ANALYTICAL METHOD DEVELOPMENT and VALIDATION Volume 27 6 Number

Volume 27 Number 6

BioPharm International BioPharm INTERNATIONAL

BioPharm June 2014 The Science & Business of Biopharmaceuticals

JUNE 2014 JUNE www.biopharminternational.com

SYNTHETIC PEPTIDES I FINISHED PRODUCT MONOGRAPHS I PROCESSING DOWNSTREAM

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION Volume 27 6 Number

PEER-REVIEWED OUTSOURCING LOT-RELEASE TESTING CONCENTRATING FEED— OUTSOURCING ASSURING EQUIVALENCY AN APPLICABLE APPROACH TO NO LONGER JUST OF ALTERNATIVE LOT- IMPROVE ANTIBODY PRODUCTION FOR COST-CUTTING RELEASE TEST METHODS

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EDITORIAL EDITORIAL ADVISORY BOARD Editorial Director Rita Peters [email protected] BioPharm International’s Editorial Advisory Board comprises distinguished Managing Editor Susan Haigney [email protected] specialists involved in the biologic manufacture of therapeutic drugs, Scientific Editor Adeline Siew, PhD [email protected] diagnostics, and vaccines. Members serve as a sounding board for the editors and advise them on biotechnology trends, identify potential Community Editor Melanie Sena [email protected] authors, and review manuscripts submitted for publication. Art Director Dan Ward [email protected] Contributing Editors Jill Wechsler, Jim Miller, Eric Langer, Anurag Rathore, Jerold Martin, Simon Chalk, and Cynthia A. Challener, PhD K. A. Ajit-Simh Jerold Martin Correspondents Hellen Berger (Latin & South America, hellen. President, Shiba Associates Sr. VP, Global Scientific Affairs, [email protected]), Jane Wan (Asia, [email protected]), Biopharmaceuticals Rory Budihandojo Pall Life Sciences Sean Milmo (Europe, [email protected]) Director, Quality and EHS Audit ADVERTISING Boehringer-Ingelheim Hans-Peter Meyer VP, Special Projects Biotechnology Publisher Mike Tracey [email protected] Edward G. Calamai Lonza, Ltd. National Sales Manager Steve Hermer [email protected] Managing Partner East Coast Sales Manager Scott Vail [email protected] Pharmaceutical Manufacturing K. John Morrow President, Newport Biotech European Sales Manager Chris Lawson [email protected] and Compliance Associates, LLC Senior Sales Executive Christine Joinson [email protected] David Radspinner Suggy S. Chrai Market Development, Classifieds, and Global Head of Sales—Bioproduction President and CEO Recruitment Tod McCloskey [email protected] Thermo Fisher Scientific The Chrai Associates Direct List Rentals Tamara Phillips [email protected] Reprints 877-652-5295 ext. 121/ [email protected] Tom Ransohoff Leonard J. Goren Vice-President and Senior Consultant Outside US, UK, direct dial: 281-419-5725. Ext. 121 Global Leader, Human Identity BioProcess Technology Consultants PRODUCTION Division, GE Healthcare Anurag Rathore Production Manager Jesse Singer [email protected] Uwe Gottschalk Biotech CMC Consultant AUDIENCE DEVELOPmENT Vice-President, Faculty Member, Indian Institute of Purification Technologies Technology Audience Development Rochelle Ballou [email protected] Sartorius Stedim Biotech GmbH Susan J. Schniepp Fiona M. Greer Vice-President Global Director, Quality and Regulatory Affairs BioPharma Services Development Allergy Laboratories, Inc SGS Life Science Services Tim Schofield Rajesh K. Gupta Managing Director Vaccinnologist and Microbiologist Arlenda, USA

Joe Loggia, Chief Executive Officer; Tom Florio, Chief Executive Officer Jean F. Huxsoll Paula Shadle Fashion Group, Executive Vice-President; Tom Ehardt, Executive Vice- Principal Consultant, Senior Director, Quality President, Chief Administrative Officer & Chief Financial Officer; Shadle Consulting Product Supply Biotech Georgiann DeCenzo, Executive Vice-President; Chris DeMoulin, Executive Bayer Healthcare Pharmaceuticals Vice-President; Rebecca Evangelou, Executive Vice-President, Business Alexander F. Sito Systems; Julie Molleston, Executive Vice-President, Human Resources; President, Denny Kraichely Tracy Harris, Sr Vice-President; Dave Esola, Vice-President, General BioValidation Associate Director Manager Pharm/Science Group; Michael Bernstein, Vice-President, Legal; Francis Heid, Vice-President, Media Operations; Adele Hartwick, Johnson & Johnson Michiel E. Ultee Vice-President, Treasurer & Controller Chief Scientific Officer Stephan O. Krause Laureate BioPharmaceutical Services, Inc. Principal Scientist, Analytical ©2014 Advanstar Communications Inc. All rights reserved. No part of this publication may Biochemistry, MedImmune, Inc. Thomas J. Vanden Boom be reproduced or transmitted in any form or by any means, electronic or mechanical including Vice-President, Global Biologics R&D by photocopy, recording, or information storage and retrieval without permission in writing from Steven S. Kuwahara Hospira, Inc. the publisher. Authorization to photocopy items for internal/educational or personal use, or the internal/educational or personal use of specific clients is granted by Advanstar Communications Principal Consultant Inc. for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. GXP BioTechnology LLC Krish Venkat Danvers, MA 01923, 978-750-8400 fax 978-646-8700 or visit http://www.copyright.com online. CSO For uses beyond those listed above, please direct your written request to Permission Dept. fax Eric S. Langer AnVen Research 440-756-5255 or email: [email protected]. President and Managing Partner Advanstar Communications Inc. provides certain customer contact data (such as customers’ BioPlan Associates, Inc. Steven Walfish names, addresses, phone numbers, and e-mail addresses) to third parties who wish to promote Principal Statistician relevant products, services, and other opportunities that may be of interest to you. If you do not BD want Advanstar Communications Inc. to make your contact information available to third parties for Howard L. Levine marketing purposes, simply call toll-free 866-529-2922 between the hours of 7:30 a.m. and 5 p.m. President Gary Walsh CST and a customer service representative will assist you in removing your name from Advanstar’s BioProcess Technology Consultants lists. Outside the U.S., please phone 218-740-6477. Professor

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magentablackcyanyellow ES445006_BP0614_003.pgs 05.24.2014 02:17 ADV INTERNATIONAL BioPharm International integrates BioPharm the science and business of biopharmaceutical research, development, Contents and manufacturing. We provide practical, peer-reviewed technical solutions Volume 27 Number 6 June 2014 to enable biopharmaceutical professionals to perform their jobs more effectively. FEATURES AnAlyticAl Method lot-releAse testing developMent And vAlidAtion Assuring Equivalency of Challenges in Analytical Method Alternative Lot-Release Test Methods Development and Validation Cynthia A. Challener Susan Haigney New test methods can provide improved quality and efficiency, Experts give insight on method transfer, QbD, and regulations but they must be validated to demonstrate equivalency. 38 for analytical method development and validation for biopharmaceuticals. 18 synthetic peptides Control Strategies for Synthetic Therapeutic peer-reviewed Peptide APIs Part III: Manufacturing Process Concentrating Feed—An Applicable Considerations Approach to Improve Antibody Production Ivo Eggen, Brian Gregg, Harold Rode, Ping Xu, Xiao-Ping Dai, Albert Kao, Rosario Scott, and Reb Russell Aleksander Swietlow, Michael Verlander, and Anita Szajek In this study, the authors evaluated different approaches to USP’s Therapeutic Peptides Expert Panel discusses manufacturing prepare highly concentrated feed media for fed-batch Chinese processes and impurity control for synthetic peptide APIs. 42 hamster ovary cell culture. 24 downstreAM processing Biopharma Moves to Integrated, Single-Use, Downstream Processing Cynthia A. Challener Suppliers see challenges to the adoption of single-use technologies for downstream processing as opportunities. 34

COLUMNS AND DEPARTMENTS

6 Guest Editorial 14 Inside EDQM 48 Product Spotlight Protecting global intellectual The European Pharmacopoeia property rights is vital to Commission re-evaluates 49 New Technology Showcase biopharmaceutical innovation. its policy on the development Jim Greenwood of monographs for 49 Ad Index/Calendar finished drug products. 50 Biologics News Pipeline 8 Global News Susanne Keitel 12 US Regulatory Beat 16 Perspectives on Outsourcing Regulators and industry With budgets growing, clients organizations explain policies see CMOs’ costs as less crucial. and standards to manufacturers Eric Langer and authorities in all regions. Jill Wechsler

Cover: Fuse/Getty Images

BioPharm International is selectively abstracted or indexed in: • Biological Sciences Database (Cambridge Scientifc Abstracts) • Biotechnology and Bioengineering Database (Cambridge Scientifc Abstracts) • Biotechnology Citation Index (ISI/Thomson Scientifc) • Chemical Abstracts (CAS) • Science Citation Index Expanded (ISI/Thomson Scientifc) • Web of Science (ISI/Thomson Scientifc)

