, Dickinson and Company. 23-18517-00 bdbiosciences.com/advbio From the Editor

Recognizing Biopharma Industry Excellence ach day, the editors of BioPharm International receive press releases promot- ing potential: announcements of new products, services, methods, and Eprocesses destined to solve a problem, cure a disease, save money, and—in some cases of extreme hyperbole—change the world. As natural cynics, the editors question the validity of these projections of great success. We also appreciate the true successes, as recognized by indepen- dent evaluations by industry experts. The CPhI Pharma Awards are one such program to recognize excellence in biopharma development and manufacturing. The awards program recognizes companies and individuals helping to accel- erate the development of biopharmaceuticals through the introduction of Rita Peters is the innovations, new technologies, and strategies that support drug development, editorial director of manufacturing, and distribution. The program is organized by CPhI Worldwide, BioPharm International. a global tradeshow for the pharma industry, scheduled for Oct. 4–6, 2016 in Barcelona, Spain. CPhI and BioPharm International are UBM plc brands. The 2016 awards are organized into 12 categories including:
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Excellence in Pharma: Bioprocessing. Technologies, products, processes, and CPhI Pharma services for the manufacture of biologic drugs.
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Excellence in Pharma: Contract Services & Outsourcing. Contracted services Awards seek and processes for the bio/pharmaceutical industry including research, development, formulation, manufacturing, analysis, and consulting. nominations
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Excellence in Pharma: Regulatory Procedures and Compliance. Technologies, products, processes, and services designed to ensure that bio/pharma com- for excellence panies comply with standards, rules, and guidances established by regula- tory and compendial authorities. in biopharma
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Excellence in Pharma: Supply Chain, Logistics, & Distribution. Technologies, products, processes, and services for ensuring the safe handling and track- development and ing of drug substances, raw materials, and drug products.
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Excellence in Pharma: Analysis, Testing, and Quality Control. Technologies, prod- manufacturing. ucts, processes, and services for the analysis and testing of drug substances, raw materials, and drug products in a laboratory or production-line setting.
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Excellence in Pharma: Packaging. Technologies, products, processes, and ser- vices related to primary and secondary packaging.
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Excellence in Pharma: Drug Delivery Devices. Technologies, products, pro- cesses, and services related to the delivery of drug products to patients.
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Excellence in Pharma: Corporate Social Responsibility. Innovation in improv- ing transparency and public outreach.
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Excellence in Pharma: CEO of the Year. The chief executive officer of an innova- tor or generic-drug company is eligible for nomination. Attributes to be con- sidered include financial performance, product performance, leadership skills, management capability, marketing, acquisitions, and corporate strategy.
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Excellence in Pharma: API Development. Technologies, products, processes, and services for the development and manufacture of APIs.
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Excellence in Pharma: Formulation and Excipients. Technologies, products, processes, and services related to the formulation of drug products.
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Excellence in Pharma: Manufacturing Technology and Equipment. Technologies, products, processes, and services for the manufacture of solid, semi-solid, parenteral, inhalation, or other dosage drugs. Applications for the 2016 CPhI Pharma Awards must be submitted by Aug. 14, 2016 via the awards website at awards.cphi.com. Finalists will be announced in September 2016. The awards will be presented on Oct. 4, 2016 during the Pharma Awards Gala at CPhI Worldwide. X

6 BioPharm International www.biopharminternational.com July 2016 Accelerate your bioprocess journey 6SHHGDQGHȻFLHQF\DUHFUXFLDODVSHFWVRIELRPDQXIDFWXULQJ 7KHULJKWVXSSOLHUFDQFRQWULEXWHWR\RXUVXFFHVV'LVFRYHU KRZRXUSLRQHHULQJWHFKQRORJLHVDJLOHVHUYLFHVDQGDELOLW\WR GHVLJQDQGFRQVWUXFWFRPSOHWHIDFLOLWLHVLPSURYHVVSHHG WRPDUNHW

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$$ Regulatory Beat

FDA and Manufacturers Intensify Concerns about Data Integrity Agency guidance and industry standards aim to reduce lapses and improve quality operations.

DA has long emphasized the importance into compliance” is an inappropriate way to Fof reliable and accurate data in ensuring achieve desired test results, a practice that is drug safety, quality, and purity, but cur- cited increasingly in inspection reports. At the rent good manufacturing practice (CGMP) vio- same time, FDA seeks to avoid adding onerous lations involving data integrity failings seem to requirements or to complicate efficient drug be on the rise, especially at overseas bio/phar- production. The regulators are all too aware maceutical operations. Current FDA policies for that a hard slap on a large producer could lead ensuring that manufacturers maintain accurate to production delays that create drug short- records and submit complete information stem ages or reduce competition that helps maintain from its Application Integrity Policy, which lower drug prices. was established in the wake of the generic-drug scandal of the 1980s. And while most data INSPECTIONS FIND PROBLEMS integrity citations tend to involve sloppy prac- FDA notes in the guidance that it is observing tices and inadvertent violations, as opposed to an increased number of violations involving outright fraud, FDA officials are taking stronger data integrity in CGMP inspections, including action to emphasize the importance of main- instances of poor records, inadequate written taining secure systems for collecting and retain- procedures, and deficient systems for ensuring ing records to document the production of effective production processes and controls at quality drugs and biologics. manufacturing facilities all over the world. FDA To highlight its concerns about the rise in inspections cite a range of serious deficiencies serious data and recordkeeping lapses in the in how employees handle important records United States and abroad, FDA issued in April and documents. There are reports of records 2016 a long-awaited draft guidance (1). The found in trash bins, data that do not match test guidance uses a Q&A format to outline key results, data manipulation, sample retesting to strategies for ensuring that manufacturing data achieve desired results, and deletion of undesir- are reliable and accurate, and that companies able results. These violations have led to warn- establish risk-based approaches for prevent- ing letters, import alerts, and consent decrees, ing and detecting problems in docu- particularly at facilities in India and China. menting processes and tests and in A scathing letter was sent in April to retaining records. FDA emphasizes Mumbai-based API producer Polidrug the need to ensure that all records are Laboratories, following an in-depth inspection complete and that effective systems in March 2015 (2). FDA cited the firm for failing are in place for retaining and tracking to record or investigate quality-related customer information and for preventing the complaints and for production deviations and deterioration and loss of stored data. inadequate controls on computerized systems The draft guidance defines key to prevent unauthorized access or changes to terms, such as “backup” and “audit manufacturing data. FDA banned all imports Jill Wechsler is BioPharm trail,” and discusses methods for from this site in September 2015, as has Health International’s Washington editor, restricting unauthorized access Canada and other regulatory authorities (3, 4). Chevy Chase, MD, 301.656.4634, to computer IT systems. There’s Another warning letter to contract manufac-

[email protected]. an explanation for why “testing turer Sri Krishna Pharmaceuticals highlights Sohm/Getty Images VisionsofAmerica/Joe

8 BioPharm International www.biopharminternational.com July 2016 Regulatory Beat

data integrity violations involv- reviewed and that appropriate con- ers in establishing mechanisms for ing incomplete laboratory records, trols can detect any problems. The detecting and remediating non- inappropriate controls on com- impact of data breaches and com- compliance situations, to encour- puter systems, a lack of written pliance problems are now “hitting age harmonized standards for procedures, plus a failure to fol- the bottom line” at pharma com- ensuring data integrity in different low those procedures that are in panies, pointed out Frances Lipp, regions, and ultimately to restore place (5). The letter cites multiple president of Lachman Consultant confidence of regulators and the situations where the firm deleted Services, at the FDLI conference. public in quality production sys- non-conforming test results and Situations involving falsified data, tems, Johnson said. PDA will dis- repeated tests to gain desired she noted, can lead to delays in cuss its guidelines for company results. FDA wants Sri Krishna to product launches, recalls, and codes and other related initiatives conduct a comprehensive inves- major overhauls of information at a workshop on data integrity tigation into the extent of record systems. in September in Washington, DC inaccuracies and to develop a (in conjunction with its annual global corrective action and pre- PDA OFFERS GUIDELINES FDA/PDA regulatory conference) vention plan. The Parenteral Drug Association and at similar workshops in Berlin, Of 28 warning letters issued by (PDA) has formed a task force to Germany, and San Diego. FDA’s Center for Drug Evaluation address the “spectrum of issues” In the old days of paper records, and Research (CDER) from January related to the complexities man- it was relatively easy to destroy or 2015 to May 2016, 21 cite data ufacturers face in ensuring the replace production files. Today’s integrity issues, reported Thomas integrity of processes generating computerized systems require Cosgrove, acting director of CDER’s key production and regulatory audit trails for every operation, Office of Compliance, at the ISPE/ information, reported PDA presi- which make discrepancies easier FDA/PQRI Quality Manufacturing dent and CEO Richard Johnson to detect—by both manufacturers conference in Bethesda, MD, in at the FDLI conference. A lack of and by FDA investigators. Thus it is June 2016. He noted that FDA is accountability in production sys- important for biopharma compa- seeing fewer problems at “top tems has led to improper data nies to ensure that all contractors tier” pharma companies, but more manipulation, adjustment of time and suppliers—for IT, manufactur- violations in China and other clocks, record backdating, exclu- ing, and clinical trials—understand foreign countries. FDA and indus- sion of adverse information, and follow the rules, and report try experts further discussed key and trashing of original records, quickly when problems emerge. components of FDA data integrity Johnson observed. requirements and industry best To remedy these problems, the REFERENCES practices at a special data integrity PDA group has developed guide- 1. FDA, Data Integrity and Compliance With CGMP Guidance for Industry, Draft workshop held in conjunction with lines to help manufacturers Guidance (CDER, CBER, CVM, April the quality conference. develop internal codes of conduct 2016), www.fda.gov/downloads/ Dealing with such problems for ensuring data integrity. Such Drugs/GuidanceCompliance can carry high legal costs, noted policies can emphasize to employ- RegulatoryInformation/Guidances/ UCM495891.pdf. attorney Neil DiSpirito of Ballard ees, suppliers, and contractors the 2. FDA, Warning Letter to Polydrug Spahr LLP at the May 2016 annual importance of meeting require- Laboratories Pvt. Ltd. Corporate Office, meeting of the Food and Drug Law ments for ensuring the accuracy April 14, 2016, www.fda.gov/ICECI/ EnforcementActions/ Institute (FDLI) in Washington, of information and records. They WarningLetters/2016/ucm496623. DC. Consequently, the importance apply to organizations that con- htm. of fixing data problems is draw- duct clinical trials and laboratory 3. FDA, Import Alert 66–40, FDA.gov, www.accessdata.fda.gov/cms_ia/ ing more attention in executive tests and that contract to provide importalert_189.html offices and prompting more corpo- services to bio/pharma companies, 4. “FDA Bans Drugs From India’s Polydrug rate initiatives to prevent and fix as well as to manufacturers. Labs, Citing GMP Issues, FDANews. data problems. The International The PDA task force also is pre- com, www.fdanews.com/ articles/173104-fda-bans-drugs-from- Society for Pharmaceutical paring a points-to-consider docu- indias-polydrug-labs-citing-gmp- Engineering (ISPE) is developing ment on the fundamental concepts issues?v=preview a white paper to make a strong for data integrity, as well as tech- 5. FDA, Warning Letter to Sri Krishna Pharmaceuticals Ltd.–Unit II, April 1, business case for investing in sys- nical reports on ensuring data 2016, www.fda.gov/ICECI/Enforcement tems able to ensure that all data accuracy in laboratory systems. Actions/WarningLetters/2016/ are appropriately recorded and The aim is to assist manufactur- ucm495535.htm. ◆

July 2016 www.biopharminternational.com BioPharm International 9 Product & Service Innovations Advertorial

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Bio/Pharma Funding Challenges Could Hurt CDMOs in 2017 CDMOs need to be aware that unfavorable public markets put emerging bio/pharma R&D spending at risk in 2017.

ontract research organizations (CROs) Cand contract development and manufac- The market has not been turing organizations (CDMOs) may not be feeling it yet, but the downturn in external friendly to young bio/pharma financing for early-stage bio/pharma companies is real. The impact on CDMOs and CROs will be delayed, but there is no doubt that service pro- companies trying to tap public viders will be feeling it in coming months. Two recent articles in the financial press markets for the first time. underscore what has been happening. An item on Bloomberg.com chronicled the challenges early-stage public companies are facing as they vs. 17 in 2015, but the amount raised, just $483 go out for further funding beyond their initial million by Bloomberg’s count, is down 75%. public offerings (IPOs) (1). The article high- Data compiled from the PharmSource Lead lights the plight of two bio/pharma companies, Sheet (Figure 1) confirms the Bloomberg analysis Aldeyra Therapeutics and Ovascience, whose and also shows that venture capital (VC) invest- stock prices took big hits when they floated ment has held up well despite the public market secondary public offerings of their stock on travails. Nevertheless, an article in the New York May 26th. The shares of Ovascience went down Times described how the balance of power has 30% on the day of the offering while those of shifted from entrepreneurs to investors in the Aldeyra fell 10%. venture capital world (2). According to the arti- The Bloomberg article noted that Aldeyra and cle, VC firms have been able to demand much Ovascience’s experiences are reflective of what tougher terms from companies they are invest- has been happening to many bio/pharma com- ing in, including lower valuations and the hir- panies trying to tap public markets. ing of more experienced executives. The article According to the article, the num- focuses on Internet companies, but the pinch is ber of secondary offerings from being felt across the start-up spectrum. bio/pharma companies is down 40% in 2016, with 64 offerings Figure 1: Venture capital investment 2014–2016. vs. 106 in 2015; but the amount

raised is down 70% from $9 bil- $12.0 lion to $2.6 billion. So it’s not just $10.0 the decline in the number of offer- $8.0 Jim Miller is president of PharmSource ings that is hurting early-stage bio/ $6.0 Information Services, Inc., and pharma; they are also raising fewer publisher of Bio/Pharmaceutical dollars per offering. $4.0

Outsourcing Report, The market has also not been US $ Billion $2.0 tel. 703.383.4903, friendly to young bio/pharma com- $- Twitter@JimPharmSource, panies trying to tap public markets 2014 2015 2016 [email protected], for the first time. There have been VC IPO Secondary Offering

www.pharmsource.com. half the number of IPOs, 8 in 2016 Images Farrall/Getty Don All figures are courtesy of the author.

12 BioPharm International www.biopharminternational.com July 2016 Perspectives on Outsourcing

IMPACT ON Figure 2: Investigational new drug filings 2009–2015. EARLY-STAGE COMPANIES It’s not surprising that CROs and CDMOs may not be feeling the 900 impact of the funding down- 800 turn quite yet. After the 2008 global financial crisis, it took 700 two years for investigational new 600 drug (IND) filings and Phase I clinical trial starts to reflect the 500 funding decline (Figure 2). That’s 400 because early-stage companies focused their remaining cash on 300 getting their lead candidates into Number of IND Filings the clinic as quickly as possible 200 in hopes of demonstrating proof 100 of concept (POC). POC is typi- cally the prerequisite for licens- 0 ing deals and other partnering 2009 2010 2011 2012 2013 2014 2015 arrangements from larger bio/ pharma as well as funding from public sources. Funding from partnering arrangements is prob- IMPACT ON CROS AND CDMOS recting their business develop- ably the most secure funding The most recent downturn in ment skills. After several years of source because large bio/pharma external financing is barely a year just answering unsolicited exter- companies now depend on in- old, so CROs and CDMOs aren’t yet nal inquiries, they will soon have licensed and acquired candidates feeling the pinch. Emerging bio/ to be prospecting for new busi- for at least half of the products pharma companies are using the ness and selling the customer on they ultimately take to commer- funds they have to get their can- why they should use them rather cial markets. didates into the clinic, as in past than a competitor. As they were in Early-stage companies try- the last downturn, new business ing to get into the clinic are an development skills will be a key to important source of business Early-stage companies sustained success. for CDMOs because most are dependent on service providers trying to get into REFERENCES to manufacture APIs and formu- 1. M. Nilsen, “Biotech’s Vicious Second- lated dose forms. Those compa- ary Cycle,” Bloomberg.com, May 26, nies represent the majority of the clinic are an 2016, www.bloomberg.com/gadfly/ar- customers at most CDMOs, but ticles/2016-05-26/biotech-secondary- they typically have just a small important source of offerings-get-punished number of new drug candidates, 2. K. Benner, “Start-Ups Once Showered so CDMOs need to constantly business for CDMOs. With Cash Now Have to Work for It,” replenish their customer port- New York Times, May 20, 2016, www. folios to thrive. That replenish- nytimes.com/2016/05/21/technology/ ment is highly dependent on a funding cycles, which is good for start-ups-once-showered-with-cash- rebound in public bio/pharma CDMO business in the near term. now-have-to-work-for-it.html?rref=col equity markets: venture capital But without public markets, the lection%2Ftimestopic%2FVenture%20 might get candidates through industry could see a sharp drop in Capital&action=click&contentCollec discovery and into preclinical, IND filings, and in the demand for but emerging bio/pharma compa- CDMO services, as it did in 2010. tion=timestopics®ion=stream&m nies need the larger tranches of One way for CDMOs and CROs odule=stream_unit&version=latest& public funding to sustain a clini- to prepare themselves for the contentPlacement=5&pgtype=collect ◆ cal development program. worst case is to focus on resur- ion&_r=0

July 2016 www.biopharminternational.com BioPharm International 13 Capacity Managing Biomanufacturing Capacity Expectations Randi Hernandez

Capacity for complex therapeutics is becoming increasingly difficult to predict. JurgaR/Getty Images

emand for any given new prod- the top things the industry must do to uct is typically only known avoid further capacity restrictions at bio- Dafter significant investments manufacturing plants (Figure 1). Analytical have already been made. testing concerns and adequate hiring of Because executives commonly plan capac- qualified personnel to manage the facili- ity requirements based on launch fore- ties are among the other top issues that are casts, there are many factors that can lead expected to create capacity limitations in to miscalculations of capacity, making the future (Figure 2). it challenging to know what capacity to build into a facility. According to a new FLEXIBLE SOLUTIONS survey by BioPlan Associates, more than Even if demand is accurately predicted, half of respondents (60%) expect facility changes to a development plan can also constraints to create biopharmaceutical pro- occur that require facility changes, says duction capacity constraints by 2021 (1). Christian Wyss, attorney at Vischer AG, BioPlan found that the development of who specializes in drafting and negotiat- more efficient single-use products, better ing contracts for clients in the life sciences. downstream purification technologies, the These developments can arise because an introduction of continuous downstream opportunity presents itself to improve a operations, and increased modularization drug or add more indications—or, scien- of production systems were identified as tific issues may have to be addressed that

14 BioPharm International www.biopharminternational.com July 2016 Biosimilar Development in CHO, NS0 & Sp2/0 with enhanced PQA assessment

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Start your project today. Visit www.abzena.com Capacity

Figure 1: The top 10 areas to address to avoid capacity constraints, according to were not planned. Wyss notes that a survey of biomanufacturers. there could also be problems with a technology transfer. “Either the man- Avoiding capacity constraints ufacturing process was not as robust “If this industry is to avoid significant capacity constraints, the as the sponsor thought it was, or the most important areas to be tech transfer failed to successfully addressed are:” convey all subtleties to the contract manufacturing organization [CMO].” Develop more cost-effective disposable, single- use products 46.0% To alter technical capacity, a facil- Develop better-performing disposable, single-use ity has to have “solution-oriented products 38.8% professionals that are willing and Develop better continuous bioprocessing - 37.4% downstream technologies able to find room for flexibility in Develop better downstream purification 36.0% a highly regulated environment,” technologies says Wyss. The change can become Develop more ‘modularized’ production systems 33.8% more complicated if there is a change in product type, which may even Standardize international regulatory processes 30.9% require a completely different facil- Streamline FDA regulatory process 29.5% ity, says Tom Ransohoff, vice-pres- Optimize cell culture systems to increase ident and principal consultant at 28.1% upstream performance BioProcess Technology Consultants. Fund more research to maximize production efficiencies 27.3% It is more difficult to respond to shift- Optimize systems to improve downstream ing demands if process equipment 26.6% purification performance and clean utility systems are hard piped into the infrastructure, says

Source: Figure adapted from the Thirteenth Annual Report and Survey of Biopharmaceutical Parrish Galliher, CTO for upstream Manufacturing Capacity and Production, BioPlan Associates Inc., 2016. Used with permission. at GE Healthcare’s Life Sciences busi- ness. Multiple closed-off cleanroom sections in facilities, numerous heat- Figure 2: The top 10 factors creating future capacity constraints, as identified by ing/ventilation/air conditioning a survey of biomanufacturers in 2016. zones, and low ceilings (which limit types and scales of new equipment) Which factors are likely to create biopharmaceutical can also serve as barriers to rapid production capacity constraints at your facility capacity expansion, Galliher states. in 5 years (by 2021)? (n=140) Forecasting long-term demand during the transition from clinical Facility constraints 60.0% to commercial is challenging, says Analytical testing and drug product release 37.1% Ransohoff. He adds that to meet uncertain or changing demands, Inability to hire new, experienced technical and production 30.7% staff one strategy is to “number up,” or Inability to retain experienced technical and production staff 29.3% use multiple single-use bioreac- tors to achieve a range of upstream Physical capacity of downstream purification equipment 27.1% scales. Galliher concurs that adding Inability to hire new, experienced scientific staff 25.0% extra operating shifts to an existing facility helps rapidly expand capac- Inability to retain experienced scientific staff 23.6% ity, as well as overlapping or “stag- Costs associated with downstream purification 20.7% gering” of batches to meet need.

Physical capacity of fermentation/bioreactor equipment 20.0% According to a report compiled by Patheon, ORC International, and Inability for me to optimize my overall system, given my 17.9% current technology and resources PharmSource, demand and capac- ity forecast inaccuracies have prompted biopharmaceutical com-

Source: Figure adapted from the Thirteenth Annual Report and Survey of Biopharmaceutical panies to embrace the use of out- Manufacturing Capacity and Production, BioPlan Associates Inc., 2016. Used with permission. sourcing with more fervor than

ever before (2). ALL FIGURES ARE COURTESY OF BIOPLAN ASSOCIATES.

16 BioPharm International www.biopharminternational.com July 2016 Capacity

FORECASTING CAPACITY NEEDS A 2007 article in Pharmaceutical HYBRID CAPACITY A multitude of unforeseen circum- Executive estimated that a launch VS. OTHER MODELS stances can skew capacity fore- delay costs an average of $15 mil- While it seems like a number of phar- casts. Some of these could include lion per drug per day (4). This num- maceutical companies still rely on a reports of a serious adverse event, ber changes, however, depending on largely in-house approach to manag- slow enrollment in clinical trials, sale the market demand of the drug in ing capacity, most large firms have of a parent company that is devel- question. “The general rule is that been open to the concept of using oping the drug, an unusually suc- a biologic will generate, on average, outside CMOs to meet short-term cessful marketing strategy, provider $300 million per year. So, each day requirements. Small firms often use motives and incentives, final cost to delayed is a loss of $1 million,” esti- a completely outsourced model to the patient, willingness for a payer or mates Galliher. “I have seen much meet capacity. The percentage of pro- pharmacy benefit manager to reim- larger numbers in print for blockbust- duction that is outsourced at each burse a drug, a change in raw mate- ers,” he adds. biomanufacturing firm depends rial availability, availability of new Including post-approval R&D largely on what type of product is therapeutic alternatives, or new regu- costs, as well as costs associated with being manufactured. According latory legislation. unsuccessful projects, the estimate to numbers from the 2016 BioPlan In a Nature Reviews Drug Discovery for the average out-of-pocket cost to report (1), approximately 59% of study from 2013, investigators develop a new compound was found respondents used at least some out- concluded that more than 60% of to be $2870 million (in 2013 dollars), sourced capacity for mammalian cell companies miss their demand fore- according to an analysis by DiMasi culture, 55% used outsourcing for casts by at least 40% (3). A signifi- et al. that appeared in the May microbial fermentation, 42% used cant number of companies were 2016 issue of the Journal of Health outsourcing for production in yeast, also overly optimistic by more than Economics (5). Even though there 33% outsourced for production in 160% of the actual peak revenues have been slight methodological plant cells, and 33% outsourced that a product could pull in. Even differences in DiMasi et al.’s studies capacity for production of therapies up to six years post-launch, fore- since 2003—when the authors began in insect cells (see Figure 3). casts were still found to be off the looking at the cost of bringing a drug Companies such as Amgen, mark by percentages as high as 45%. to market—this cost of development Bristol-Myers Squibb, and Roche use The researchers found that demand has still increased substantially since hybrid approaches for the production for oncology drugs was most com- 2003. Additionally, said the authors of their medications. Wyss estimates monly underestimated, most likely of the study, “clinical success rates that almost all biotech companies because of the additional indica- are substantially lower for the stud- that have several products on the tions for which these drugs earned ies focused on more recent periods” market use a mixed approach to approval by FDA after initial launch. (5). Thus, because failure rates have manufacturing, but most companies This demand underestimation is an increased and the cost of developing keep the number of CMOs that they important finding considering the a drug has also increased so mark- work with to a minimum. An excep- large number of biologic, immune- edly, it is increasingly difficult to tion would be a small biopharma oncology therapeutics (with various accurately predict the demand for a company with few products, says proposed indications) that are cur- drug—as well as that drug’s associ- Wyss. “Drug development companies rently in the pipeline. The authors ated capacity requirements. with no product on the market or found that analyst forecasts for Indeed, many industry experts one-product companies often rely on generic therapies were also mark- agree that predicting capacity will CMOs only and do not use in-house edly off-target (3). These findings become even more problematic for manufacturing. When the date for could have implications for future pharmaceutical manufacturers in the market launch is set, these companies demand calculations for biologics, as future because of market access issues. will often look for additional CMOs well as biosimilars with numerous In Europe, because physicians seem to back up their supply chain.” market competitors—especially if the to be more accepting of biosimilars, Centers for Medicare and Medicaid market penetration forecasts may be EXCESS CAPACITY Service’s proposal to use reference a bit more clear—but in the United As mentioned, it is common to over- pricing for all groups of therapeu- States, physician acceptance and pre- estimate or underestimate demand tically equivalent drugs under scribing practices (as well as the inter- for a drug. Overestimating can lead Medicare Part B (even for biosimilars changeability status of a biosimilar) to the manufacture of too much that are not interchangeable) comes may make launch and capacity pre- product, which would then have into effect. dictions increasingly challenging. to be disposed at the manufactur-

July 2016 www.biopharminternational.com BioPharm International 17 Capacity

Figure 3: Current percent production outsourced; by system. products and biosimilars strictly separate the supply chain man- agement for original products “What percent of biomanufacturing organization’s production and biosimilars.” currently outsourced for each?” 2016 CAPACITY REDUCTIONS: 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% DECREASING VOLUMES WITHOUT COMPROMISING QUALITY OR REVENUE Mammalian 13.7% 23.5% 7.8% 5.9% Cell Culture 2.0% 5.9% In general, a decrease in capacity is viewed as a negative event, and com- panies are more reticent to announce Microbial 3.2% capacity reductions. “Decreases in 19.4% 3.2% 12.9% 3.2% 12.9% Fermentation capacity/moth-balling facilities are generally not positive developments for companies since they represent Yeast 8.3% 8.3% 8.3% 16.7% inefficient utilization of capital, often resulting from a failure of a product candidate (or candidates) in clinical trials or of a manufacturing business Plant Cells 16.7% 16.7% model to develop as planned,” states Ransohoff. “By contrast, increases in capacity signify optimism for the

Insect Cells 11.1% 22.2% future of the company’s products or manufacturing business model.”