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Bureaucratic Roadblocks Threaten Biopharma Growth here is a clear link between a country’s rate of economic development and Tthe strength of its intellectual property laws. This is particularly true in knowledge-intensive sectors such as biopharmaceuticals. The good news is that some mature and emerging economies are making growing use of patent systems to facilitate biotechnology research and commercialization. The bad news is that a number of countries have established bureaucratic and burdensome hurdles to patentability. This reality stands to harm innovation everywhere. The Biotechnology Industry Organization (BIO) is concerned over a series of patent revocations including a decision by India’s Supreme Court to deny patent protection for a novel therapy on the grounds it did not demonstrate enhanced Jim Greenwood is president efficacy. Brazil’s unprecedented review of patents by the health regulatory and CEO of the Biotechnology authority, China’s increased data requirements, and Canada’s increased utility Industry Organization (BIO). requirements all ultimately undermine the drug development and patenting process and may impede the delivery of novel medicines to market. Despite the 2010 declaration by the then-President of India that the next 10 years will be a “Decade of Innovation,” the use of a compulsory license, a series Protecting global of patent revocations, and weak enforcement efforts raise serious concerns about India’s commitment to promote innovation. intellectual property The tragedy underlying this anti-intellectual property position is not simply that it diverts attention away from the real problems of access to healthcare that rights is vital to millions of Indians face, but that it undermines the nation’s goal of becoming a healthcare and science innovator. There are companies around the world inter- biopharmaceutical ested in collaborating in research, science, and the development of medicines in India. India’s anti-intellectual property position makes it difficult, and often innovation. impossible, for such deals to happen. It is imperative for governments that want to foster a robust biotech innovation environment to create a set of strong intellectual property standards—particularly those governing data protection, patents, and trade secret protection—that are relevant to biological products. The biotechnology industry is a dynamic, job-creating industry and presents opportunities for every country. Indeed, the vast majority of biotechnology companies are small- and medium-sized enterprises. What these companies share is a philos- ophy that is crucial to the task of developing biotechnology products—a willingness to take huge risks and invest private capital in development of new technologies that will lead to new products and services that will improve people’s lives. With no source of product revenue and long development times before market launch, biopharmaceutical companies must leverage the strength of their global patent portfolio to raise the large amounts of capital required to get their innovations to the market. Upholding the system of intellectual property rights is essential to guarantee- ing future innovation and future jobs not just for US biopharmaceutical companies, but also for other countries and other industries around the world. BIO believes that strong intellectual property laws create an environment that promotes collaboration and innovation across borders. We will continue to work around the world to champion intellectual property rights as a vital means of creating jobs, saving lives, advancing global economic growth, and generating breakthrough solutions to global challenges. The 2014 BIO International Convention, taking place June 23-26 in San Diego, will host an Emerging Opportunities in Global Markets Forum specifically designed to highlight biotech initiatives in global markets, looking at both emerging economies and global initiatives and trends that affect the industry’s landscape. The Forum will overview developments in key markets, cross-cutting policy trends, unique collaborations, and cross border initiatives to further innovation. To learn more, visit convention.bio.org. ◆

6 BioPharm International www.biopharminternational.com June 2014

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magentablackcyanyellow ES446151_BP0614_007_FP.pgs 05.28.2014 03:13 ADV magenta black cyan yellow Though the Bruneian pharmaceutical sector is at its infancy, it it infancy, is at its sector pharmaceutical Bruneian the Though Though this has attracted the attention of international international of attention the this attracted has Though There are no personal income tax, as well as export, sale, payroll, payroll, sale, as export, as well tax, income personal no are There and sultanate’s biodiversity the rich identified has government The 8 Affairs issued the guidelines for the manufacturing and and manufacturing the for guidelines the issued Affairs (CAGR) of 9% between 2013(CAGR) between 9% of 2018, and US$150 of avalue reaching (R&D) and training grants, as well as tax incentives and exemption exemption and incentives as tax as well grants, (R&D) training and stable most world’s as the Recognized says. Singapore (PwC) Simpor Pharma says. In addition, the country is also tapping tapping is also country the addition, In says. Pharma Simpor which offers an excellent platform for the exploitation of rich rich of exploitation the for platform excellent an offers which with universities and relevant departments to explore what what to explore departments relevant and universities with US$100 2013 in million rate growth annual acompound with scientists, more efforts will have to be put into inviting experts experts into inviting have put will to be efforts more scientists, products. forest useful other and plants medicinal of sources such as Azerbaijan, Iran, Yemen, and United Arab Emirates (UAE). Emirates Yemen, Arab Iran, United and as Azerbaijan, such and manufacturing taxes (1). Africa, Europe, in it be community Muslim the amongst acceptance of key manager accounts Rumpun, Paul Bin Jason advantage, the country, and Brunei to be the hub for halal pharmaceutical pharmaceutical halal for hub the to be country,Brunei the and to Business Monitor International (BMI), the sector is valued at at is valued (BMI), sector the International Monitor to Business to five months. Rumpun adds, “The halal brand will gain strong strong gain will brand halal “The adds, Rumpun months. to five a$26 In government. the million from certification halal full the collaboration in studies more conducting and country to the Brunei possesses ‘pull’ factors such as political stability and low low and stability as political such factors ‘pull’ possesses Brunei handling of halal medicinal products, thus setting the stage stage the thus setting products, medicinal halal of handling 2010, In market. Religious of halal growing into the Ministry the Pharmaceutical Market Growth Market Pharmaceutical Brunei’s Potential exports, Cher Boon Piang, analyst of BMI Asia, says. says. Asia, BMI of analyst Piang, Boon Cher exports, joint venture between Canadian-based Viva Pharmaceutical and and Pharmaceutical Viva Canadian-based between venture joint industry’s to the used be can they how and there is out capsules for exports by February 2014, by February three in to them ship aiming exports for capsules corporate income corporate The tax. income rate tax has been to investors overseas attracting for as means workforce educated in growing countries and are thatstrong commented halal sales who Pharma, Simpor of director Ko, by Edward managing echoed is market.” This global to the out reach and to expand opportunity this of advantage take will Bruneians the Hopefully, America. or million by 2018.million According development. and growth for potential huge possesses manufacturing plant produced 25 million Syariah-compliant Syariah-compliant million 25 produced plant manufacturing drug Brunei’s first Aureos (Brunei) Capital, fund equity private reduced to the current rate of 22% compared to 30% in 2008. 2008. in to 30% 22% compared of rate current to the reduced macroeconomy by the World Economic Forum (GCI 2010/11), pharmaceutical and healthcare leader of PricewaterhouseCoopers location as astrategic Brunei use and projects specific in partner Rich biodiversity Rich for the establishment of halal pharmaceutical companies in in companies pharmaceutical halal of establishment the for for supporting their regional expansion plans, Abhijit Ghosh, Ghosh, Abhijit plans, expansion regional their supporting for The country is located in a region with great biodiversity, biodiversity, great with aregion in is located country The Ghosh adds, “The country offers research countryGhosh offers and adds, development “The In October 2013, Simpor Pharma was established after acquiring acquiring 2013, after October In established was Pharma Simpor B i o P ha r m Gl I n t e o r nat b i o al na l w