Source: Figure adapted from the Thirteenth Annual Report and Survey of Biopharmaceutical Indeed, as facilities age, they may Manufacturing Capacity and Production, BioPlan Associates Inc., 2016. Used with permission. have to be updated or taken offline entirely. “Retirement of very old plants is being exceeded by new er’s cost. Or, if an overestimation points out that some large companies capacity growth, [and] overall capac- becomes apparent prior to produc- also purposely keep some capacity ity needs are growing,” notes Greg tion, gaps in revenue could appear, available to account for “unantici- Guyer, leader in biologics develop- and the company must somehow pated surges in demand.” ment and operations at Bristol-Myers fill capacity. Ransohoff notes that he has Squibb (BMS). Announcements of Idle capacity at CMOs can some- seen some companies using their capacity reduction are indirectly seen times be handled without disruption excess capacity for the production when sites are sold between compa- (provided that notice is given to the of biosimilars, citing Biogen’s nies, he says. In fact, the sale of these CMO in advance), and the CMO can manufacture of Biogen/Samsung types of facilities can have a posi- use the capacity for other custom- Bioepis’ etanercept biosimilar tive spin, notes Galliher. When the ers, says Wyss. If little to no notice is Benepali as an example of this sale of an older facility occurs, “fis- given, Wyss says that costs for equip- trend. Sometimes CMOs use pro- cal responsibility is also being dem- ment remaining idle “can be 80–90% visions that are built into con- onstrated by closing unnecessary of the costs of manufacturing, at least tract agreements to resell unused capacity and selling off underutilized for a couple of weeks or months.” capacity, which Ransohoff says assets, in which case positive finan- While costs to maintain an idle facil- helps CMOs mitigate the “costs cial/investor outcomes can result.” ity may be high, says Ransohoff, of typical ‘take or pay’ provi- There is new evidence that a “they are much lower than the eco- sions for clients,” or the costs to capacity decrease may not neces- nomic losses associated with failing reserve facility time regardless of sarily have negative connotations. to supply the market demand for a if capacity was used. For example, concentrated fed-batch highly profitable biopharmaceutical.” Wyss argues, however, that he (CFB) cell culture has been shown Facilities can become idle for vari- does not expect excess capacity in early experiments to yield prod- ous reasons, including the failure of in-house to be used for the pro- ucts of similar quality compared a late-stage product to get approval duction of biosimilars too often: with those made through traditional by FDA or the failure of a new prod- “To my knowledge, even pharma fed-batch culture (6). Not only could uct to gain market share. Ransohoff companies having both original these concentrated fed-batch runs

18 BioPharm International www.biopharminternational.com July 2016 Capacity

be manufactured at lower volumet- If a CMO/CDMO has its own pro- about keeping IP secure often prevail, ric capacities (meaning smaller facili- prietary manufacturing platform, a says Wyss, and as a result, pharma ties could accommodate volumes biopharma company cannot easily companies rarely outsource to coun- typically seen at larger facilities), the transfer the process back in-house tries with perceived weak patent pro- resulting products were also shown or to another CMO, Wyss points tection or in areas where national to enhance cell-line charge hetero- out. “If the contract manufacturing laws provide for mandatory licenses geneity, proving that concentrated agreement does not give the sponsor to local generic drug manufacturers. fed-batch could be associated with a license to this technology solely “It seems that originator companies “both process and product quality for the continued production of this have been able to solve all quality benefits” (6). specific biologic drug, the manu- related issues when manufacturing Despite these benefits, concen- facturing process will have to be in those countries, but are still reluc- trated fed-batch used more perfusion partially re-designed, which is prac- tant to expose themselves to these and feed media, required numerous tically not feasible from a time and legal risks before patent expiry.” Wyss filters, and also overloaded down- cost perspective.” tells this publication that intellec- stream processes, causing filter tual property issues come into play fouling in some cases. The yields Intellectual property most when a manufacturer is decid- obtained in the Yang study (6) were barriers to capacity outsourcing ing between different CMOs in vari- not sufficient enough to suggest Manufacturers typically choose an ous parts of the country. He adds, totally replacing larger facilities, but in-house model to keep better con- “many countries have regulations the technique has potential for some trol of their supply chain, handle requiring that at least a part of the slow-growing cell lines. While it may development risks, manage speed manufacturing of the drugs sold is not be economically feasible for a leg- of development and launch, and for accomplished within that country, acy system to be converted to CFB, tax purposes, says Ransohoff—but either directly by relevant legislation Yang et al. wrote that new compa- another important reason to keep (such as in Russia), or indirectly by nies seeking flexibility in capacity production in house is to ensure pro- making this a requirement to obtain operations might want to consider tection of a company’s intellectual research funding or collaborate with trying CFB to meet their production property (IP). public academic institutions (e.g., needs. The authors concluded, “The There seems to be mixed responses the standard Cooperative Research key to unlocking the cost and capac- about whether or not the protection and Development Agreement in the ity savings of concentrated fed-batch of IP is a significant problem when United States requires manufactur- is increasing the specific productivity working with CMOs. Guyer says ing in the US).” On the other hand, of the process through cell line and when BMS outsources, it establishes Galliher mentions that it is also rela- process development.” clear contract provisions to protect its tively common for some companies IP, and if it cannot reach agreeable IP to choose a foreign CMO to handle WORKING WITH CMOS terms with CMO partners, it simply capacity specifically to avoid local The use of CMOs can be helpful does not work with that CMO. He IP legislation. when there are fluctuating capac- says BMS rarely finds IP to be a bar- ity and demand conditions, but rier to successful relationships with REFERENCES sometimes a biopharma company outsourcing partners. Conversely, 1. BioPlan Associates, Inc., Thirteenth Annual Report and Survey of may be wary of the CMO model. A GE’s Galliher sees IP as a “major Biopharmaceutical Manufacturing biopharma company may initially issue” for CMOs when joint owner- Capacity and Production, April 2016. choose an “in-house” approach ship of technology platforms and/ 2. ORC International, Patheon, and because it fears the leverage a CMO or inventions exist. “The customer PharmSource, “Impact of Incorrect Forecasts on New Product Launches,” can gain over its business. “The usually wants to retain its rights to Industry Report, 2016. leverage stems from the fact that the drug and cell line and how it is 3. M. Cha, B. Rifai, and P. Sarraf, Nat. Rev. the CMO/contract development made, especially if proprietary tech- Drug Disc. 12, pp. 737–738 (2013). and manufacturing organization niques are used. The CMO wants the 4. T. Noffke, “Successful Product Manager’s Handbook”, a supplement [CDMO] has all manufacturing business freedom to use the process to Pharmaceutical Executive (March knowledge, and there is always a techniques for other customers and 2007), www.pharmexec.com/no-time- substantial risk that technology its own cell line if it is providing it.” delay, accessed April 30, 2016. transfer will not be successful imme- Although there many be signif- 5. J.A. DiMasi, H.G. Grabowski, and R.W. Hansen, J. Health Econ. diately,” states Wyss. Thus, he says, icant advantages in terms of labor 47, pp. 20–33 (2016). detailed technology transfer plans costs when using foreign outsourc- 6. W.C. Yang et al., J. Biotechnol. are crucial. ing operations, specifically, concerns 217, pp. 1–11 (2016). ◆

July 2016 www.biopharminternational.com BioPharm International 19 Quality: Microbiological Testing Microbiological Testing: Time is of the Essence Cynthia A. Challener

Pressures to accelerate current and next-gen therapies are challenging traditional microbiological testing methods. Stockbyte/Getty Images Stockbyte/Getty

ffective microbiological test- ing to Marian McKee, senior director of ing during biopharmaceutical BioReliance operational development Edrug development and manu- services at MilliporeSigma. Traditional facturing is crucial for ensur- microbiological methods for sterility ing sterility, determining antimicrobial testing take 14 days from inoculation to effectiveness, detecting microbial con- detection and conclusive results, while tamination or bioburden levels, ana- culture-based methods for mycoplasma lyzing endotoxins, and implementing take 28 days. “The time to obtain results environmental monitoring programs. for these traditional microbiological test Growing pressures to increase produc- methods is lengthy,” McKee notes. tivity, flexibility, and cost-effectiveness For release of traditional biologics that and the unique properties of many are produced in large lots with longer next-generation therapies are challeng- stability profiles, the turnaround time on ing today’s microbiologists. conventional microbiological tests, which are sensitive and robust, is not a concern CONVENTIONAL PROTOCOLS NO LONGER with respect to the ability to obtain reli- SUITABLE FOR TRADITIONAL BIOLOGICS able results. The faster release of bulk drug Microbiology testing of pharmaceutical substances and batches during in-pro- Cynthia A. Challener, PhD products is performed with a view to cess testing, however, is desirable. “More is a contributing editor to detection, enumeration, and identifica- rapid alternatives to traditional methods BioPharm International. tion of microbial contaminants, accord- are needed to speed the manufacturing

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July 2016 BioPharm International 21 Quality: Microbiological Testing

process while ensuring process and extremely time consuming. In addi- of patient cells followed by their product safety,” says McKee. tion, the many different matrices of injection back into the patient, Protein-based biologic drugs can the different in-process samples lead the process is typically completed also present real technical challenges to challenges with recovery of differ- within just a few days. As a result, with respect to reading assay results. ent organisms depending on the pro- Lovatt points out that the use of “Occasionally the therapeutic mol- cess step,” he explains. contract research organizations/ ecule can have toxic effects on the third-party testing laboratories may detector cells that are used to detect NEXT-GEN THERAPIES not be practical. “Microbiological viruses in in-vitro assays that comply HAVE UNIQUE NEEDS testing for these therapies may with ICH Q5A (1) and related regu- The lengthy time to obtain results need to be performed at the pro- latory guidelines,” observes Archie for traditional microbiological test duction site in order to avoid addi- Lovatt, scientific director of biosafety methods, while not a technical issue tional delays due to the need for for SGS. He does add, however, that for more stable proteins, antibodies, the shipment of samples,” he says. this issue can usually be overcome and other older types of biologics, Ortiz also notes that developing by dilution of the sample. Results of is typically unacceptable for newer and implementing appropriate tech- the tests performed on diluted sam- cell-based therapeutics. “Some next- nologies for the detection of organ- ples can also be supported by viral generation products have short shelf isms in many of the new types of clearance data generated during pro- lives and require novel technologies biologic drug substances and drug cess development. to detect microbes that are more products classified as next-genera- For biopharmaceutical manufactur- rapid than traditional methods,” tion therapies is also a challenge. ers including contract manufacturers says Lovatt. Cellular- and gene-ther- that produce many different prod- apy products, which not only have VACCINES DRIVE ucts, the time and resources involved short shelf lives, but also nontradi- TEST DEVELOPMENT in performing required microbial tional lot sizes, are driving the need The need for a rapid response to a testing is a challenge, says Aaron for rapid microbiological methods possible influenza pandemic in the Ortiz, QC manager of microbiology (RMMs) to reduce testing times and first decade of the 2000s spurred at GSK Biologics’ GMS Rockville site. sample volumes, agrees McKee. the development of RMMs, accord- “Qualifying the bioburden method In addition, for cellular thera- ing to McKee. Alternative strategies for each step of each process is pies that involve the modification for in-process release of vaccine batches were needed to accelerate production of flu vaccines, with the 14-day sterility test seen as a US and EU Regulators Seek to Reduce Inspections prime target for decreasing the turnaround time. FDA is assessing an information exchange with the European Medicines Agency The high cell content in many (EMA) that would identify facilities with strong records of compliance with good vaccine batches can also interfere manufacturing practices based on inspections by competent local inspectorates, with the ability to visually read potentially reducing the number of facility inspections. traditional test results. “Alternative Although FDA has been receiving inspection reports from EMA for years, current test methods based on detection of FDA policy prevents sharing of trade secret information that appears in field inspection metabolites or luminescence have reports, explained Dara Corrigan, associate commissioner for global regulatory policy been developed that can overcome at FDA at the ISPE/FDA/PQRI conference in Bethesda, MD, in June 2016. Other US this obstacle.” McKee notes. government agencies share sensitive, classified information with European Union (EU) Viral vaccines in particular can authorities, offering a precedent for FDA to act similarly. Legislation enacted in 2012 cause challenges due to the need permits FDA to share confidential information in situations where the agency can to generate neutralizing antisera to certify that the other country can keep this information secret. neutralize the cytopathic effect of FDA has been negotiating for three years to devise a system for mutual reliance the vaccines on the cells that are on inspection reports by local regulators found to meet acceptable standards. The used to detect viruses/mycoplasma, situation is complicated due to each of the 28 EU member states conducting its such as MRC-5 and Vero, etc. “The own pharma facility inspections following different practices and standards. generation of the antisera can sig- Currently, regulatory officials from FDA, EMA, and EU member states are auditing nificantly extend the time required inspection programs by other authorities, with the goal of building confidence in the for the testing process and, con- capabilities of the other inspectorates and their inspection findings. sequently, manufacturing of viral vaccines,” says Lovatt.

22 BioPharm International www.biopharminternational.com July 2016 Quality: Microbiological Testing

Influenza vaccines, for example, tive impact on the development methods are still culture-based, but must be manufactured in a very of more rapid, advanced microbio- with automation to help accelerate limited time due to their seasonal logical methods (2). Amendments the process. Others involve com- nature, and it is not possible to gen- to the Sterility Test for Biological pletely new testing technologies. erate antisera quickly enough. As a Products rule (21 Code of Federal “Some automated RMMs use result, microbiological testing using Regulations 610.12), which estab- a culture phase coupled with an rapid nucleic acid methods is allowed lishes FDA’s microbiological testing automated end-point to reduce the in the pharmacopeias, while poly- requirements for biological products, overall testing time, but are still merase chain reaction (PCR) methods have been particularly important, in keeping with the traditional for mycobacteria detection, which according to McKee. “The changes tests described in the compendia. typically take one week, can be used to the rule provide manufacturers Alternative methods, on the other as alternatives to traditional culture- of biologic products greater flexibil- hand, are different from traditional based methods, which take up to 56 ity. Biopharmaceutical manufactur- methods and often do not incorpo- days, according to Lovatt. Nucleic ers are in fact encouraged to use the rate a culture phase,” McKee says. acid testing is also an effective alter- most appropriate and state-of-the-art The results for alternative methods native for microbial testing of viral test methods to assure the safety of are also often reported in units other vaccines for which the viral drug sub- biologic products,” she explains. than colony forming units (CFUs), stance is difficult to neutralize. In addition, McKee notes that the which is the typical format for cul- changes to the rule promote innova- ture-based assays. The results of these REGULATORY FLEXIBILITY tion in the development of sterility tests, therefore, are not directly com- CAN BE BENEFICIAL testing and are thus paving the way parable with those obtained using Significant changes to regulatory for the use of novel methods such traditional or automated growth- requirements for microbial testing as adenosine triphosphate (ATP) bio- based methods, according to McKee. have, in fact, been crucial for acceler- luminescence, chemiluminescence, “The prevailing concern is that ating the adoption of more advanced and even non-growth based RMMs. these alternative methods require methods. As mentioned previously, “Not only was the use of a mandated extensive validation studies to dem- the pharmacopeias have added chap- method for sterility testing removed, onstrate equivalency to traditional ters to address the needs of next-gen- the change in the rule allows greater microbiological methods. The need eration therapies for RMMs that can flexibility in sampling, putting the for such extensive method vali- assure product safety. McKee points onus of sample size on the manu- dation in addition to the capital to the European Pharmacopoeia (Ph. facturer; the sample must be appro- expenditures necessary to develop Eur.) chapters 2.6.7 Mycoplasmas and priate to the material tested both in and implement these new technolo- 2.6.27 Microbiological Control of volume and representation of the lot gies poses a hurdle for most bio- Cellular Products as two important size,” she says. pharmaceutical manufacturers that examples. Ph.Eur. 2.6.27 outlines an There are some changes to regula- wish to employ rapid microbial alternative to traditional compendial tions that have created challenges, methods,” McKee concludes. The sterility testing. “The 2.6.27 method however. Ortiz points to the rela- strong value proposition that RMMs is suitable for qualification and vali- tively recent low endotoxin recovery offer, however, extends to the devel- dation of rapid methods for sterility (LER) phenomenon. For biologics, opment of assays targeting other testing and incorporates conditions FDA is requesting studies to deter- adventitious contaminants includ- and control organisms that yield a mine if drug substances or drug ing viruses, mycoplasmas, residual more sensitive and broader range of products demonstrate LER when DNA, and residual proteins. Assays detection for cellular products,” she performing endotoxin analyses (3). that can be performed near the bio- explains. Ph.Eur. 2.6.7 Mycoplasmas, Due to the recent discovery of this reactor location and in real time are meanwhile, includes specific guid- phenomenon and the various formu- definitely the assays for the future. ance for validation of nucleic acid lations of drug substances and drug amplification techniques (NAT) that products, there have been difficulties REFERENCES 1. International Council for Harmonisation, may be used as alternatives to the in developing proper studies for LER. Q5A (R1) Viral Safety Evaluation 28-day culture and indicator cell of Biotechnology Products Derived methods for detection of adventi- RAPID SOLUTIONS: from Cell Lines of Human or Animal Origin (September 1999). tious mycoplasmas. AUTOMATED VS. ALTERNATIVE 2. FDA, “FDA issues final rule on sterility In addition, FDA has made The development of new test meth- testing of biological products,” Press Release, May 3, 2012. changes to its microbial testing ods has been pursued using two 3. K. Williams, BioPharm International, requirements that have had a posi- fairly different strategies. Some 28 (7) 28-33 (2015). ◆

July 2016 www.biopharminternational.com BioPharm International 23 Analytical Testing Forced Degradation Studies for Biopharmaceuticals Anette Skammelsen Schmidt The author addresses critical issues to consider prior to performing forced degradation studies and provides best practice recommendations for these types

of studies. Photo by Masakazu Matsumoto/Getty Images

orced degradation studies are cule is different, and certain freedoms performed by means of various for selecting stress conditions for bio- F stressing agents such as pH, pharmaceuticals are inherent (4, 5). temperature, light, chemical Hence, conditions should be carefully agents (e.g., oxidizing, deamidating selected on a case-by-case basis (3). agents, etc.), and mechanical stress to Regulatory guidance documents speed up the chemical degradation, specify the following expectations on physical degradation, or instability forced degradation: of a molecule. Currently, there are no t
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July 2016 BioPharm International 25 Analytical Testing

Figure 1: The purpose of forced degradation studies (FDS). some degradation products may retain biological activity (1–3). An Establish and Determine example of this is illustrated in understand Identify likely intrinsic stability degradation of molecule Figure 2 and describes a situation degradation pathways Support process products development in which oxidation is not associ- ated with a decrease in activity. Validation of Information to analytical methods, process robustness including stability FDS studies DEGRADATION PATHWAYS indicating power OF BIOPHARMACEUTICALS Biopharmaceuticals can usually Provide samples for Impurity / variant analytical development degrade in many different path- characterization Support formulation Support formal ways following different kinet- stability studies development ics. The extent of stress needs to provide a measurable change and confirm the most relevant Figure 2: Determined oxidized forms (A) and activity (B) of drug substance (DS) degradation pathways. Too much and drug product (DP) after exposure to 0.003% H2O2 at 22 °C for 0, 2, 4, 6, and stress, however, might form sec- 24 hours. ondary degradation products not seen in formal stability studies,

60 and the level of stress might not AB 50 reflect actual potential stress- 40 DS DS ors. An extent of degradation of 30 DP 2 mg DP 2 mg 20 DP 1 mg DP 1 mg approximately 5–20% is assumed Activity 10 to be suitable for most purposes Oxidized forms (%) 0 0102030 and for most analytical meth- 0102030 Time (hours) Time (hours) ods. An adequate level of stress should be carefully selected on a case-by-case basis (3). The selection of the degradation tions may be useful in determin- information regarding the likely pathways to be investigated dur- ing whether accidental exposures degradation products of a specific ing forced degradation should be to conditions other than those biological drug. This informa- based on known and anticipated proposed (e.g., during transpor- tion can be useful for many pur- degradation pathways—as well as tation) generate changes in the poses, and can help to establish prior knowledge from similar mol- molecule. Stress studies are also the degradation pathways and the ecules, if such knowledge exists. useful for evaluating which spe- intrinsic stability of the molecule. The degradation pathways are typ- cific test parameters may be the Challenging the analytical proce- ically either physical (e.g., aggrega- best indicators of product sta- dures helps validate the method’s tion) or chemical (e.g., oxidation) bility and should be monitored stability-indicating power (4, 5). in nature. under proposed storage condi- Prior to performing a forced Aggregation can be noncovalent tions (3). degradation study, the goal of the in nature, such as an association of study needs to be defined. Several monomers that are dissociable at THE PURPOSE OF purposes might be addressed the right conditions (e.g., solvent, FORCED DEGRADATION in one study. When relevant, a temperature). These noncovalent Forced degradation study is forced degradation study can be aggregates are mainly formed by defined as an intentional break- performed at different develop- denaturation and unfolding of the down of a molecule to an ment stages. Figure 1 shows exam- molecule, or by an interaction with appropriate extent by means of ples of the various reasons that interfaces such as liquid-air, liquid- various stressing agents (includ- forced degradation studies are per- solid, or even liquid-liquid. These ing mechanical stress) to speed formed. associations are typically a result of up the chemical and physical Degradation products for bio- mechanical stress such as shaking, degradation and instability of a pharmaceuticals may be either stirring, rotation, pumping; freeze- biopharmaceutical. A forced deg- product-related substances or thaw cycles; heating; or exposure

radation study can give a range of product-related impurities, as to acidic pH. ALL FIGURES ARE COURTESY OF THE AUTHOR

26 BioPharm International www.biopharminternational.com July 2016 Analytical Testing

Table I: Examples of selected analytical methods for evaluation of degradation Due to the complexity pathways. RP– SE– IE– Peptide SDS– Pathway Activity* of biopharmaceuticals, HPLC HPLC HPLC mapping PAGE Aggregation - xxx - x x x

there is no single Fragmentation/ xx x x x xxx x clips stability-indicating Oxidation xxx - - xxx - x method that can Deamidation x - xxx xx x x Disulfide bridge xx-x -x profile all its stability exchange *Effect depends on location and extent of change in molecule - indicates no effect, x indicates small effect, xx indicates significant effect, xxx indicates severe effect characteristics. RP–HPLC: reversed-phase high-performance liquid chromatography SE–HPLC: size-exclusion high-performance liquid chromatography Aggregation can also be cova- IE–HPLC: ion-exchange high-performance liquid chromatography SDS–PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis lent in nature, such as chemical bonding between the molecules, and is non-dissociable during buf- specific role the residue has in [DTT]) and oxidation of cysteine fer change. These chemical bonds the functionality and active site residues such as oxidation by Cu are often formed by rearranged of the protein. (II) or Fe (II) ions. disulfide bridges or other altered Photolysis by exposure to light Several biopharmaceuticals intramolecular bridges. They are involves a free radical mecha- contain ligands or conjugates. typically a result of reactions of the nism that affects many functional Such bound moieties (e.g., acyla- amino acid residues with trace met- groups (e.g., carbonyl groups). The tion and conjugation) might be als (copper or iron) or an incom- free radicals can result in oxida- lost due to chemical or physical plete reduction of the protein. tion, aggregation, or peptide bond stress on the molecule. Side chains of methionine, cleavage. The photolysis is due cysteine, histidine, tryptophan, to exposure to photo-irradiation, SELECTION OF MATERIALS FOR or tyrosine residues are suscep- which is typically in the form of A FORCED DEGRADATION STUDY tible to oxidation, where methio- ultraviolet irradiation. When performing forced degra- nine is the most reactive residue. Hydrolysis (fragmentation) dation studies, it is important Oxidation can alter the physico- is a cleavage of peptide bonds to use a single batch of material. chemical properties of a protein, between amino acid residues Forced degradation studies usu- such as folding and subunit asso- releasing smaller peptide chains. ally require a large amount of ciations. The oxidation is mainly The peptide bonds of Asp-Pro material. However, the material due to exposure to atmospheric and Asp-Gly are the most suscep- could be non-GMP, a test batch,

O 2 under conditions of light, tible to hydrolysis. Hydrolysis is or even an out-of-specification heat, moisture, agitation, or to mainly a result from exposure to batch (if such is available), as long exposure to oxidizing agents. acidic or alkaline pH. as the choice of batch is justified. Deamidation is a hydrolytic con- Disulfide bridge exchange All relevant sample types version of asparagine or gluta- might cause incorrect paired should be included in the forced mine to a free carboxylic acid disulfide bridges, which affects degradation study. Drug prod- residue and is typically due to the tertiary structure of a protein. uct at both high- and low-dose changes in pH, ionic strength, Such incorrect disulfide bridges levels can be included for drug temperature, and humidity in might be a result of partial cleav- product-specific methods. If the the case of lyophilized proteins. ing and reformation of disulfide molecule is modified (e.g., by The overall effect of a chemical bonds as a result from denatur- acylation, glycosylation, or con- modification of a single amino ing/reducing conditions (expo- jugation), the inclusion of the acid residue depends on its posi- sure to reagents such as GnHCl, intermediate is highly recom- tion in the protein and on the urea, and 1,4-Dithiothreitol mended to aid understanding of

July 2016 www.biopharminternational.com BioPharm International 27 Analytical Testing

Figure 3: Examples of common stress conditions, including light for forced able to detect the change if/ degradation studies. when it occurs. Examples of ana- lytical methods to evaluate deg- radation pathways are shown in Oxidation Light 8 e.g., 0.003% H O overnight 25 ºC, or Table I. 2 2 8
Exposure - ICH Q1B conditions atmospheric O 2 8 ≥ 1.2 million lux hours and ≥ 200 W 8
pH Examples of common methods hours per m2 (25 ºC); ICH Confirmatory 8 e.g., overnight at 5 ºC and 25 ºC conditions 8
Elevated temperature to employ for analysis of biophar- 8 Dark control (25 ºC); wrapped in 8 e.g., 1-2 weeks or even up to 4 weeks aluminium foil and placed next to maceuticals during forced degra- 8 Depends on normal storage temperature exposed sample (and accelerated studies) 8
Exposure - stressed conditions dation are appearance (i.e., color, 8 Normal storage (e.g., 25 ºC), then 8 ≥ 2.4 million lux hours and ≥ 400 W stressed study (e.g., 50 ºC to 60 ºC) hours per m2 (25 ºC); stress conditions clarity, particulate matter); activ- 8
Mechanical stress 8 Dark control (25 ºC); wrapped in 8 e.g., rotation, shaking, agitation, aluminium foil and placed next to ity measurement; sodium dodecyl freeze-thaw exposed sample 8 Freeze-thaw (e.g., 5-10-15 cycles) sulfate polyacrylamide gel electro- 8 Depending on the biologics, even 8
Reduction harsher conditions can be employed 8 e.g., 0.01 M DTT overnight 25 ºC phoresis (SDS–PAGE); microchip gel electrophoresis; size-exclusion DTT: 1,4-Dithiothereitol; ICH: International Council for Harmonization high-performance liquid chro- matography (SE–HPLC) (e.g., for Figure 4: (A) Determined high-molecular weight proteins (HMWP) and (B) total protein content and aggregates); protein content after storage at elevated temperature (5 °C, 25 °C, and 37 °C) reversed-phase high-performance at one and two weeks. The chromatogram (C) from the high-performance liquid liquid chromatography (RP–HPLC) chromatography illustrates the increasing HMWP peak at high temperature. (e.g., for purity and specific impu- rities); isoelectric focusing (IEF)/ imaged capillary isoelectric focus- 45 A 40 ing (iCE)/ion-exchange HPLC (IE– 35 30 HPLC) (e.g., for deamidated forms); 25 T=0 (as is) 1.2 20 peptide mapping; and physico- 15 1 week C HMWP (%) 10 2 weeks 1.0 chemical analysis (e.g., differen- 5 0 0.8 tial scanning calorimetry [DSC], 52537 Temperature (ºC) HMWP circular dichroism [CD], and fluo- 0.6 rescence). Additional analysis can 37ºC 2 weeks 12 B 0.4