Ne w w . b w i o p ha s r m i n t e r nat i o na l . c o m

J Japanese-based Japanese-based Thailand (18th)Thailand for issues the of is one (2). tape red Bureaucratic (NITE) and Bruneian scientists are researching the possibilities of of possibilities the researching are scientists Bruneian and (NITE) 1. Embassy of Brunei Darrussalam in Tokyo, in Darrussalam of Brunei Embassy 1. 2. Doing Business Report 2014, The World Bank, www.doingbusiness. 2014, World The Bank, Report Business Doing 2. stages of manufacturing, the government has been willing to to willing been has government the manufacturing, of stages starting a business, enforcing contracts, and property registration. registration. property and contracts, enforcing abusiness, starting Ghosh shares a similar view that the lack of adequate infrastructure infrastructure adequate of lack thatthe view asimilar shares Ghosh also does not possess high quality infrastructure to facilitate the the to facilitate infrastructure quality high possess not does also are in place, there is a market for more companies to open to open companies more for is amarket there place, in are certain to eliminate policies its liberalizing on emphasis its place also and Evaluation Evaluation and machinery and the Bruneian pharmaceutical industry. He says, “Given the shortage shortage the “Given says, He industry. pharmaceutical Bruneian the 2014, Report Business the World’s Doing the on Bank Based National Institute Institute National expansion of manufacturing facilities, Ko says. facilities, manufacturing of expansion to years six Pharmaceutical Viva took it Forexample, companies. essential to bring about development,” he says. he development,” about to bring essential thatis investment direct foreign potential for lookout aconstant on systems and processes the Once business. to do companies and experts pharmaceutical developing for facilities training on investing start should government the workforce, askilled of 59 189 ranked of was out behind and country countries exploring theexploring idea with of partnering the country for developing Technology of on raw materials up the value chain to develop its pharmaceutical sector. sector. pharmaceutical its to chain develop value the up using microbes for pharmaceutical purposes. Germany is also is also Germany purposes. pharmaceutical for microbes using build its facilities due to bureaucratic slowdowns. The country country The slowdowns. to bureaucratic due facilities its build manufacturing facility in the country. Moreover, the government is is country. Moreover, the in government the facility manufacturing for to easier make it processes existing streamline and review experimental the in is still Brunei “Though industry. pharmaceutical move and infrastructure its to develop as Singapore such neighbors established from help seeking of idea the may to want explore country The Brunei.” in business to do easier make it and tape red should It industry. the in to succeed wants it if researchers biotech (first), Malaysia countries Singapore (sixth), neighboring and raw ingredients for Chinese traditional medicines. traditional Chinese for raw ingredients plant Sultanate’s the diverse from culled products pharmaceutical Currently, projects.” Future investment References facilities for R&D and manufacturing could be stumbling blocks for for blocks stumbling be could manufacturing and R&D for facilities field. China is also looking to partner with Brunei for the supply of of supply the for Brunei with to partner looking is China also field. for qualifying duties import from u ES444955_BP0614_008.pgs 05.24.2014 02:14 ADV n e Rumpun maintains a positive outlook for the Bruneian Bruneian the for outlook apositive maintains Rumpun However, Brunei will need to improve on areas including including areas to on improve However, need will Brunei 2 org/rankings, accessed May 9, May 2014. accessed org/rankings, bruemb.jp/business-in- brunei, accessed May 9, May 2014. accessed brunei, bruemb.jp/business-in- 0 14

—Jane Wan is writerSingapore afreelance in based Business in Brunei Business , www. .

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BIO and ViS Research of the pediatric patient population with live feeds of local patient demographics, reduce the site burden from Help Streamline feasibility assessments, and decrease site start-up time. “Our partnership with ViS Research Institute is Pediatric Clinical Trials helping streamline pediatric clinical research worldwide,” The Biotechnology Industry Organization (BIO) and ViS said BIO President and CEO Jim Greenwood in the Research are expanding the Pediatric Clinical Research press release. “ViS Analytics will help drug developers Group, launched in 2013, to include pediatric research identify pediatric research sites, making it faster and sites. BIO and its member companies work with ViS easier to conduct pediatric clinical trials and, ultimately, to use its online analytics platform to evaluate global deliver treatments and cures to children suffering from pediatric clinical research infrastructure, identify life threatening and debilitating diseases.” pediatric patient populations, and empower clinical “Innovative research approaches through analytics research collaboration. The aim of the Pediatric Clinical are needed to improve success in these most challenging Research Group is to help member companies more pediatric drug development programs with more timely efficiently assess the global pediatric clinical research access for these new drugs in children,” said Dr. Ron landscape and to engage sites more easily for pediatric Portman, Immediate Past Chair of BIO’s Pediatrics clinical studies, according to a BIO press release. The Committee. initiative also hopes to enable comprehensive mapping

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BioPharm International integrates the science and business of biopharmaceutical development and manufacturing. We provide practical, peer-reviewed technical solutions to enable biopharmaceutical professionals to perform their jobs more effectively. We are currently seeking novel research articles for our peer-reviewed journal as well as manuscripts for our special issues. Submitted manuscripts should be sufficiently novel to be of interest to an experienced audience. Articles should be data driven and provide sufficient technical detail to support the main thesis or should offer a novel synthesis of existing data. Topics should be timely and useful and should focus on the development of peptides, monoclonal antibodies, fusion proteins, other therapeutic proteins, nucleic acids, vaccines, cells for cell therapy, and any other class of biotechnologi- cally generated molecular class. For peer-reviewed papers, members of BioPharm International’s Editorial Advisory Board and other industry experts review manuscripts on technical and regulatory topics. The review process is double-blind. Manuscripts are reviewed on a rolling basis. Our single-themed issues, which include literature reviews and tutorials, cover a range of topics. Upcoming issues address analytical and bioanalytical testing and quality by design.

BioPharm International readers are involved in product and process development, manufacturing, quality control/quality assurance, analytical technologies, regulatory affairs, plant and project engineering and design, and corporate management for the entire scope of biopharmaceutical products, including therapeutic peptides, proteins, nucleic acids, and cells for cell therapies and regenerative medicine, as well as both therapeutic and prophylactic vaccines. Please visit our website, www.BiopharmInternational.com, to view our full Author Guidelines. Manuscripts may be sent to Editorial Director Rita Peters at [email protected]. www.biopharminternational.com

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magentablackcyanyellow ES444951_BP0614_010.pgs 05.24.2014 02:13 ADV The Parenteral Drug Association presents the... 2014 PDA/FDA Joint Regulatory Conference Connecting Regulatory, Quality, Science & Compliance: Assuring Customer-Focused Outcomes throughout the Product Lifecycle September 8-10, 2014 RENAISSANCE WASHINGTON HOTEL | WASHINGTON, DC TOP REASONS WASHINGTON, DC WILL BE THE PLACE TO BE FROM SEPTEMBER 8-12: 1. From September 8-10, the conference will address relevant topics, regulatory trends and information with sessions devoted to Compliance Updates and Center Initiatives. You’ll hear from experts like: • Ilisa Bernstein, Deputy Director, Offi ce of Compliance, CDER, FDA • Bernadette Dunham, Director, CVM, FDA • Martine Hartogensis, Branch Chief, International Compliance, CVM, FDA • Mary Malarkey, Director, Offi ce of Compliance, CBER, FDA • Steven Silverman, Director, Offi ce of Compliance, CDRH, FDA • Doug Stearn, Deputy Director, Policy & Analysis, ORA, FDA • Janet Woodcock, MD, Director, CDER, FDA 2. The 2014 PDA Drug Shortage Workshop, held on September 10-11, will focus on the technological improvements that can have a positive impact on preventing drug shortages, and discuss economic and regulatory barriers to implementation as well as potential incentives or regulatory changes that could improve the business case for quality improvements. 3. PDA’s Training and Research Institute will be hosting six training courses on September 11-12 to complement what you learned at the conference on subjects such as: • GMPs for Manufacturers of Sterile and/or Biotechnology Products • Role of the Quality Professional in the 21st Century • Application of a Quality Systems Approach to Pharmaceutical CGMPS • Preparing for Regulatory Inspections for the FDA and EMA • Quality by Design for Biologics: A Practical Approach – New Course • Managing the QC and R+D Laboratory in a GMP Compliant Manner – New Course Visit www.pda.org/pdafda2014 to register for more information. EXHIBITION: SEPTEMBER 8-10 | POST CONFERENCE WORKSHOP: SEPTEMBER 10-11 | COURSES: SEPTEMBER 11-12

magentablackcyanyellow ES446134_BP0614_011_FP.pgs 05.28.2014 03:12 ADV Regulatory Beat

Global Expansion Shapes Drug Oversight Regulators and industry organizations explain policies and standards to manufacturers and authorities in all regions.

ew FDA officials discuss pharmaceuti- designed to provide consistency and clarity to Fcal regulation these days without refer- QbD policies. ence to how the international reach of the At a London meeting in April, senior leadership biopharmaceutical industry has altered drug from FDA, EMA, and the European Commission research, production, and regulation. At the reviewed efforts to leverage each other’s inspec- annual meeting of the Food and Drug Law tion resources; collaboration to support global Institute (FDLI) in April 2014, FDA commis- development of biosimilars; pediatric cluster sioner Margaret Hamburg concluded her key- meetings to encourage more common policies for note address by describing a “dramatically pediatric studies and applications; information changing global marketplace” and its “huge exchange by the EMA-FDA pharmacovigilance implications” for FDA’s ability to ensure the cluster; and broader cooperation related to vet- safety and quality of products manufactured erinary medicines. The participants also dis- elsewhere. To better assess product risks, cussed the creation of a EU-US Identification of Hamburg proposed “enhanced intelligence” Medicinal Products task force to explore adoption and more collaboration with regulatory part- of US substances registration software in the EU, ners through bilateral and multilateral agree- with an eye to forming a global identification sys- ments and through international organizations. tem for medicinal products. In addition to conducting more inspections And last month, Howard Sklamberg, deputy and oversight of foreign manufacturers, FDA commissioner for Global Regulatory Operations is working with international organizations and Policy (GO), Janet Woodcock, director of to communicate its policies and standards the Center for Drug Evaluation and Research, more clearly and to enhance regulatory capac- and Karen Midthun, director of the Center for ity in emerging countries. In a March 2014 Biologics Evaluation and Research, unveiled a lecture to the United Kingdom’s Medicines new initiative to collaborate more with EMA and Healthcare Products Regulatory Agency and European governing bodies to enhance (MHRA), Hamburg described efforts pharmaceutical quality. FDA will be repre- underway to form an International sented by a European-based team, with the Coalition of Medical Regulatory aim of moving beyond information-sharing to Authorities (ICMRA), which would “strengthen mutual reliance” on trusted regula- work to coordinate and enforce regu- tors outside the US. latory standards to ensure product safety and quality around the world. Challenges in india and China At the same time, FDA and the At the MHRA meeting, Hamburg commented European Medicines Agency (EMA) on her visit to India in February and FDA efforts annouced plans to expand their to expand inspections and develop stronger Jill Wechsler is BioPharm many collaborative programs, includ- ties with local regulatory authorities. The com- International’s Washington editor, ing renewal of a pilot program that missioner acknowledged that FDA has imposed Chevy Chase, Md, 301.656.4634, allows for parallel review and con- import alerts on multiple drugs produced by [email protected]. sultation on quality-by-design (QbD) leading Indian pharmaceutical companies such Read Jill’s blogs at components to new drug applica- as Wockhardt Ltd. and Ranbaxy Laboratories,

PharmTech.com/wechsler. tions and supplements, an initiative but emphasized that FDA was not “targeting” Sohm/Getty Images VisionsofAmerica/Joe

12 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445011_BP0614_012.pgs 05.24.2014 02:18 ADV Regulatory Beat

Indian manufacturers, but apply- ished drugs need to do more to a plan that addresses shortage- ing consistent inspection and monitor suppliers and contractors. related quality issues relevant to quality evaluation standards to all the European market. products imported into the US. gloBal ouTReaCh Global expansion has been a FDA is expanding its presence in While inspections and import prominent theme at the United China even more to monitor the alerts attract public attention, FDA States Pharmacopeia (USP), which rising volume of APIs and medical also emphasizes its role in China now has offices and laboratories products produced for US markets. and other regions in educating in India, China, and Brazil to pro- At a hearing before the US-China public officials and industry lead- vide local manufacturers with Economic and Security Review ers on US policies and standards. access to reference standards, test Commission, held in Washington, Sklamberg’s GO organizes a range methods, and training programs D.C. in April 2014 to assess the of capacity building programs and on correct procedures for test- safety of medical products collaborative activities designed ing and ensuring product qual- imported from China and other to strengthen regulatory systems ity. USP’s Promoting the Quality health-related trade issues, this in developing countries and to of Medicines initiative, which Congressionally appointed panel explain US and international man- is funded by the US Agency for queried experts on the difficulties ufacturing policies and standards International Development, also faced by FDA and manufacturers to local authorities. assists manufacturers in develop- in ensuring the quality of Chinese Similarly, leading trade and pro- ing nations produce medicines drug products and components. fessional organizations are broad- that meet quality and safety stan- Christopher Hickey, the direc- ening international outreach and dards; a recent five-year extension tor of FDA’s China office, reported educational programs to support of the program will provide up on increased FDA inspections in these efforts. The Parenteral Drug to $75 million for USP to provide China—up to 84 in 2013 from 46 Association (PDA) has a European technical assistance to government in 2010. Hickey acknowledged that subsidiary and chapters around regulatory systems in establishing his office has only two full-time the world, including a new one in domestic quality control laborato- field inspectors, but has plans to Singapore. These entities hold con- ries, quality monitoring systems, double FDA’s China staff, pending ferences and training courses on and effective domestic regulatory resolution of delays in obtaining quality manufacturing and testing, authorities. necessary visas. He also noted for- as seen in a program this month Efforts to harmonize phar- mation of an FDA-China working on parenteral manufacturing in macopeial standards in differ- group on economically motivated Istanbul. ent regions over several decades adulteration of medical products. The Drug Information have been stymied by differences With some 4000 establishments Association (DIA) recently estab- in legal authority and traditional producing medical products and lished its global headquarters in practices in different countries. substances intended for the US, Washington, D.C. to better serve Pharmacopeial harmonization now however, Commission members 18,000 members in 80 countries is shifting to a more prospective concluded that FDA’s operation through operations in Europe, approach, working with the World seems “woefully, inadequately Japan, India, China, and Latin Health Organization to develop staffed” for its job. Numerous API America. DIA’s chief executive, common testing practices and samples are falsified or substan- Barbara Lopez Kunz, came from standards-setting processes. dard, according to research by the Battelle with strong international These activities will be on the American Enterprise Institute. Alan management experience to over- agenda at the next USP convention Coukell of The Pew Charitable see DIA’s worldwide growth, which in April 2015, which will set the Trusts described rising concern will be highlighted at its 50th stage for USP programs through over reliance on Chinese sources annual meeting this month. 2020, when the organization for key ingredients in older pain- The International Society of marks its 200th anniversary. To relief medicines and antibiot- Pharmaceutical Engineers (ISPE) prepare for these and other chal- ics. He noted that more frequent is unveiling a Drug Shortages lenges, USP’s new chief executive FDA inspections of foreign drug Prevention Plan this month, along officer, Ron Piervincenzi, is con- establishments are important in with plans to collaborate on a simi- sulting with stakeholders, review- prompting manufacturers to meet lar proposal for the EMA. ISPE and ing the organization’s operations, quality measures and agreed with PDA are working with European and examining options for future Hickey that manufacturers of fin- associations and regulators to craft growth and change. ◆

June 2014 www.biopharminternational.com BioPharm International 13

magentablackcyanyellow ES444997_BP0614_013.pgs 05.24.2014 02:16 ADV Inside EDQM

Finished Product Monographs in the European Pharmacopoeia The European Pharmacopoeia Commission re-evaluates its policy on the development of monographs for finished drug products.

he European Pharmacopoeia (Ph.Eur.), Twhich is celebrating its 50th anniversary the same general principles in 2014, has a worldwide reputation for its monographs on APIs and excipients. Owing to its close collaboration with European regula- for monograph elaboration tors, the Ph.Eur. is well aligned with regulatory developments and needs, and reflects state-of- will be applied. the-art technologies and requirements. While the Ph.Eur. includes amongst its texts a wide range of general dosage-form mono- Working Party, the Ph.Eur. Commission graphs and has taken measures to stay abreast decided in 2012 to initiate a pilot phase on the with scientific and technical developments, feasibility of finished product monographs. A for example, by fostering the implementa- dedicated working party composed solely of tion of quality-by-design principles and rel- representatives of licensing authorities and evant modern analytical technologies, the official medicines control laboratories was set Ph.Eur. Commission has been somewhat reluc- up and assigned two tasks: to elaborate draft tant in the past to initiate extensive devel- monographs on two finished products, one for opment of specific dosage-form monographs. which the API is still under patent in Europe Until recently, such monographs have only (a so-called “single-source” product) and a sec- been established in certain specialized areas ond one for which generic drugs are already (e.g., for vaccines, blood products, insulin, and on the market (“multi-source” product), and radiopharmaceuticals). to draft a guidance document to explain the It has to be acknowledged, however, that rationale for finished product monographs and there is a clear demand for these monographs. how to use them, intended for both regulators They are needed by official medicines control and the industry. laboratories for their market surveillance studies; they support and facilitate the Finished product monographs development of generic drugs, which Since then, the pilot phase has concluded are crucial to ensure the sustainabil- and the outcome was presented to the Ph.Eur. ity of today’s healthcare systems, and Commission for approval at its March 2014 ses- they may also be useful to regula- sion. Based on the commission’s unanimous tory authorities by facilitating the decision, the draft monograph on sitagliptin assessment of respective quality dos- phosphate tablets—a text elaborated by the siers. Based on the feedback received working party in close collaboration with the from stakeholders in the context of innovator—is now published in Pharmeuropa, its tri-annual scientific conference the Ph.Eur.’s free online-forum, for public com- Susanne Keitel, PhD, is head of held in Prague in October 2010, the ments (1). For this monograph, like all other the european directorate for the Ph.Eur. Commission has reviewed its monographs based on a single-source product, Quality of medicines of the council policy. Following intensive discus- broad public consultation is even more impor- of europe. edQm is responsible for sions with the European Medicines tant than for multi-source products to ensure

the european pharmacopoeia. Agency’s Joint CHMP/CVMP Quality that the resulting acceptance criteria and test Images 123render/E+/Getty