Absorbance Units 25ºC 2 weeks be employed based on the results 10 0.2 8 Monomer 5ºC 2 weeks obtained by the initially selected 6 T=0 (as is) T = 0 (as it) 0.0 4 1 week analytical methods. 2 weeks 2 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Content (mg.ml) 0 Retention time (minutes) 52537 SUITABLE CONDITIONS FOR Temperature (ºC) FORCED DEGRADATION STUDIES All molecules can be degraded by some chemical or physical means. Figure 3 shows examples the changes seen in the underly- that can profile all its stability of common stress conditions ing structure of the molecule. characteristics (2, 3). The nature known to induce different deg- Solution/buffer blanks and con- of biopharmaceuticals will dic- radation pathways for biophar- trols (excipients) are included for tate which test methods to use. maceuticals. The conditions used evaluation of peak profile regard- In general, methods that are in forced degradation have to be ing occurrence of new peaks used in stability studies should harsher than conditions used in as a result of stress conditions. be included in forced degrada- accelerated studies. If the condi- Always include reference samples tion studies, as well as methods tions result in no change, longer in each experiment. that determine identity, purity, exposure time is recommended, content, and methods for moni- rather than the use of a more SELECTION OF ANALYTICAL toring impurities. The methods extreme temperature. When METHODS FOR FORCED should provide reliable data—as selecting the relevant stress con- DEGRADATION STUDIES measured by a satisfactory selec- ditions, the following points Due to the complexity of bio- tivity between the main peak must be considered: pharmaceuticals, there is no sin- and impurities—an adequate t
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radation pathways of the molecule. Results from forced degrada- new methods and character- By performing such studies early tion studies should be pre- ization) were used? in development, this knowledge sented graphically and should The total protein content should about the molecule will be avail- include compare plots for chro- be measured for all samples (as able for optimal process and for- matographic methods. A result shown in Figure 4) to evaluate mulation development. matrix is an excellent way to the presence of insoluble aggre- The degraded samples can aid show results, as such a matrix gates. As the determined total the development of stability-indi- will be able to indicate which protein content is constant under cating analytical methods by dem- forced degradation conditions the applied conditions, insoluble onstrating if the current methods resulted in changes for which aggregates are not formed under are sufficient to evaluate stabil- degradation pathway. Statistical these conditions. Conditions of ity (e.g., use oxidized samples to and kinetic tools should be used high temperature and long peri- develop method for determination for evaluation of data when pos- ods of time, however, lead to high of oxidized forms) and by identify- sible to aid the understanding amount of high-molecular weight ing which test parameters are the of the degradation kinetics. A proteins (HMWP). Reference best indicators of stability. forced degradation study reveals the most important degrada- tion pathways. Such pathways can, for example, be indicators Forced degradation studies can be performed of aggregation or the formation of specific impurities that could in early development or late development cause concern. The forced deg- radation study also indicates which analytical methods are depending on the purpose of the study and the most concerning and whether these methods are able to detect amount of material available. the change that occurs. In sum- mary, degradation pathway stud- ies can help investigators predict samples have to be placed next Degraded samples are also use- whether an analytical package to forced degradation samples in ful during analytical validation, is sufficient for a molecule or order to evaluate the cause for an as they can be spiked in valida- whether a need exists for the observed effect. All samples from a tion samples. However, a limited development of other analytical specific study need to be analyzed amount of material is usually methods. in the same analytical series to available at the early stage of exclude the effect of possible ana- development and the analytical REFERENCES lytical variation. package might be incomplete. 1. ICH, Q6A, Specifications: New During development, the process Chemical Drug Substances and FORCED DEGRADATION DURING steps and the formulation might Products, Step 4 version (1999). THE DEVELOPMENT PHASES change. Additionally, the analyti- 2. ICH, Q6B, Specifications Test Forced degradation studies can be cal methods might change due to Procedures and Acceptance Criteria performed in early development further optimization of the ana- for Biotechnological/Biological Products, Step 4 version (1999). or late development depending on lytical conditions. Hence, forced 3. ICH, Q5C, Stability Testing of the purpose of the study and the degradation studies most likely Biotechnological/Biological amount of material available. The need to be repeated or extended Products, Step 4 version (1995). health authorities expect forced at a later stage of development. In 4. ICH, Q1A(R2), Stability Testing degradation studies to be carried conclusion, a limited forced degra- of New Drug Substances and out during development Phase dation study should be performed Products, Step 4 version (2003). III at the latest, but no guide or as early as possible during develop- 5. EMEA, Guideline On Stability Testing: specific requirements exist about ment, and a more comprehensive Stability Testing of Existing Active when to perform forced degrada- forced degradation study during Substances and Related Finished tion studies. Phase III should be performed. Products (London, October 2003). ◆

July 2016 www.biopharminternational.com BioPharm International 29 Peer-Reviewed Bioprocessing Technology Trends of RNA-Based Therapeutics and Vaccines

Claire Scanlan, Priyabrata Pattnaik, Ruta Waghmare, Elina Gousseinov, Mikhail Kozlov, Aaron Hammons, Ling Bei, Youssef Benchek, and Karim Pirani

ABSTRACT In 2014, the monoclonal antibodies market had the highest growth rate (19%) for the number of new molecules in the pipeline. DNA and RNA therapeutics were not far behind, achieving 12% year-over-year growth. Industry analytics data suggest that the RNA-based therapeutics market will reach $1.2 billion by 2020. LAGUNA DESIGN/GettyLAGUNA Images

his article reviews the current 60% of the nucleic acid-based therapeu- dynamics in the RNA thera- tic pipeline is in preclinical development. Tpeutics/vaccines market as well It is interesting to note that 35% of such as differences between small- pipeline is focused on oncology (2, 3). interfering RNA (siRNA), RNA interfer- Several companies (approximately 160) ence (RNAi), microRNA (miRNA), and and many academic institutes (approxi- messenger RNA (mRNA). In addition, mately 65) are developing RNA-based the authors outline the general produc- therapeutics. Table I provides a non- tion processes for these platforms, the comprehensive list of a few (4). Two challenges encountered during process companies have marketed RNA- development and production, and the based therapies: NeXstar and Ionis strategies to overcome them. Pharmaceuticals. There are 12 mRNA vaccines in development, seven MARKET OVERVIEW of which are being developed by RNA-based therapeutics target the treat- Curevac (Germany). Based on current ment of diseases such as diabetes, cancer, outlook, the RNA therapeutics market tuberculosis, and some cardiovascular seems more promising than the mar- Elina Gousseinov, Mikhail Kozlov, conditions. There is currently a great ket for DNA therapeutics. Claire Scanlan, Aaron Hammons, Ling Bei, deal of money being put into this rela- From a partnership perspective, Ionis Youssef Benchek, Karim Pirani, and Ruta tively new class of therapeutics and vac- Pharmaceuticals has entered into a global Waghmare are all from MilliporeSigma. cines, which is projected to grow 12% collaboration with Janssen Biotech, Inc. Priyabrata Pattnaik works in the in 2016 and reach $1.2 billion by 2020 to discover and develop antisense drugs Biomanufacturing Sciences and Training (1). The 2015 research and develop- to treat autoimmune disorders of the gas- Centre at Merck Pte Ltd., Singapore. ment (R&D) biotech pipeline is shown trointestinal tract (5), and Merck & Co. PEER-REVIEWED in Figure 1. There are more than 700 (MSD) has bet $100 million on Moderna’s Article submitted: Nov. 9, 2015. nucleic acid-based therapeutics (DNA mRNA technology (6). Moderna also has Article accepted: April 5, 2016. and RNA) in the pipeline and more than previously announced collaborations

30 BioPharm International www.biopharminternational.com July 2016 Advertorial Product & Service Innovations

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July 2016 BioPharm International 31 Peer-Reviewed

Figure 1: R&D biotech pipeline expansion. RNA interference (RNAi) and RNA anti- sense technologies appear to be dominating 9765 the market. RNAi is a gene-silencing technol- ogy in which RNA molecules inhibit gene 8490 2209 expression by targeting and destroying spe- cific mRNA molecules. RNA antisense tech- Bioengineered vaccine 2009 nology involves synthesizing an RNA strand 917 that binds to a specific mRNA or to a splic- Cell therapy 733 727 ing site on a pre-mRNA molecule to prevent 487 DNA & RNA therapeutics 660 translation. The major challenges associated Gene therapy 377 with the commercialization of these RNA- 2331 based therapies are toxicity and drug delivery. Monoclonal antibody 1952

427 RNA-BASED THERAPEUTICS Antibody-drug conjugates 295 Other biotechnology product 976 1104 With the advent of RNA-based therapeu- tics and their potential in treating a variety Recombinant product 1488 1563 of chronic diseases, it is important to note the number of enabled technologies used to 2014 2015 exploit the RNA mechanism/pathway, some of which are discussed in the following.

Figure 2: An insight into the RNAi pathway. Small hairpin RNA RNAi (shRNA) is a class of double-stranded RNA (dsRNA). The dsRNA is RNAi technologies work by “silencing” or cleaved or degraded by a “dicer” enzyme into oligonucleotide segments turning off a gene through the use of its own called small interfering RNA (siRNA), which then enter a cell to form DNA sequence (Figure 2). The process is initi- the RNA-inducing silencing complex (RISC). The siRNA strands then ated by double-stranded RNA (dsRNA) that separate or unwind to form the activated RISC complex, which can then expresses either as a small or short hairpin target messenger RNA (mRNA), bind to it, and cleave it. RNA (shRNA) or as a microRNA (miRNA) tran- script. Using this silencing mechanism, RNAi Dicer is commonly used to gain a better under- dsRNA standing of gene function, which can then or shRNA be used to generate additional targeted thera- dsRNA cleavage peutics (9). Small interfering RNA (siRNA) and siRNA miRNA are the core elements of RNAi tech- nology based therapeutics.

siRNA RISC Assembly RNAi utilizes a “dicer” enzyme to cut dsRNA into 21 oligonucleotide segments, called siRNA. These siRNAs can then bind to a specific family of proteins called Argonaute Target mRNA RISC Complex proteins, of which there are two classes: Ago Unwound siRNA and Piwi. Ago proteins bind to siRNAs or miRNAs, while Piwi proteins bind to Piwi- interacting RNA (piRNA) and are used to silence mobile genetic elements. The siRNA, Target mRNA cleavage miRNA, or piRNA complex bound to the Argonaute protein is called the RNA-induced silencing complex (RISC). Once bound to the Argonaute protein, one strand of the dsRNA with Alexion, AstraZeneca, and the Defense is removed and the remaining strand binds Advanced Research Projects Agency (DARPA) to and directs the degradation of the comple- totalling $450 million. Moderna has raised $625 mentary RNA target sequence, which then

million in equity funding (7, 8). leads to the loss of protein expression (10). ALL FIGURES ARE COURTESY OF THE AUTHORS

32 BioPharm International www.biopharminternational.com July 2016 Peer-Reviewed

Table I: Biopharmaceutical companies developing RNA-based therapeutics and vaccines. siRNA=small interfering RNA, miRNA=microRNA, and mRNA=messenger RNA.

siRNA miRNA mRNA Kyowa Hakko Kirin Andes Biotechnologies CureVac Silence Therapeutics Mirna Therapeutics Biontech RNA Pharmaceuticals Debiopharm miRagen Therapeutics Boehringer Ingelheim Marina Biotech Marina Biotech Johnson & Johnson Ipsen Moderna Therapeutics Ludwig Institute for Cancer Research Alnylam Pharmaceuticals Alnylam Pharmaceuticals BioNTech Sanofi Pasteur Sanofi Pasteur Sanofi Pasteur Tekmira Pharmaceuticals Tekmira Pharmaceuticals NanoCarrier Regulus Therapeutics Dicerna Pharmaceuticals Biogen Idec BioCancell Therapeutics GlaxoSmithKline Samyang Group AstraZeneca Silenseed Ionis Pharmaceuticals siRNAsense Les Laboratoires Servier Reference Biolabs Celsion Avena Therapeutics Rosetta Genomics Lipella Pharmaceuticals Santaris Pharma Arrowhead Research Shire InteRNA Technologies Alexion Pharmaceuticals t2cure Rigontec Microlin Bio

It has been reported that synthetic siRNA Though siRNA silencing requires exact is able to knock down targets in various dis- match between target and small interfering eases in vivo, including hepatitis B, human RNA, miRNA are non-specific and can exert papilloma virus, ovarian cancer, bone cancer, action through imperfect base pairing. In hypercholesterolemia, and liver cirrhosis. Only addition, miRNA triggers translation inhibi- a few molecules of siRNA per cell are required tion (i.e., prevents the RNA from synthesiz- to produce effective gene silencing (11). siR- ing protein from amino acids), while siRNA NAs are most commonly delivered into cells triggers mRNA degradation. using microinjection or a transfection agent. Many companies now offer siRNA-delivering mRNA reagents to simplify this process (12). mRNA, which codes for protein, is an essen- tial component of the central dogma of life miRNA (DNA­mRNA­protein). mRNA is tran- miRNA do not code for proteins, as they scribed from a DNA template. mRNA takes belong to specific class of non-coding RNAs. the genetic code from DNA to the ribosome miRNA are 19–25 nucleotides in length and where the mRNA is translated to protein. are encoded within introns (i.e., the portions There has been a significant increase in of the gene sequence that are not expressed in mRNA-based therapies in large part due to the protein) (13). miRNA acts as a guide strand the many advantages that mRNA has over for the RISC complex to its mRNA target in DNA in relation to gene expression and vertebrates. Approximately 30% of genes in the transfer. While RNAi and antisense RNA human genome are regulated by miRNA (14). technologies are used primarily for gene

July 2016 www.biopharminternational.com BioPharm International 33 Peer-Reviewed

silencing, mRNA technologies are often used als, the demand for efficient and cost-effec- in vaccines or gene therapy (15). In both tive manufacturing strategies will grow (19). cases, after injection into the human body, RNA-based biopharmaceuticals are inher- mRNA is translated to protein, which can ently susceptible to endonucleases, so spe- ultimately replace a missing protein (ther- cial handling is required for production apeutic) or induce an immune response and purification. Degradation of product (preventive approach). The production of during manufacturing adds heterogeneity synthetic mRNA for therapeutic use is rela- and chemical instability to the product. tively straightforward, and the challenges Therefore, the manufacturing and purifi- associated with its stability and delivery have cation methods used in RNA-based thera- been tackled through scientific advances in peutics differ from that of DNA and other recent years (16). proteins (20). mRNA purification (post-chemical syn- RNA-BASED VACCINES thesis) includes concentration precipitation, Conceptually, mRNA-based vaccines are a extraction, and chromatographic methods simple approach to inducing an immuno- (including high-performance liquid chroma- logical response by delivering the coding tography) (19). The purpose of the upstream genetic element as a translation-ready mol- concentration and diafiltration step is to ecule. Upon direct vaccination with mRNA concentrate (if lower titer) and change the molecules, dendritic cells (antigen-present- buffer to the necessary pH and conductiv- ing cells) take-up, process, and encode the ity for the first chromatography step. The target antigen, which in turn induces an objective of the final concentration and dia- immune response. Typically, mRNA vaccines filtration step is to de-salt and achieve the are produced by in-vitro synthesis through necessary final concentration prior to ster- an enzymatic process. Such a synthetic pro- ile filtration. A 5-kD membrane cut-off is cess can be tightly controlled, resulting in generally used for concentration and diafil- a quality and predictable product profile. tration in mRNA processes. Because siRNA mRNA can be easily tailored to offer a spe- are smaller than mRNA, a 1-kD membrane cific immunogenic profile and pharmacoki- cut-off is used for adequate retention of the netics (17). mRNA’s stability and antigenic siRNA product (21). properties can be easily manipulated by changing codon or modifying base pairs. CHROMATOGRAPHIC PURIFICATION STEPS Ongoing clinical trials show that mRNA Since the breakthrough discoveries of cat- can be delivered as naked mRNA; immobi- alytic RNAs in the early 1980s and RNA lized on particles or in liposome nanopar- interference in the late 1990s, more than ticle; or transfected in dendritic cells in vitro 50 RNA or RNA-derived therapeutics have resulting in a discernible immune response reached clinical testing. In RNA purifica- and protective efficacy. mRNA can also act tion, despite the different techniques such as an adjuvant and mRNA also has been as arginine-affinity, ion-pairing reversed- explored to stimulate the innate immune phase, or pellicular anion exchange, the system through toll-like receptors (18). RNA- traditional ion-exchange (IEX) media— based vaccines are comparatively simple especially anion exchange (AEX)—remains to produce and can be developed, manu- the most popular technique used in both factured, and administered in a short time pure RNA and RNA packaged for delivery period, therefore, they are suitable for pan- (22, 23, 24, 25). demic situations. Thermostability of mRNA Sm and Sm-like proteins, which can form vaccines can also significantly contribute to heteromeric complexes or bind to vari- their low cost, as they do not require cold- ous RNAs, were proven to contain ancient chain distribution. RNA-binding motifs (Sm domain) with oligo(U) specificity (26). Fractogel TMAE MANUFACTURING (MilliporeSigma), a strong anion-exchange RNA-BASED BIOPHARMACEUTICALS resin, was used for the purification of small As more experimental RNA drugs move nuclear ribonucleoproteins (snRNP). The through the clinic and into large-scale tri- snRNP molecule was eluted with Tris/HCl

34 BioPharm International www.biopharminternational.com July 2016 Peer-Reviewed

and 300 mM NaCl. Ribonucleoprotein and Chemical modification is one method for uncoupled RNA were separated from free pro- hardening the RNA against such enzymatic tein, and the sample was immediately used attacks. Modifications to the molecule can for negatively stained electron microscopy. also increase its target affinity, decrease its Both AEX and reversed-phase (RP) tech- undesired immunogenicity, and improve its nologies are widely used in the RNA puri- overall efficacy. Hardening strategies include fication process. Quarternary amine (Q) modifications to the backbone, sugar, or base and Dimethylaminoethyl (DMAE) chemis- of the RNA molecule. try are among the choices for AEX (27, 28, Conjugation of the RNA therapeutic is 29, 30). One study proved that a few AEX a strategy that is increasingly being used resins can be used for RNA purification for improved delivery and uptake. Alnylam with optimized experimental conditions to Pharmaceuticals has adopted a method of achieve high dynamic binding capacity. In conjugating an amino sugar derivative of this study, among the 18 AEX medias that galactose, N-Acetylgalactosamine (GalNac) were screened, only four resins—Q Sepharose to improve the delivery of siRNA therapies FF (GE Healthcare), POROS 50HQ (Applied to the liver. The GalNac-conjugated siRNA Biosystems), Q Ceramic HyperD F (Pall), and is taken up by asialoglycoprotein receptors Fractogel DEAE (MilliporeSigma)—showed in the liver resulting in a fivefold increase in baseline separations of RNA and plasmid efficacy versus the parent molecule (33). DNA (31). After optimized loading and elut- Arrowhead Research is developing a com- ing conditions, Fractogel DEAE had a wider peting conjugation strategy. Arrowhead’s range of operation, higher dynamic binding delivery technology, termed Dynamic capacity, and complete separation of RNA Polyconjugates (DPCs), is a siRNA bound to in the breakthrough from plasmid in the an endosomolytic polymer backbone via a elute. The high recovery, robustness, and disulfide bond. The endosomolytic polymer reproducibility also met the requirement for enables the quick and efficient release of the large-scale manufacturing. These binding siRNA from the endosome. Arrowhead’s most and elution conditions can be utilized as a recent strategy includes attaching cholesterol starting point for optimal experimental con- to the siRNA and GalNac to the endosomo- ditions in RNA purification. lytic polymer, ensuring they are both deliv- Overall, many biochromatography res- ered to the hepatocytes. The co-injection ins are suitable for RNA purification similar therapy was shown to increase the efficacy to use in other biomolecule separations. In of siRNA-cholesterol 500-fold with a 90% many cases, the Fractogel resins showcased knockdown (34). superior capacity and efficiency, largely due to the “tentacular” structure whereby func- Encapsulation tional groups are located at the end of long The dominant and most-studied strategy for arms grafted to the bead surface, which cir- the delivery of RNA-based therapeutics is cumvent the steric hindrance caused by large lipid-based delivery systems. One successful biomolecules (32). platform is the use of stable nucleic acid lipid particles (SNALPs), which are lipid particles FORMULATION AND DELIVERY formed from a fusogenic lipid, cationic lipid, The most challenging aspect of RNA-based and PEG-lipid mixture. The SNALP deliv- therapeutics is its delivery to target cells. ery system has been developed and champi- Several methods have been explored and oned by Tekmira Pharma; the company now tested in clinical trials. Some of the most refers to it as LNP technology. According to promising approaches are explained in the Tekmira, the LNP “encapsulates siRNAs (also following passages. mRNA) with high efficiency in uniform lipid nanoparticles that are effective in delivering Polymer conjugation/ RNAi therapeutics to disease sites in numer- chemical modification ous preclinical models” (35). Native RNA and RNA-based therapies are Another promising lipid delivery tech- vulnerable to degradation from the many nology is the proprietary Smarticles deliv- ribonucleases found within the cell. ery platform developed by Novosom and

July 2016 www.biopharminternational.com BioPharm International 35 Peer-Reviewed

now owned by Marina Biotech. Similar to 3. EvaluatePharma, World Preview 2015, SNALPs, the Smarticles technology can Outlook to 2020 (8th Edition, June 2015). change their surface charge to facilitate both www.evaluategroup.com/public/reports/ stability and endosomal release. Smarticles EvaluatePharma-World-Preview-2015.aspx, are capable of encapsulating both single- accessed May 24, 2016. and double-stranded nucleic acid therapies. 4. E. Gousseinov et al., Genetic Engineering & Smarticles are comprised of cationic, anionic, Biotechnology News (Sept. 15, 2015), www. and neutral lipids. The negatively charged genengnews.com/insight-and-intelligence/rna- Smarticles avoid the often seen toxic effects based-therapeutics-and-vaccines/77900520/, of positively charged lipids at physiologi- accessed May 24, 2016. cal pH but convert to a positive charge in 5. Ionis Pharmaceuticals (n.d.), www.ionispharma. the acidic environment of the endosome, com/, accessed May 24, 2016. facilitating its release. Other interesting 6. D. Garde, “Merck Bets $100M on Moderna and encapsulation techniques involve PLGA its Pioneering RNA Tech,” www.fiercebiotech.com/ nanoparticles (36, 37). partnering/merck-bets-100m-on-moderna-and-its- pioneering-rna-tech, accessed May 24, 2016. CONCLUSION 7. Moderna Messenger Therapeutics, “Our RNA-based therapeutics are a relatively Core ‘Expression’ Platform: Messenger RNA new class of therapies that has bright pros- Therapeutics,” www.modernatx.com/mrna- pects in the treatment and prevention of expression-platform, accessed May 24, 2016. difficult-to-treat chronic and rare diseases. 8. B. Fidler, “With Massive Venture Round, Moderna RNAi work by interfering with the tran- Has $450M Reasons to Stay Private,” www. scription process, and thereby inhibit pro- xconomy.com/boston/2015/01/05/with-massive- tein translation. Though such a therapeutic venture-round-moderna-has-450m-reasons-to- approach is highly selective and targeted, stay-private/2/, accessed May 24, 2016. special care is required during the produc- 9. UMass Medical School, “How RNAi Works,” www. tion of these therapies and vaccines because umassmed.edu/rti/biology/how-rnai-works, of their susceptibility to ubiquitous RNAse- accessed May 24, 2016. induced degradation. Large-scale manufac- 10. J. Höck and G. Meister, Genome Biol. 9 (2):210. turing of new class of therapeutics would doi:10.1186/gb-2008-9-2-210. Feb. 26, 2008). require bioprocessing components, chemi- 11. Gene Link, “What is RNAi and siRNA?”, www. cals, and tools free from RNAse. Technology genelink.com/sirna/RNAiwhatis.asp, accessed and tool providers need to consider making May 24, 2016. such products available to enable large-scale 12. M. Gujrati and Z.R. Lu, “Targeted Delivery of production of RNA-based therapeutics. The Therapeutic siRNA,” in Gene Therapy of Cancer: surmounting challenges related to poten- Translational Approaches from Preclinical Studies to tial toxicity and drug delivery need to be Clinical Implementation, E.C. Lattime and S.L. Gerson, addressed before such products can be com- Eds. (Academic Press, 3rd ed., 2013), pp. 47–65. mercialized. However, new technologies are 13. Sigma-Aldrich, “miRNA (microRNA) Introduction,” emerging to overcome some of these chal- www.sigmaaldrich.com/life-science/functional- lenges, and the future of RNA-based thera- genomics-and-rnai/mirna/learning-center/mirna- peutics is very promising. introduction.html, accessed May 24, 2016. 14. Qiagen, “MicroRNA–Why Study It and How,” www. REFERENCES sabiosciences.com/pathwaymagazine/pathways7/ 1. Allied Market Research, “RNA Therapeutics microrna.php, accessed May 24, 2016. Market is Expected to Reach $1.2 Billion, 15. R. Scott McIvor, Mol. Therapy 19 (5), pp. 822– Globally, by 2020,” Press Release, www. 823 (2011). prnewswire.com/news-releases/rna-therapeutics- 16. U. Sahin, K. Karikó, Ö Türeci, Nat. Rev. Drug market-is-expected-to-reach-12-billion-globally-by- Discov. 13 (10), pp. 759 –780 (October 2014). 17. T. Kramps and L. Probst, Wiley Interdisp. Rev. 2020---allied-market-research-274471461.html, RNA 4 (6), pp. 737–749 (July 25, 2013). accessed May 24, 2016. 18. S. Pascolo, Handb. Exp. Pharmacol. 183, pp. 2. Personal communication, Donia Slimani, EMD 221–235 (2008). Millipore (now MilliporeSigma). Contin. on page 48

36 BioPharm International www.biopharminternational.com July 2016 Functional Comparability Studies for Biosimilar Development

ON-DEMAND WEBCAST Aired June 21, 2016

Register for free at www.biopharminternational.com/bp/development

EVENT OVERVIEW: Who should attend Regulatory authorities require applicants for biosimilar ■ Laboratory managers therapies to demonstrate that the proposed product is ■ Product development scientists biosimilar to the reference product using analytical and ■ Process development leaders other studies. Comparative analytical testing that evaluates ■ Quality control professionals factors including structure and function can be used to make decisions about the scope of subsequent studies and could result in a shortened clinical development process. Presenters Hoss A Dowlat, PhD This webcast will review key factors in developing, Vice President, optimizing and implementing comprehensive func- Regulatory Affairs, Global Strategy tional comparability studies to provide data needed to PharmaBio Consulting advance the development process. Experts will discuss the regulatory requirements and guidelines for functional Abhi Saharia, PhD comparability studies and the types of studies that should Director, Cell-based be conducted; review approaches to understand multiple Assays and Biologics DiscoverX mechanisms of action and assay development; and the qualification of assays used in these studies. Nicolas Fourrier, PhD Director Biomarker Key learning objectives and Biopharmaceutical Testing ■ Review the regulatory requirements for comparability studies SGS Life Sciences ■ Learn which functional comparability studies should be conducted Moderator ■ Understand the role of reference products for Rita Peters comparability studies Editorial Director ■ Learn about development & qualification of assays for BioPharm International comparability studies

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For questions contact Kristen Moore at [email protected] Cleanroom Standards Revised ISO Cleanroom Standards Improve Air Cleanliness Classification

Jennifer Markarian he International Organization particles, not other factors crucial to for Standardization (ISO) cleanroom qualification, such as smoke Tpublished the long-awaited tests to determine airflow patterns. Revised versions revisions to its standards for The introduction of Part 1 explains, classification and monitoring of air “This part of ISO 14644 specifies classes of ISO 14644 cleanliness in cleanrooms on Dec. 15, of air cleanliness in terms of the num- 2015. ISO 14644-1:2015 “Cleanrooms ber of particles expressed as a concentra- Parts 1 and and associated controlled environments tion in air volume. It also specifies the Part 1: Classification of air cleanliness standard method of testing to deter- 2 introduce by particle concentration” (1) replaces mine cleanliness class, including selec- changes to ISO 14644-1:1999, and ISO 14644- tion of sampling locations” (1). 2:2015 “Cleanrooms and associated con- sampling trolled environments Part 2: Monitoring SAMPLING CHANGES to provide evidence of cleanroom per- The primary changes to Part 1 involve the procedures formance related to air cleanliness by number of samples and the selection of particle concentration” (2) replaces ISO sampling locations. “The number of sam- and monitoring 14644-2:2000. ples will increase from what was required plans for The 2015 editions are the result of a previously,” explains Marsha Stabler systematic review and include changes Hardiman, senior consultant at ValSource. cleanrooms and made in response to requests by users and “The minimum number of samples is now experts in the cleanroom community. In determined from a lookup table (instead clean zones. particular, the requests for reviewing Part 1 of an equation), and that number is set to were related to “the basis for the number of be statistically significant.” sampling locations and, most importantly, According to ISO, the new method the whole statistical basis of classification for selecting the sites and number of of cleanliness using the Student T-test for sampling collections uses a more con- one to nine sampling locations,” notes sistent statistical approach based “where Gordon Farquharson, convenor of the ISO samples are drawn randomly without TC209 working group 1, which performed replacement from a finite population. the review and revisions. Because Part 2 is The new approach allows each location closely aligned with Part 1, the committee to be treated independently with at least reviewed both parts together. a 95% level of confidence that at least 90% of the cleanroom or clean zone CLASSIFICATION BY areas will comply with the maximum PARTICLE CONCENTRATION particle concentration limit for the target The addition of “by particle concen- class of air cleanliness. No assumptions tration” to the title of the standard is are made regarding the distribution of a long-overdue clarification, com- the actual particle counts over the area ments Karen Ginsbury, CEO at PCI of the cleanroom or clean zone; while in Pharmaceutical Consulting. “For years, ISO 14644-1:1999 an underlying assump- I have heard cleanroom contractors and tion was that the particle counts follow practitioners alike wrongly describe ISO the same normal distribution across the 14644-1 and 2 as cleanroom ‘validation’ room” (1). The sampling locations are or ‘qualification’ standards.” She notes to be chosen representatively, meaning that the standards only address airborne that “features such as cleanroom or clean