14 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445005_BP0614_014.pgs 05.24.2014 02:17 ADV inside edQm

methods are indeed robust and stability. As analytical procedures While the pilot phase clearly applicable to a wide range of prod- may be affected by the presence of demonstrated the feasibility of ucts. During the elaboration of excipients and/or the manufactur- developing monographs for both the finished product monograph, ing process selected, demonstra- single-source and multi-source a monograph for the active sub- tion that the testing procedures products, the Ph.Eur. Commission stance sitagliptin phosphate mono- described in a finished product felt it would be prudent to gather hydrate was also drafted, for which monograph are suitable for the more experience with single- the consultation period ended in specific product will need to be source products and has decided March 2014. included in the marketing autho- to start with the elaboration of It is the Ph.Eur. Commission’s rization dossier. However, this is single-source monographs on policy that each finished prod- not an unusual requirement as products that are potential future uct monograph, taken as a whole, any applicant referring to a com- generic drugs. This does not, how- should provide a reliable basis for pendial API has to demonstrate ever, exclude the possibility of making an independent judge- that the pharmacopoeial mono- also including multi-source prod- ment as to the quality of the graph is indeed capable of ade- ucts in the work program. Such a preparation. Finished product quately controlling the quality decision will be based on a critical monographs will cover differ- of the substance, manufactured assessment of the usefulness of ent formulations and strengths according to the submitted route having a Ph.Eur. monograph and (whenever possible) of the same of synthesis. its impact on products already on dosage form containing the same the market. API. The Ph.Eur. Commission, as regulatory reQuirements While the decision of the it does for all its API and excipi- The EU pharmaceutical legisla- Ph.Eur. Commission to be more ent monographs, will only elab- tion, in directive 2003/63/EC active in the field of finished orate monographs on products detailing the requirements of product monographs will help to that have been authorized in at marketing authorization appli- further broaden the scope of the least one of its member states and cations (2), already provides for Ph.Eur., it is not a change in para- that contain an API for which a a feedback mechanism between digm. The same general principles monograph has already been regulators and pharmacopoeia for monograph elaboration will be published in the Ph.Eur. or which authorities in case a compendial applied as for API and excipient is on the work program. As for product is not adequately con- monographs to ensure they are of all monographs, the elaboration trolled by the respective mono- the same high scientific quality. and revision of finished product graph. This provision allows In addition, public consultation monographs will be subject to assessors to make sure that the will provide feedback from a wide public consultation and take into specifications for a given prod- range of stakeholders and contrib- account current scientific knowl- uct are indeed adequate for the ute to the texts’ usefulness and edge and relevant medicinal prod- specific purpose and provides the adequacy. ucts authorized at the time. Ph.Eur. Commission with valu- Based on the promising out- In addition, finished prod- able information on the need come of the pilot phase and its uct monographs, like all other to review and potentially revise decision to transform this into a API and excipient monographs, the monograph. In addition, the routine work program, the Ph.Eur. while legally binding and reflect- Ph.Eur. itself in its general notices Commission is looking forward ing the state of the art, are not offers much flexibility to users, to its future collaboration with intended to be a “regulatory from the notion of alternative sponsors who are interested in straitjacket” that stifles innova- methods—provided they lead to contributing to the elaboration of tion. In line with the require- the same “pass/fail” result and finished product monographs in ments for all compendial APIs, a are approved by the competent the Ph.Eur. full dossier will need to be sub- authority—to the possibility of mitted to the licensing authori- using modern control strategies, reFerences ties for a finished product covered including parametric release and 1. EDQM, Sitagliptine tablet monograph, Pharmeuropa, http://pharmeuropa.edqm. by a Ph.Eur. monograph, detailing real-time release testing or even eu/TextsForComment/, accessed May 9, its composition, pharmaceutical using manufacturing flexibility, 2014. development, manufacture, con- for example, that is allowed by 2. European Commission, Commission Directive 2003/63/EC, Official Journal of trol, container/closure system, and the design space. the European Union (June 25, 2003). ◆

June 2014 www.biopharminternational.com BioPharm International 15

magentablackcyanyellow ES445002_BP0614_015.pgs 05.24.2014 02:17 ADV Perspectives on Outsourcing

Outsourcing No Longer Just for Cost-Cutting With budgets growing, clients see CMOs’ costs as less crucial.

iopharmaceutical companies have tra- grams, each by more than 4 in 10 respondents. Bditionally considered outsourcing as a Against that backdrop, the responses found way to control costs and manage their specific to outsourcing were much less signifi- internal resources to make the core activities cant. In fact, of the 19 actions identified, the left in-house more efficient. In recent years, five specific to outsourcing were among the six surveys of biopharmaceutical manufacturers by least-taken. What’s more, after a couple of years BioPlan Associates have shown that cost cutting of increases, the percentage of respondents this is, by far, not the primary reason companies year having used outsourcing as a cost-cutting outsource anymore. In fact, this factor ranks far mechanism was for the most part either flat or below other measures. New data from the 11th down from last year’s study, for example: Annual Report and Survey of Biopharmaceutical After doubling from 7% of respondents in Manufacturing Capacity and Production (1) indi- 2011 to 14% last year (2), the share in this year’s cate that cost control is becoming less of a respondents outsourcing manufacturing to driver of outsourcing activity. This reduction domestic service providers specifically to cut is due in part to outsourcing becoming more costs fell to below 10%. highly interwoven with higher-end, technical After more than doubling from 5.7% of activities for which the right partner is more respondents in 2011 to 12.6% last year, the important than an extra dollar saved. This percentage of respondents in this year’s study trend also dovetails with evidence of a sig- who off-shored manufacturing to cut costs was nificant expansion in planned funding for out- essentially flat, at 13.1%. sourcing activities. Those trends weren’t unique to outsourcing As part of the study, BioPlan evaluated how of manufacturing. Fewer respondents reported companies are addressing cost issues in bio- having outsourced jobs in process development pharmaceutical manufacturing through their and R&D to cut costs, while the percentage that actions. As with the past three years, the most outsourced jobs in manufacturing remained flat common action being taken by respondents after two years of increases. These results indi- was to implement programs to reduce operat- cate that while outsourcing can, and does, play ing costs. This action was done by 70.3% of the a role in cost containment, it is no longer a top more than 200 global respondents priority when facilities are looking to cut costs. to this year’s study. The numbers are consistent with last year’s study, Fewer clients evaluate cMOs On their cOst though down slightly from prior The data concerning cost-cutting changes spe- years, when budgets may have been cific to outsourcing is particularly interesting a little tighter. in light of separate results from BioPlan’s study Other common actions taken where we evaluated the importance of vari-

by companies to cut costs include ous attributes that clients consider when they s e reducing process development partner with CMOs. When companies were g ma I y t times and costs, implementing asked to rate the importance of 19 attributes t Ge / l Eric Langer is president of BioPlan operational excellence programs when considering outsourcing to a CMO, only l ra r a

associates, tel. 301.921.5979, such as Six Sigma, and implement- 22% this year said that cost was “very impor- F n o

[email protected]. ing “lean manufacturing” pro- tant.” Essentially, CMOs are less expected to D

16 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445029_BP0614_016.pgs 05.24.2014 02:19 ADV Perspectives on Outsourcing

demonstrate the cost-effectiveness Figure 1: Budget change comparisons, 2009-2014, selected areas. of their services. We found this attribute in the bottom quartile of -7% -5% -3% -1% 1% 3% 5% 7% 4.4% 5.5% the list. 4.1% Process development 5.0% 2.8% That is a significant departure 3.8% 4.4% 4.7% 5.2% from last year’s study in which New capital equipment 6.0% 1.6% -0.6% this attribute was in the top 4.2% 4.9% New technologies to improve effciencies/costs 6.0% third—cited by almost twice as for upstream production 3.3% 6.2% 2.4% many respondents (42%) as being 4.0% 4.9% New technologies to improve effciencies/costs 6.4% very important. Additionally, it for downstream production 4.2% 6.4% 2.5% 3.9% was one of only a few attributes 1.7% Outsourced biopharmaceutical manufacturing -0.4% -1.2% 0.8% that saw a decrease in perceived -1.3%

level of importance this year Source: 11th Annual Report and Survey of Biopharmaceutical Manufacturing, April 2014, www.bioplanassociates.com/11th relative to last, and the 22% of respondents seeing it as such was the lowest share seen in BioPlan’s process development, budgets for will the trends hOld? annual studies going back at least outsourcing of biopharmaceutical A common complaint made by to 2006. manufacturing are growing, year- CMOs in past editions of BioPlan’s What could be the cause of this on-year, far more quickly than industry study has been that cli- change? It’s possible that the bio- other areas (see Figure 1). ents want to contain costs by pharmaceutical community is Overall, the study reveals that doing limited development runs, increasingly looking at CMOs as more than one-third (37.3%) of but still expect successful full-scale partners that can provide highly- respondents are projecting an manufacturing. Biomanufacturers’ specialized equipment, expert increase in their funding of out- focus on cost-cutting has no doubt technical knowledge and flex- sourced manufacturing to some affected their interactions with ibility beyond what is available in- degree. As a result, on average, it CMOs, and relationships can easily house. That’s especially the case is estimated that budgets for out- be strained when clients are look- when companies look to outsource sourced manufacturing will ing to cut corners. higher-value activities, such as pro- increase by 3.9% this year. That’s a Data indicate that these con- cess development or quality-by- marked uptick from budget projec- cerns might ease this year as design services. From that context, tions made over the prior 5 years, clients take a more balanced selecting a CMO is less about going which ranged from a low of -1.3% approach to outsourcing that val- with the lowest bidder and more to a high of 1.7%. ues benefits beyond simple cost about finding the right fit from a Other results from the study containment. This is likely a reflec- technical and relationship stand- confirm this shift. Separately, tion of financial situations, which point. respondents were asked to indicate have eased somewhat after the how their spending on outsourcing dire conditions seen a few years OutsOurcing Budgets will change over the following 12 ago. It remains to be seen whether are trending uP months in manufacturing or R&D. or not clients will retain this new Another reason why biomanufac- Budget enthusiasm was even more approach to outsourcing when dif- turers may be paying less attention pronounced: Almost half (49.9%) ficult times strike again. to outsourcing from a cost stand- of respondents (vs 43.7% last year) point is that their budgets for out- said that their spending on out- reFerences sourcing are expanding relatively sourcing of R&D or manufacturing 1. BioPlan Associates, 11th Annual Report and Survey of r