38 BioPharm International www.biopharminternational.com July 2016 Cleanroom Standards

zone layout, equipment disposition, were formerly defined as ultrafine sources. It is also very important to and airflow systems should be con- particles in ISO 14644-1:1999, but understand the people, material, and sidered when selecting sampling will address these under a new waste flows,” concludes Hardiman. locations” (1). Part 12, notes Farquharson, who Pharmaceutical cleanrooms typi- This representative selection of explains, “These particles are mea- cally already have monitoring plans, sample locations is a big change from sured using a different particle which are required by Annex 1 of the previous random selection, says counter, and industries such as the EU GMPs, says Ginsbury (3). She Hardiman. “A company now has to semi-conductor monitor for con- notes that users should, however, have a rationale and justification for centration of these very small air- check with their cleanroom contrac- sample site location to ensure that borne particles at critical control tors to determine whether a contrac- the sample locations selected are rep- points. These particles are not gen- tor is qualified and familiar with resentative of the characteristics of erally of interest to the pharmaceu- the revisions, because the regulators that section. Companies will have to tical and life sciences industries.” reference the ISO standards and they look at the new number of sample must be followed, says Ginsbury. “I locations and then determine where MONITORING AND TESTING would recommend having the dis- the representative sample locations ISO 14644-2:2015 now requires cussion now so that, by 2017, your will be collected. If contracting out monitoring to provide evidence of contractors and in-house staff are the classification activities, you cleanroom performance, explains fully up to speed and following the should make sure that your contrac- Farquharson. The standard new standard,” she notes. “Even if tor is now using your new, represen- addresses airborne particle concen- you use contractors, the responsi- tative sample locations.” tration, airflow, and device pres- bility for review and approval of sure difference. New topics include their results and compliance with CLASSIFICATION OF MACRO monitoring of critical parameters regulatory standards lies with you. AND NANO-PARTICLES and setting action and alert alarms. ISO may not require 5 μm particles The revision makes a change The revised standard now allows and may let you use risk assessment regarding large (≥ 5 μm) particles, companies to use risk management to determine frequency of classifi- which are required to be measured to set their periodic classification cation. However, EU Annex 1 still for some classifications in the EU testing schedules, notes Hardiman. requires measuring 5 μm particles Annex 1 GMP guidelines (3) and “In the past, the retesting was pre- as part of classification, and current others. “The experts working on scribed in a table and the timing was industry practice (EU [3] and FDA the revision of ISO 14644-1 were based on the ISO class of the clean- [4]) is to perform cleanroom classi- of the opinion that particles ≥ 5 room or clean zone. Now, compa- fication twice a year for aseptic core. μm diameter should not be used nies can put more emphasis on the We haven’t heard the regulators to classify ISO class 5 and cleaner day-to-day data that they generate in on this one but I wouldn’t rush to environments because of the their own facilities to help determine reduce that frequency based on risk uncertainty associated with par- the appropriate testing and frequen- assessment,” cautions Ginsbury. ticle collection efficiency and cies needed for continued cleanroom accuracy of counting low con- compliance. If a company is generat- REFERENCES centrations,” says Farquharson. ing great data and the risk to contin- 1. ISO, ISO 14644-1:2015, Cleanrooms and associated controlled “In order that the European ued cleanroom compliance is low, environments – Part 1: Classification Union (EU), the Pharmaceutical then they can set a longer periodic of air cleanliness by particle Inspection Convention and classification frequency,” explains concentration (Geneva, 2015). Pharmaceutical Inspection Hardiman. She notes that risk-based 2. ISO, ISO 14644-2:2015, Cleanrooms and associated controlled Co-operation Scheme, World sample site selection is crucial for environments – Part 2: Monitoring Health Organization, and Chinese environmental monitoring. “The key to provide evidence of cleanroom GMPs are not left without a clas- is understanding your unique prod- performance related to air cleanliness by sification tool for their Grades A (at ucts and processes and selecting sites particle concentration (Geneva, 2015). 3. EC, EudraLex Volume 4: Good rest and operational) and B (at rest), that best address the risks that your manufacturing practice Guidelines, ISO 14644-1:2015 provides a mech- products and processes present. It is “Annex 1, Manufacture of Sterile anism of extrapolating the macro- important to be able to identify all of Medicinal Products,” (Brussels, 2008). particle descriptor for class limits of the potential contamination sources 4. FDA, Guidance for Industry: Sterile Drug Products Produced 20 and 29 particles ≥ 5 μm.” in each cleanroom and to select by Aseptic Processing—Current The new document does not environmental monitoring sample Good Manufacturing Practice address nano-scale particles, which locations in close proximity to these (Rockville, MD, 2004). ◆

July 2016 www.biopharminternational.com BioPharm International 39 Packaging Trends Raw Materials Packaging Innovations for Biopharmaceutical Manufacturing

Nandu Deorkar iopharmaceutical manufactur- tinuously for anywhere from ing is a complex process involv- 15–35 days, to newer generation single- Bing many unit operations, use technologies, with multiple 2000-L precise amounts of materials, and bioreactors operating in overlapping Recent trends numerous variables. These elements must sequences to achieve similar or greater be addressed properly to ensure consistent productivity. in raw product quality and drug product yield. In general, two kinds of packaging While a strong focus on quality, safety, systems are in use today: Traditional materials and drug efficacy is absolutely essential, steel 100 kg drums with one or two plas- biopharmaceutical manufacturers are tic liners, and smaller cardboard boxes packaging also committed to finding ways to reduce with plastic liners holding 50 kg. Both costs and improve manufacturing efficien- bulk-packaging systems are part of stan- may impact cies. One production step with significant dard practices that most raw materials costs (and potential risk to quality) is the suppliers have established for their sup- manufacturing, buffer and cell-culture materials prepara- ply chain systems. The end user (i.e., tion process: it is labor-intensive, requires the biopharmaceutical producer) typi- quality, and investment in storage and environmental cally orders, receives, and stores enough resources, and involves repeated quality salts, buffers, and other cell-culture cost of assurance (QA) testing as bulk materials powdered materials to last several weeks are subdivided for individual process runs. or months. The material consumption biopharma- New innovations in raw materials rates can be substantial: a typical buf- packaging technologies can directly fer media preparation/usage cycle may ceuticals. impact this process—streamlining oper- consume between 150 and 400 kg of ations, mitigating risks, and contribut- dry products per run, and uses multiple ing to operational excellence (OpEx). In raw materials per cycle. These materials this article, the author examines new are then subdivided by the biopharma- methods of raw materials packaging ceutical producer and used in smaller and how they may lead to manufac- amounts depending on the processes turing efficiencies, elimination of raw they are running. In essence, this bulk material yield losses, reduction in QA material packaging and delivery meth- testing time and costs, and lower costs odology satisfies the raw material sup- in weigh and dispense production. plier’s operational requirements, without full consideration of how that material is TRADITIONAL RAW MATERIAL used by the biopharmaceutical producer. DELIVERY METHODS Nandu Deorkar, PhD, MBA, is Upstream biopharmaceutical processes OPERATIONAL vice-president of R&D for Avantor consume various raw materials, includ- INEFFICIENCIES AND RISKS Performance Materials. He has ing cell-culture media, carbohydrates, Following the more traditional method more than 25 years of experience in amino acids, and buffers, which are typ- described above, the biopharmaceuti- materials technology research and ically supplied in powder form. The bio- cal manufacturer must follow multiple development, and has worked on reactors and medium preparation tanks processing steps to properly manage and various aspects of chemical/polymer that use these materials often oper- use these bulk raw materials. R&D, drug development, formulation, ate around the clock. This operation t
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drug delivery technologies, process includes both large-scale reactors with inventoried in storage areas that development, and technology transfer. 10,000 L capacity that run con- must have the appropriate temper-

40 BioPharm International www.biopharminternational.com July 2016 Packaging Trends

ature and humidity controls process of caking when free mois- kept in storage and the number of to maintain the material’s ture migrates onto the surface of times a container was subdivided. integrity. the crystals and dissolves a small Subdividing was still required, how- t
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result in complete solidification of control on process parameters to the drum are subdivided by the entire package. Caked materi- protect the final drug’s safety and hand according to manufactur- als must be completely broken up quality, and finding ways to strictly ing requirements. For example, in order to measure out the precise control the amount of raw mate- if 45 kg are initially required, amounts needed for bioreactor pro- rial that goes into bioreactors can that quantity is removed and the cesses. This is a time-consuming enhance operational excellence and remainder is put back into stor- manual activity with an open con- process yield. age. For a 2000-L bioreactor, a tainer, which is at risk for cross- manufacturer may need to sub- contamination and absorption of NEW PACKAGING FOR PRE-WEIGHED, divide a 100 kg drum of material additional moisture, potentially FREE-FLOWING MATERIALS between two and five times. extending the problem. This prac- Innovative chemicals suppliers t
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July 2016 www.biopharminternational.com BioPharm International 41 Packaging Trends

Figure 1. A common problem with packaging, storing and sub-dividing powdered the bag to obtain a product sample. materials in bulk containers is material caking or clumping. The tailgate sample process must be validated to ensure that the material in the tailgate sample is equivalent to the materials packed in the bag. DIRECT DISPENSE SYSTEMS: TIME AND COST SAVINGS Expanding the use of these direct dispense systems can help advance operational excellence initiatives and reduce costs within the bio- pharmaceutical industry’s supply chain. By adopting flexible manu- facturing technology, such as the direct dispense systems, biophar- maceutical manufacturers may be able to reduce their operating costs in certain areas by up to 40%. There are multiple savings associ- ated with the use of these systems: t
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The inner layer uses a vapor per- In addition to near Raman test- Raman testing and tailgate sam- meable material already in use in ing, some suppliers also will pro- ples greatly simplifies the testing/ other container-lining applica- vide a tailgate sample along with validating step. Products do not tions. Any moisture that develops the bags. Biopharmaceutical pro- have to be re-validated each time within the bag passes through this ducers that are required to conduct material is sub-divided from a material and is controlled by the full analyses of all materials used in bulk container. In addition, the

desiccant, to maintain the cor- their processes don’t need to open primary packaging material typ- ALL FIGURES ARE COURTESY OF THE AUTHOR

42 BioPharm International www.biopharminternational.com July 2016 Packaging Trends

ically used for these bags is the Figure 2. To help ensure a free-flowing dispensing system and eliminate same as standard drum liners, so clumping, some bags have outer and inner layers with special desiccant materials contact material does not have to installed between the two layers. The inner layer uses a vapor permeable material; be re-validated. any moisture that develops within the bag passes through this material and is t
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tal, health, and safety practices, BLANKING CAP as employees no longer need to engage in the potentially unsafe 4” TC CLAMP practice of breaking up clumps that can form in large drums. Figure 3. With new developments in raw material packaging, even after four t
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1SF weeks at 40 °C and 90% relative humidity, the material in this 10 kg bag remains weighed amounts in direct dis- free-flowing. pense bags that match specific biopharmaceutical process requirements eliminate the need to buy and store material in bulk, reducing overages, out-of-date materials, and disposal costs. ADVANCES IN BULK MATERIAL PACKAGING FOCUS ON IMPROVING OVERALL OPEX By evolving raw material packag- ing and delivery options to align more closely with the operational requirements of biopharmaceutical producers, raw materials suppli- ers are helping to eliminate inef- ficiencies and drive down costs within the overall supply chain and production environment. The success of these bag-based, pre- weighed, free-flowing direct dis- to deliver ready-made liquid solu- solutions are more complex than pense systems is also encouraging tions to customers, which would solids, there are opportunities to the development of streamlined eliminate the biopharmaceutical apply innovations to raw-materi- systems for other types of materi- manufacturing step of taking solid als packaging designs to improve als beyond bulk powders. Some materials and creating solutions. the efficiency, productivity, safety, raw-materials suppliers are inves- While the transportation and and quality of biopharmaceutical tigating new packaging methods storage considerations for liquid manufacturing. ◆

July 2016 www.biopharminternational.com BioPharm International 43 Troubleshooting

Design and Qualification of Single-Use Systems The author provides a review of the concepts of design and qualification that apply to single-use systems.

ingle-use technologies (SUT) have made sig- beyond the official or other established require- Snificant inroads in biopharmaceutical and ments” (1). While not specifying those require- vaccine manufacturing. Greater adoption in ments, this requirement highlights the need for the years to come can be anticipated, given their SUT equipment designers to consider operational broad use in pre-clinical and clinical manufactur- performance criteria, as well as potential chemical ing and expanding use for approved therapeutics. interactions and equipment-derived impurities or With increasing regulatory oversight of SUT pro- particulate contaminants that may be introduced cesses, it’s worthwhile to review basic concepts of into applicable processes and potentially impact design and qualification that apply to single-use intermediates or final dosages. components and systems (SUS). Much of SUT use occurs in therapeutic pro- tein API production. International Council EQUIPMENT DESIGN on Harmonization (ICH) Q7, European REGULATIONS AND GUIDANCE Medicines Agency (EMA) Q7, and FDA Q7A While drug and vaccine manufacturers are sub- Good Manufacturing Practice Guidance for Active ject to regulatory review and inspection of how Pharmaceutical Ingredients incorporate essen- equipment is used, that is not the case for the tially the same design requirement statement: manufacturers of process equipment, including “Equipment should be constructed so that surfaces SUT components and systems, even when sold for that contact raw materials, intermediates, or APIs use under good manufacturing practice (GMP). do not alter the quality of the intermediates and Suitability of design and qualification for use must APIs beyond the official or other established speci- be determined by the therapeutic manufacturer. fications” (2–4). The US Code of Federal Regulations (21 CFR 211.63), Fortunately, established SUT manufacturers Section 211.63 states, “Equipment used in the have significant experience working with drug and manufacture, processing, packing, or holding of a vaccine companies and have learned to adopt SUT drug product shall be of appropriate design, ade- equipment designs to meet user needs in GMP- quate size, and suitably located to facilitate opera- regulated processes. Adoption of established good tions for its intended use and for its cleaning and engineering practices for design and deployment maintenance” (1). While this is the responsibility of SUTs is crucial and requires close collaboration of the user, equipment manufacturers need to between users, component designers and system understand and design to user requirements for engineers having experience in polymer materials, what may be considered appropriate, device manufacturing, human interface engineer- adequate, or suitable. ing, and other related practices (5). US 21 CFR does provide some information for process equipment SUT DESIGN CONSIDERATIONS designers: Section 211.65, paragraph Design considerations for SUT manufacturers can (a) states, “Equipment shall be con- be divided into six categories: structed so that surfaces that contact t
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components, in-process materials, or and performance of the materials of construc- drug products shall not be reactive, tion and finished component under antici- Jerold M. Martin is an independent additive, or absorptive so as to alter pated use conditions, with reasonable safety consultant and Chairman Emeritus, the safety, identity, strength, qual- factors. These properties can include pressure

BPSA, [email protected]. ity, or purity of the drug product requirements (e.g., operating, burst, creep, pres- Demidoff/GettySveta Images

44 BioPharm International www.biopharminternational.com July 2016 Troubleshooting

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as integrity (i.e., retention prop- (OQ) (i.e., does it operate according requirements (e.g., operating, erties of filters, leak absence of to the manufacturer’s specifications?), thermal sterilization, melt, cryo- containers, connector and hose fit- and performance qualification (PQ) genic, impact of temperature on ments, microbial barrier, etc.) bio- (i.e., does it consistently perform pressure performance), mechani- burden, endotoxins, and particle to meet the user’s requirements?). cal properties (e.g., flexibility, contamination or generation. Additional terms that combine these rigidity, and tensile strength), t
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July 2016 www.biopharminternational.com BioPharm International 45 Product & Service Innovations Advertorial

Distek, Inc.

Company Description Distek, Inc., headquartered in North Brunswick, NJ, is a leading manufacturer of laboratory testing instruments for the pharmaceutical and biotechnology industry, as well as an experienced provider of validation and qualification services. Distek’s robust product portfolio includes water bath and bathless dissolution, dissolution media heating, degassing, dispensing and disposal, in-situ fiber optic UV, bathless tablet disintegration, content uniformity, digital video monitoring, programmable automated sampling, and now a single-use bioreactor system for mammalian cell culture applications. Founded in 1976, Distek has an excellent reputation for innovation, product reliability, and customer support, and we believe that these, as well as continual improvement, are essential to our current and future growth and crucial to ongoing success. use bioreactor. Simply remove your existing headplate For technological advancements that offer reliable and place the preassembled and irradiated Distek BIOne and consistent performance, the pharmaceutical industry System into the glass vessel. The “patent pending” trusts Distek. bioreactor liner will mold to and mimic your existing glass system allowing you to continue using your existing Markets Served cabinet, probes, motor, heating jacket and recipes. Since Distek’s core market includes brand name and generic the liner conforms to the dimensions and aspect ratio pharmaceutical drug manufacturers, CROs, CMOs, of your existing vessel, there is no need to change your Nutraceuticals, government agencies, and universities. process. All materials are USP Class VI, animal derivative To ensure consistent quality and to meet the international free and utilized in existing single-use products. standards of our world market, Distek has been ISO certified since 2002. Services & Innovations Distek does not simply design ‘me too’ products. Distek instruments are considered to be highly innovative, which is exemplified in the product portfolio. Distek engineers technically modern and user-friendly instruments that have provided many innovative features to customers, worldwide. Distek holds several instrumentation and accessory patents and has been ISO certified since 2002. Distek’s robust product portfolio includes water bath and bathless dissolution, dissolution media heating, degassing, dispensing and disposal, in-situ fiber optic UV, bathless tablet disintegration, content uniformity, digital video monitoring, programmable automated sampling, Distek, Inc. and now a single-use bioreactor system for mammalian cell 121 North Center Drive culture applications. North Brunswick, NJ 08902 Phone: 732.422.7585 Major Product Innovations Fax: 732.422.7310 You don’t need to make a large capital investment to www.distekinc.com convert your existing benchtop glass bioreactor to a single- Email: [email protected]

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Contin. from page 45 qualification of extractables, steril- REFERENCES ization, and particulates testing (7). 1. FDA, US Code of Federal Regulations (21 the user requirements specifica- Additional tests for SUS qualification CFR 211.63), Section 211.63, Current Good Manufacturing Practice for Finished tion should be verified during the have been described by users (8–12). Pharmaceuticals (FDA, April 2014). design qualification” (6). The United States Pharmacopeial 2. ICH, Q7 Good Manufacturing Practice Convention (USP) has published a Guide forActive Pharmaceutical Ingredients (ICH, November 2000). SUPPLIER QUALITY SYSTEM draft standard on extractables test- 3. EMA, Note for Guidance on Good AND QUALIFICATION PRACTICES ing for plastic process equipment Manufacturing Practice for Active SUT and SUS are unique from tra- (13). A BPSA guide on SUS leak test- Pharmaceutical Ingredients (CPMP/ ICH/4106/00), November 2000. ditional stainless equipment in that ing is also under development. In 4. FDA, Guidance for Industry, Q7A Good the user depends on the supplier’s addition, BPSA provides an indus- Manufacturing Practice Guidance for Active quality system. Review of the suppli- try consensus-generated Quality Pharmaceutical Ingredients, August 2001 5. M. Botterill and B. Rawlings, BioProcess er’s quality system should be part of Agreement Template that can be Int. (December 2008). supplier qualification. Furthermore, used to document accountabili- 6. EMA, EU Guidelines for Good users cannot normally perform ties (7), and audits should be used Manufacturing Practice for Medicinal Products for Human and Veterinary Use, incoming testing prior to using SUSs to review these under the supplier’s Annex 15: Qualification and Validation, intended for implementation in pro- quality management system. February 2014. duction, so are also more dependent Also important for SUS users is 7. BPSA, Component Quality Test Matrices, Gamma Irradiation and Sterilization, on suppliers’ product qualification to qualify the SUT equipment sup- Extractables and Leachables, Particulates, practices. plier’s reliability and delivery his- Quality Agreement Template, www. Supplier qualification of SUT tory. Potential supplier performance bpsalliance.org 8. PDA, Technical Report No. 66,Application products can be summarized in can be assessed by the experience of Single-Use Systems in Pharmaceutical product qualification and process of integrators and users who have Manufacturing (PDA, 2014). validation reports suitable for user 9. D. M. Stephenson, J. Val. Technol. previously worked with them. (February 2003). documentation and submittal to Attendance at industry meetings, 10. M. A. Petrich, Amer. Pharm. Rev. 16 (7) regulatory authorities. Applicable participation on scientific asso- (November/December 2013). 11. W. Ding, BioPharm Int. 28 (9) (September test methods for pre-use qualifica- ciation SUS committees, or trade 2015) pp. 32–39. tion are detailed in Bio-Process associations such as BPSA pro- 12. D. Riedman and J. Martin, BioProcess Int. Systems Alliance (BPSA’s) Quality Test vide important access to suppli- 9 (S2) (2011) pp. 28-35. Reference Matrices 13. USP, <661.3> (draft): Plastic Systems document, along ers, integrators, and users to aid in Used for Manufacturing Pharmaceutical with supplemental BPSA guides EFUFSNJOJOH
BQQSPQSJBUF
TVQQMJFS Products, USP—Pharmacopoeal Forum with specific recommendations on integrator qualification practices. 42(3) (USP, March 2016). ◆

Peer-Reviewed—Contin. from page 36

19. R. Martins, J.A. Queiroz, and F. Souza, J. Chrom. A 1355, 30. W.H. Pan et al., Mol. Ther. 9 (4), pp. 596–606 (April 2004). (August 2014). 31. A. Eon-Duval et al., J. Chrom. B Analyt. Technol. Biomed. Life 20. T. Schlake et al., RNA Biol. 9 (11), pp. 1319–1330 (Nov. 1, 2012). Sci. 804 (2), pp. 327–335 (2004). 21. Personal communication with BSN team, EMD Millipore (now 32. I. Theodossiou, M. SØndergaard, and O.R. Thomas, MilliporeSigma). Bioseparation 10 (1–3), pp. 31–44 (2001). 22. R. Martins, J.A. Queiroz, and F. Sousa, Anal. Bioanal. Chem. 33. J.K. Nair et al., J. Am. Chem. Soc. 136 (49), pp. 16958– 405 (27), pp. 8849–8858 (November 2013). 16961 (Dec. 10, 2014). 23. P.M. Swiderski et al., Anal. Biochem. 216, pp. 83–88 (1994). 34. S.C. Wong et al., Nucleic Acid Ther. 22 (6), pp. 380–390 24. J.R. Thayer et al., Chrom. Today, (March 2011), www. (December 2012). chromatographytoday.com/article_read/985/, accessed 35. Arbutus Biopharma, “Tekmira Presents Recent Advances in May 24, 2016. mRNA Delivery at Scientific Symposium,” Press Release, Feb. 25. F. Wincott et al., Nucl. Acids. Res. 23 (14), pp. 2677 –2684 (1995). 25, 2014, http://investor.tekmirapharm.com/releasedetail. 26. T. Achsel, H. Stark, and R. Luhrmann, Proc. Natl. Acad. Sci. cfm?ReleaseID=827952, accessed May 24, 2016. U.S.A. 98 (7), pp. 3685–3689 (March 27, 2001). 36. P. Pantazis et al., “Preparation of siRNA-Encapsulated PLGA 27. C. Miller et al., Mol. Syst. Biol. 7 (458), doi: 10.1038/ Nanoparticles for Sustained Release of siRNA and Evaluation of msb.2010.112 (Jan. 4, 2011). Encapsulation Efficiency,” in Nanoparticles in Biology and Medicine: 28. L.E. Easton et al., RNA 16 (3), pp. 647–653 (2010). Methods and Protocols, Methods in Molecular Biology, M. Soloviei, 29. W.J. Issa et al., “Ion Exchange Purification of mRNA,” US Ed. (Springer Science+Business Media, 2012), pp. 311–319. patent WO2014144767 A1, Sept. 18, 2014. 37. M. Chen et al., ACS Nano 6 (6), pp. 4835–4844 (2012). ◆

48 BioPharm International www.biopharminternational.com July 2016 New Technology Showcase

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July 2016 www.biopharminternational.com BioPharm International 49 BIOLOGICS NEWS PIPELINE

IN THE PIPELINE

Using a New CRISPR Effector to Edit RNA Study: HIV-1 Neutralizing Antibodies A naturally occurring CRISPR (clustered regularly in Infants May Impact Vaccine Development interspaced short palindromic repeats) system that spe- A new article in Cell that studied HIV-neutralizing antibod- cifically can be used to modify the RNA of an organ- ies in an infant may have important implications in the ism is the newest development in the technology’s development of an HIV vaccine. The novel study exam- evolution. A new study, published in Science, identifies ined broadly neutralizing antibodies (bnAbs) in a Nairobi C2c2, a bacterial protein that can be used as a tool infant prior to receiving antiretrovirals and approximately to cleave single-stranded RNA sequences at desired one-year post infection. Researchers isolated and character- locations. As an RNA-guided RNAse, C2c2 can be har- ized infant HIV-1 neutralizing antibodies in order to fur- nessed to defend against viral intruders and turn off ther understand their impact on the HIV virus. gene expression of certain conditions, according to Multiple studies have been done on HIV-1 bnAbs in researchers. adults, but little is known about infant bnAbs contributing Typically, gene silencing is a gene expression manip- to broad plasma responses. While a subset of adults with ulation performed by small interfering RNA (siRNA), HIV-1 develop bnAbs, these antibodies exhibit high levels but the study researchers attest that the C2c2 complex of somatic hypermutation (SHM) and only neutralize the is even more efficient when it comes to editing RNA. disease after years of affinity maturation. By comparison, Specifically, C2c2 can be used to add to or delete infor- infant’s bnAbs develop broad responses early, and “HIV-1- mation from existing RNA sequences, and can also be specific neutralization breadth can develop without pro- used to tag RNA to learn more information about the longed affinity maturation and extensive SHM.” Therefore, function of certain sequences. In addition, C2c2 can infants may have the ability to neutralize the disease more be programmed to knock out certain messenger RNA quickly than adults. (mRNA), disrupting the DNA-->mRNA-->protein tran- “Most studies of adults have looked at responses many scription process. C2c2 requires only a single RNA to years after infection and these have suggested that broad function, and is “genetically encodable,” the researchers and potent HIV neutralizing antibodies take years to said in a Broad Institute/Harvard press release accompa- develop and that they require extensive hypermutation to nying the study. As an RNA-targeting immune system be effective,” study author Julie Overbaugh, PhD, member mechanism, they say C2c2 is a promising tool for future of the Human Biology Division at the Fred Hutchinson RNA manipulation. Cancer Research Center, told BioPharm International. “This The authors concluded that the CRISPR-C2c2 complex is the first example of broadly neutralizing antibodies is probably not the only system that can be programmed detected this early in infection. These infant antibodies to alter RNA sequence, and other, patentable editing tools have much less hypermutation and yet, they are nonethe- may be on the horizon. They wrote, “It is likely that other, less broad and potent.” broadly analogous Class 2 RNA-targeting immune systems Overbaugh notes the infant studied had a polyclonal exist, and further characterization of the diverse members response to the virus, meaning, the infant’s antibodies of Class 2 systems will provide a deeper understanding of targeted multiple epitopes, making it more difficult for the bacterial immunity and provide a rich starting point for virus to survive. This presents an interesting comparison the development of programmable molecular tools for in to studies done on adult antibodies, which target only one vivo RNA manipulation.” dominant epitope, Overbaugh says. Although the research on CRISPR-C2c2 was conducted The challenge is now finding ways to harness these by various teams at MIT, the Broad Institute, Harvard unique attributes of infant bnAbs for practical applications University, the National Institutes of Health, Rutgers such as vaccine development. The goal, researchers say, University-New Brunswick, and the Skolkovo Institute is to better understand infant bnAbs that develop early of Science and Technology, the Broad Institute’s Feng in natural infection in order to develop vaccines that can Zhang is at the helm of the research and serves as one of elicit neutralizing antibodies more quickly. the paper’s senior authors. Zhang is notorious for being “Infants mount a more rapid potent antibody one of the founders of the CRISPR-Cas9 complex and its response to HIV than adults—understanding how they applications for editing DNA, however, there is currently do this is important, both in terms of how they do it an ongoing legal battle between the Broad Institute and more rapidly and whether there are fundamental dif- the University of California (involving work by researcher ferences in the pathway,” said Overbaugh. “Adults take Jennifer Doudna and by French researcher Emmanuelle a long time to develop these responses [naturally], lon- Charpentier) for the patent rights to the technology. ger than is possible to imagine for a vaccine regimen.”

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