o quickly. The BioPlan study asked would increase to some extent over

h Biopharmaceutical Manufacturing t u respondents to consider the cur- the next 12 months Capacity and Production (Rockville, e a

h rent economic situation and fore- Based on these data, BioPlan Md., April 2014), www. F t cast to what degree their funding estimates that, on average, budgets bioplanassociates.com/11th y o s

e 2. BioPlan Associates,10th Annual

t would change over the next 12 for outsourcing at individual facili- r

u Report and Survey of

o months for 12 separate areas. In ties will increase by 15.6% over the Biopharmaceutical Manufacturing s c comparison to areas such as new next 12 months, compared to esti- Capacity and Production (Rockville, e 1 I

r capital equipment (where budget mated increases of 10.4% last year

u Md., April 2013), www. G I

F increases are not keeping pace) or and 9.3% the year before. bioplanassociates.com/10th ◆

June 2014 www.biopharminternational.com BioPharm International 17

magentablackcyanyellow ES445031_BP0614_017.pgs 05.24.2014 02:18 ADV Analytical Method Development and Validation Challenges in Analytical Method Development and Validation Susan Haigney

Experts give insight on method transfer, QbD, and regulations for analytical method development s

and validation e ag m I y t t

for biophar- e G e/ s u maceuticals. F

he manufacture of biophar- poses a challenge. According to Yong maceuticals presents some Guo, professor of pharmaceutical Tunique challenges when sciences at the School of Pharmacy, ensuring product quality and Fairleigh Dickinson University (FDU), patient safety. Analytical testing can analytical development and cross- provide the data needed to produce a functional development teams should safe and effective drug product. The work together to anticipate changes development and validation of ana- when creating the development time- lytical methods is crucial in drug devel- line. Alice Krumenaker, manager, QC opment. While the biopharmaceutical Stability Administration, at West- industry has had success in reducing Ward Pharmaceuticals, suggests that risk by incorporating analytical plat- an analytical method may need to be form technologies for monoclonal modified and/or updated during the antibody products, new molecules and drug-development process to mini- drug conjugates pose additional chal- mize inaccurate data, and the modified lenges, according to Stephan Krause, method may need to be revalidated. principal scientist, analytical biochem- Technologies, such as FDA’s quality- istry, at MedImmune. by-design (QbD) initiative, may have a The extended drug development positive impact on analytical method timeline of biopharmaceuticals also development and validation according

18 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445008_BP0614_018.pgs 05.24.2014 02:17 ADV WWW.IVTNETWORK.COM

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magentablackcyanyellow ES446150_BP0614_019_FP.pgs 05.28.2014 03:12 ADV Analytical Method Development and Validation

to Paul Smith, EMEAI laboratory analytical development. Often As far as making changes to the compliance productivity specialist times, the development timelines method goes, if the changes are at Agilent Technologies, because are compressed for various rea- necessary, they must be made. methodologies are identified early sons; however, insufficient con- Sometimes, changes to a process, in the development process. sideration is given to analytical necessary reagents no longer being BioPharm International spoke method development and valida- manufactured, improvements with Krumenaker, Krause, Guo, tion. People expect the analytical in technology or other circum- and Smith about the challenges methods to be ready all the time stances out of the lab’s control involved in analytical method regardless of timeline or resource may result in a method no lon- development and validation in bio- changes. To handle this challenge, ger being suitable as written and/ pharmaceutical manufacturing. the analytical development team or as validated. Depending on needs to be closely working with the extent of the changes, some BiophArMACeutiCAl the cross-functional development form of revalidation is likely to ChAllenges team to anticipate the changes be needed. This may range from BioPharm: What are the challenges and actively participate in any a simple verification, demonstrat- involved in analytical method relevant discussion regarding the ing that the method still performs development and validation for timeline. It is also important for as intended, to a full-blown val- biopharmaceutical manufacturing? analytical development to educate idation for significant changes. Krause (MedImmune): The use of and work with the project manag- In addition, this may impact the analytical platform technologies ers to be more visible during the regulatory submission; however, for common product types, such development process. the bottom line is if the original as monoclonal antibodies, has method isn’t accomplishing what lowered the uncertainty/risk for BiophArMACeutiCAl it needs to accomplish, it needs to the manufacturer; therefore, less DeVelopMent tiMeline be modified and updated to mini- development, qualification, and/ BioPharm: How is analytical mize the possibility of inaccurate or validation work is required. method development incorporated results. In that event, all appropri- More challenges typically exist into the long development time- ate amendments must be made to for new types of molecules and/or line of biopharmaceuticals? Can the original submission. conjugate products. For example, methods be changed mid-stream? Krause (MedImmune): The timing patient-specific cancer vaccines At what point should analytical and effort spent on method devel- require conceptually a different methods be validated? opment correlates with the degree approach in that some (routine) Krumenaker (West-Ward): Method of novelty of the product type test methods should test for non- development and validation are and manufacturing process. The patient-specific manufacturing typically done in the product sooner methods are optimized, (batch-to-batch) consistency ver- development stage. While the the lower the risk will be to over- sus those that are patient-specific method may go through various all product development. critical quality attributes. A direct iterations during product devel- Methods can be changed any potency test method may not opment, as the final formula- time during and/or after prod- exist, and instead, several surro- tion emerges, the method will be uct development. The change gate test methods may need to optimized and ready for valida- to faster, more sensitive, accu- be used for potency. Obviously, tion. The analytical method will rate, and/or reliable test methods whenever new test methods are become part of the submission is encouraged by the regulators. to be used throughout product package and, once a submission Besides providing sufficient quali- development, more focus should is approved, there is usually great fication or validation results for be on the development, optimiza- resistance to submit changes to the new method, method com- tion, and validation of these new FDA. So at this point, it is impor- parability results should also be methods. tant to have confidence in the provided. In some cases, product Guo (FDU): As in any drug-devel- method and its ability to be specifications may need to be re- opment program, there are, of transferred from R&D to qual- evaluated and potentially adjusted course, many challenges involved ity control or, in some cases, to to the established bias and/or in analytical method development a contract organization that will increased sensitivity of the new and validation for biopharma- be doing the testing. A successful method. ceuticals. I would like to men- validation will provide this level Regulatory expectations vary tion one particular challenge to of confidence. a little bit depending on prod-

20 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445000_BP0614_020.pgs 05.24.2014 02:16 ADV Analytical Method Development and Validation

uct type and/or quality attribute PDA TR 57’s content for their cur- ing reference standard with a pri- tested. For example, for blood or rent draft guidance. mary reference standard so that it blood plasma products, valida- Guo (FDU): The International is linked to clinical trial material tion of potency and some safety Conference on Harmonization and the current manufacturing test methods is expected much (ICH) has a general guidance on process’ (1). The new draft guid- earlier (clinical Phase 1). For method validation, ICH Q2(R1): ance does not address specific most other products, method Validation of Analytical Procedures: method validation recommenda- validation is typically executed Text and Methodology (3). This is tions for biological and immuno- against the to-be-commercial the guidance that the industry chemical assays. specifications prior to process generally follows in performing validation, which is typically ini- analytical method validation. QuAlity By Design tiated during the pivotal clini- The ICH guidance, however, does BioPharm: How can method val- cal phase. Method validation is not specifically address method idation benefit from a QbD usually completed one to two validation for biopharmaceu- approach? years prior to commercial license ticals. Interestingly, FDA issued Krause (MedImmune): For a QbD application(s), depending how a new draft guidance for indus- approach for analytical meth- much real-time stability data are try on Analytical Procedures and ods, the analytical method life- desired following the process Method Validation for Drugs and cycle stages should consider the qualification runs (process vali- Biologics early this year (1). The desired method performance at dation stage 2). new draft guidance supersedes the each product development stage. Guo (FDU): The analytical meth- 2000 draft guidance for indus- Starting with the appropriate, pre- ods need to be validated for any try on Analytical Procedures and established method performance GMP activities. This is a GMP Methods Validation, and covers expectations (Analytical Target requirement as well as FDA expec- both small-molecule drugs and Profile), the selection of the test tation. The analytical meth- biologics. In comparison to the methodology and instrumenta- ods should be properly validated 2000 guidance, the new guidance tion are the first step. Depending even to support Phase I studies. specifically addresses analytical on the intended use of the Appropriate approaches should method development and sug- method, typical performance cri- be considered to validate the ana- gests the parameters that should teria (accuracy, reliability, specific- lytical methods to support differ- be evaluated during method ity, sensitivity, range, robustness/ ent clinical phases. The concept development, including specific- maintenance, as well as speed and of ‘phase appropriate validation’ ity, linearity, limits of detection throughput capacity, but possibly has been proposed and applied to (LOD) and quantitation limits also costs and ease of operations) method validation. (LOQ), range, accuracy, and preci- should be established early and sion. The robustness of methods is re-evaluated as needed throughout regulAtory ConsiDerAtions particularly discussed in the new product development. BioPharm: What regulatory draft guidance, and a systematic The use of analytical platform parameters exist for analytical approach for method robustness technology (APT) methods can method development and valida- study (e.g., design of experiments) also greatly reduce the selection, tion for biopharmaceuticals? should be adopted to ‘fully under- development, and validation Krause (MedImmune): A F DA stand the effect of changes in effort and lower the uncertainty/ draft guidance for development method parameters on an analyti- risk(s) for unsuitable method per- and validation was recently made cal procedure’ (1). The new draft formance during product develop- available for public commenting guidance refers to the ICH guid- ment. Since method performance (1). Biopharmaceuticals are in ance Q2(R1) for more details on criteria are well established, only the scope of this draft guidance. each method validation character- product-specific suitability would A more comprehensive practi- istics and also removes the recom- need to be confirmed. cal guidance, Technical Report 57, mended validation characteristics Guo (FDU): The QbD approach published by the Parenteral Drug for various types of tests. is more applied to method devel- Association (PDA) and specific For the qualification of new ref- opment than method validation for biopharmaceuticals, provides erence standards, the new draft since method validation is the the best practical guidance cur- guidance recommends a two- process of demonstrating that a rently available to industry and tiered approach, which involves well-developed analytical method regulators (2). FDA used much of ‘a comparison of each new work- is suitable for its intended pur-

June 2014 www.biopharminternational.com BioPharm International 21

magentablackcyanyellow ES445012_BP0614_021.pgs 05.24.2014 02:18 ADV Analytical Method Development and Validation

pose. The QbD concept is often robust. Issues may not develop Krause (MedImmune): We should narrowly interpreted in literature until a larger change has been distinguish a validation deviation for analytical method develop- made. The goal in method vali- from a validation failure. Both ment, and the method robustness dation isn’t just to document can occur, but the frequency of study is often used as an exam- that the method is suitable for these events should be low so ple of the application of the use. It should be to truly confirm that the manufacturer can main- QbD approach. Well, the QbD that it would work as expected, tain a state of control. In general, approach is certainly applicable regardless of the analyst, the lab, an unplanned deviation is typi- to the robustness studies and also and within reason, the environ- cally easier to resolve and may consistent with the FDA expec- mental conditions. It may take not hold up the completion of tation. The results of the robust- longer to incorporate ‘real-world’ the validation study. For example, ness studies do not actually define parameters into the process, but the use of an incorrect analyte the design space. A broader design there will be more value in the spike or a confirmed sample mix- space can be evaluated using the day-to-day use after validation up may require an execution rep- QbD approach during analytical is complete and the method is etition but may not need to be method development. This would transferred. treated like a (potential) valida- mean a significant more upfront Smith (Agilent): The application tion failure once the deviation effort in method development. of QbD principles to analytical has been confirmed. Often, a Krumenaker (West-Ward): QbD is method development and vali- lack of clarity in the validation not always employed in method dation will have a significant protocol, poor preparation and development and validation positive impact. Fundamentally, planning, and/or insufficient because it is often considered analytical QbD requires that the analyst training can result in to build time into the process. analytical target profile (ATP) unplanned deviations. Often, labs are working under is identified before the analyti- A validation failure results aggressive timelines to develop cal technology is considered. from failing to pass the protocol and validate methods. Using This means that fundamental acceptance criteria (or test method a pre-established set of parame- requirements of the methodol- performance specifications). ters for method validation may ogy are identified ‘up front.’ For Validation failures are always be helpful in expediting the HPLC methods, systems are avail- serious and difficult to deal with validation process, but it doesn’t able that integrate experimental under current interpretation of necessarily provide relevant infor- design software with the chro- regulatory compliance. PDA TR mation about how the method matography data system (CDS) 57 provides good ideas and sug- will perform in ‘real world’ condi- and analytical instrument, so that gestions on how to systematically tions. When QbD is utilized in analytical method screening can deal with such events and to set the development stages, critical be performed in an automated up an appropriate quality system product attributes are identified. manner to identify a lead column, (similar to dealing with out-of- Why not utilize this information gradient, and mobile phase com- specification results). in regards to analytical meth- bination. This ‘Lead’ system is ods? For small molecule products, then subject to additional optimi- MethoD trAnsfer light or humidity may affect the zation experiments and final veri- BioPharm: What are the chal- product, so there may be poten- fication of the analytical design lenges in method transfer? tial problems related to these con- space. This approach has the Krause (MedImmune): L i k e ditions that could arise during potential to significantly speed up method validation deviations testing. The same is true for bioanalytical method development and failures (see ‘Deviations and pharmaceuticals. and result in HPLC methods that Variations’), similar root causes Until the method has been are more robust, and therefore, may also exist for method trans- stressed to the point where a analytical transfer problems are fers (validation extensions). problem occurs, you don’t know reduced. The readiness of the receiving what the true boundaries are. laboratory should be evaluated. Don’t just assume that by modi- DeViAtions AnD VAriAtions Consideration should be given to fying a mobile phase by 2% or BioPharm: What deviations or the availability of required ana- varying a pump speed by 0.1 variations may occur during analytical and supporting equipment, mL that you have demonstrated lytical method validation in bio- software, critical reagents, stan- that the method is sufficiently pharmaceutical manufacturing? dards, controls, and analysts who

22 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES444998_BP0614_022.pgs 05.24.2014 02:16 ADV Analytical Method Development and Validation

are skilled in the relevant analyti- shipping the reference standards experience of the ‘receiving’ lab- cal techniques as well as the qual- and samples overseas. Proper oratory have a significant impact ification status of all materials, documentation including import on the success of the transfer. equipment, and analysts. licenses, if necessary, should be Differences in instrumentation, If gaps are identified (e.g., obtained in advance. To ensure culture, and ways of working the receiving laboratory has a the success of method transfer, also contribute to possible chal- similar analytical instrument), the analytical methods should lenges, and therefore, need to be a risk assessment should be per- be evaluated by the receiving considered. The analytical tech- formed before execution of the laboratory before the initiation nology transfer (ATT) usually formal transfer studies. Shipment of method transfer. The samples follows an analytical protocol, and receiving procedures are for method transfer should also where results obtained between needed to allow transfer of criti- be carefully selected. The new the ‘receiving’ laboratory are cal reagents, standards, and FDA draft guidance recommends compared to those obtained by samples between laboratories. that forced degradation samples the ‘donor’ laboratory transfer- Incorporation of the test method or samples containing pertinent ring the technology. Subjective procedure into the receiving lab- product-related impurities should tests such as color and appear- oratories quality system is also be analyzed at both labs for a ance can cause disproportionate part of the transfer process. stability indicating method. For problems unless the specification The sending laboratory biopharmaceuticals, it might be is carefully worded. Tests with should provide hands-on train- difficult to ship the forced degra- low level impurities require care- ing in the specific test method dation samples to the receiving ful evaluation using % absolute to analysts at the receiving labo- lab. A suitable protocol may be differences, rather than student’s ratory, if needed. The type and needed to guide the receiving labs t-tests in the ATT protocol. In amount of training needed will to generate similar forced degrada- part, cultural and ‘ways of work- vary depending on the analyti- tion samples themselves. ing’ differences can be overcome cal method transferred and the Krumenaker (West-Ward): When through training and short-term existing experience of the receiv- transfers are being done between secondment, so that analysts in ing lab and its personnel. The sites, or with contract facilities, the ‘receiving’ laboratory acquire evaluation of the capability of the issues may arise with different as much knowledge of the meth- receiving laboratory to execute the makes of instruments. While an odology as possible. system suitability requirements of HPLC has the same basic com- For transfer of HPLC meth- the method successfully during ponents, regardless of manu- ods, generally, fewer problems the training is recommended. It is facturer, there may be subtle are experienced where both labo- important that all responsibilities differences in performance. For ratories have the same analyti- between sending and receiving example, the construction of the cal equipment. However, this is laboratories are established. PDA optics in the detector may be not always possible and ‘hidden’ TR 57 provides additional practi- just different enough to present differences in instrumentation cal tips on what to consider for unexpected issues with linear- (such as gradient formation, analytical method transfers. ity or sensitivity. In some cases, delay volume, or signal to noise) Guo (FDU): Method transfer is older models from the same can contribute towards problems a very challenging exercise and manufacturer may have differ- with transfer. Testing or qualify- can become more challenging if ences in construction that will ing the equipment in the same the receiving laboratories are in challenge a method in ways that way can help identify differences different countries/continents. will prevent a successful transfer. in performance between analyti- Planning is definitely the key to Some exploratory testing may cal equipment. a successful method transfer. The be valuable, prior to transfer, to timelines for method transfer determine the compatibility of referenCes need to be discussed and clearly the instruments in the receiving 1. FDA, Guidance for Industry, Analytical Procedures and Methods Validation understood by both the originat- lab and the method. for Drugs and Biologics, Draft ing and receiving labs. The meth- Smith (Agilent): Method transfer Guidance (CDER, Rockville, Md., ods transfer protocols should be can go smoothly or can result in February 2014). carefully reviewed and approved significant problems. The robust- 2. PDA, Technical Report 57 (PDA, 2012). 3. ICH, Q2(R1): Validation of Analytical by both labs. Logistics should be ness of the analytical methodol- Procedures: Text and Methodology well coordinated especially when ogy being transferred and the (ICH, November 2005). ◆

June 2014 www.biopharminternational.com BioPharm International 23

magentablackcyanyellow ES445010_BP0614_023.pgs 05.24.2014 02:18 ADV Peer-reviewed: antibody Production concentrating feed— an applicable approach to improve antibody Production

Ping Xu, Xiao-Ping Dai, Albert Kao, Rosario Scott, and Reb Russell

ABSTRACT In this study, the authors evaluated different approaches to prepare highly concentrated feed media for fed-batch Chinese hamster ovary cell culture. By raising the pH or by adding surfactants, the feed media could be concentrated s

e by 1.3- to 2.5-fold. The feed medium concentrated by pH adjustment led to g a an increase in monoclonal antibody productivity at a 5000-L manufacturing Im y t scale. The addition of surfactant Poloxamer 188 not only reduced the feed et G /

e volume by 2.5-fold but also increased the titer by 10 to 50%. The improvement c ur

o in productivity could be due to the enhanced protein expression and secretion S e

g machinery. These simple approaches did not affect product quality and were a Im

/ effective for multiple cell lines and applicable for large-scale manufacturing. s e g a Im B E R ecombinant protein produc- be increased by adjusting the pH (3). tion in the biopharmaceutical Recently, surfactants have been used Rindustry mostly relies on fed- to increase the solubility of chemicals batch mammalian cell culture in the feed media (4). In this study, the (1, 2). Often in fed-batch processes, the authors compared strategies including feed volume can be more than 1000 raising the pH and adding surfactant(s) Ping Xu* is a scientist at Bristol-Myers L for a 5000-L bioreactor with 30 to in the preparation of concentrated feed squibb, Process development, 519 route 40% (v/v) feed. To alleviate workload media. In the experiment, the authors 173 west, Bloomsbury, nJ 08804, ping.xu@ in media preparation and simplify the fed less volume of concentrated feeds bms.com; Xiao-Ping Dai is director of Process manufacturing process, a reduction in to the cell cultures, as opposed to the development, Biologics at celgene corporation, feed volume is greatly desired. Feed- constant volume feeding strategy used 200 connell drive, Berkeley heights, nJ 07922, volume reduction can also reduce the by Hossler et al. (4). Among the surfac- and previously manager of cell culture sciences cost of consumables, such as dispos- tants evaluated, Poloxamer 188 (P188) at Bristol-Myers squibb when this work was able bags and medium storage space. (i.e., Pluronic F68) is a nonionic surfac- done; Albert Kao is an undergraduate student Because the feed dilutes the culture tant that is an additive in mammalian at the university of Michigan, ann arbor, Mi and reduces product concentration, the cell culture media and drug substance 48109; Rosario Scott is a senior scientist, and use of lower feed volumes can improve formulation (5–7). It was found that Reb Russell is site head and head of Biologics overall process productivity as well. the feed volume could be reduced by Process development, both at Bristol-Myers squibb, Process development, 519 route 173 One approach of decreasing feed vol- 2.5-fold with the concentrated media west, Bloomsbury, nJ 08804. umes but still stoichiometrically supply- supplemented with higher levels of ing nutrients to cells is to concentrate P188, while volumetric productivity was *to whom all correspondence should be the feed media. The preparation of improved by 10–50%. The improve- addressed. highly concentrated feed media is, how- ment of productivities was likely due PEER-REVIEWED ever, challenging because of the lim- to the enhancement of protein fold- article submitted: March 2, 2014. ited solubility of medium components. ing and secretion machinery suggested article accepted: March 13, 2014. The solubility of many amino acids can by quantitative polymerase chain

24 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445123_BP0614_024.pgs 05.24.2014 03:41 ADV magentablackcyanyellow ES446147_BP0614_025_FP.pgs 05.28.2014 03:13 ADV Peer-reviewed: antibody Production

Table I. Concentrated feed media.

Concentrated Osmolality Total feed Media PS80 (v/v) P188 (w/v) pH media (mOsm/kg) volume

1X Control 0 0.10% 835 8.58 Vo

1.30X 0 0.13% 1128 8.67 0.77 Vo

1.60X 0 0.16% 1413 8.98 0.63 Vo

1.75X 0 0.17% 1703 9.99 0.57 Vo 1.90X 0 0.18% 1889 10.14 0.53 V M1 o 2.50X 0 0.24% 2432 10.41 0.40 Vo

2.50X+0.01% PS80 0.01% 0.24% 2425 9.69 0.40 Vo

2.50X+0.05% PS80 0.05% 0.24% 2174 9.97 0.40 Vo

2.50X+0.5% P188 0 0.50% 2261 9.18 0.40 Vo

2.50X+2.5% P188 0 2.50% 2286 9.15 0.40 Vo 1X Control 0 0 1252 8.8-9.5 V M2 o 1.33X 0 0 1668 8.8-9.5 0.75 Vo

PS80 is Polysorbate 80; P188 is Poloxamer 188; Vo is the original feed volume.

reaction (qPCR) array assay. Supplementing an osmometer (Advanced Instruments). This surfactant and raising pH approaches were medium is referenced as M1 in Table I. successfully used in the preparation of con- To prepare the feed medium for cell line centrated feed media for 7-L and 5000-L biore- C, the formulated powders were added into actor runs, respectively. These straightforward WFI or cell-culture-grade water sequentially approaches can be applied to large-scale pro- at room temperature. 10 N NaOH was used duction of recombinant proteins. to raise the pH until the solution was clear. After mixing well, the solution was made up Materials and Methods to the required volume and filtered through Cell lines. Three recombinant Chinese hamster a 0.22 µm filter (Millipore). The pH and ovary (CHO) cell lines, developed in-house to osmolality of the medium were measured. produce three different monoclonal antibod- This medium is referenced as M2 in Table I. ies, were used in this study. Cell lines A and B Fed-batch studies in shake flasks and biore- are dihydrofolate reductase-deficient (DHFR- actors. Fed-batch studies in shake flasks were deficient) DG44 cell lines. Cell line C is a CHO carried out in 250-mL Erlenmeyer flasks in o glutamine synthetase (GS) cell line. Frozen vials incubators at 36.5 C with 5% CO2. Applikon of the cell lines were thawed and grown in 7-L benchtop bioreactors were used for scal- shake flasks containing Bristol-Myers Squibb’s ing up shake-flask studies. The process for proprietary basal media. The flasks were shaken cell line C was scaled up to the 5000-L pro- o in incubators at 36.5 C with 5% CO2. duction bioreactor. Media preparation. To prepare feed media Cell counts, antibody titer, protein quality for cell lines A and B, the formulated powder analysis, and qPCR array. Cell-culture sam- was added into water for injection (WFI) or ples were counted by a Vi-CELL (Beckman- cell-culture-grade water at 37 oC, followed Coulter) using the trypan blue exclusion by addition of Polysorbate 80 (PS80) or P188. method. The pH and metabolites in spent 10 N sodium hydroxide (NaOH) was used media were measured by a NOVA FLEX to raise the pH until the solution was clear. instrument (Nova Biomedical). Antibody After the chemicals were completely dis- titer was measured using high-performance solved, the medium was made up to the liquid chromatography (HPLC) (Waters required volume by adding WFI or cell-cul- Corporation) equipped with a protein A ture-grade water and filtered through a 0.22- column (Applied Biosystems). The protein µm filter (Millipore). The medium pH was charge profile was analyzed by imaged measured by a pH meter (Thermo Scientific), capillary isoelectric focusing (iCIEF) and the medium osmolality was measured by (ProteinSimple, Santa Clara, California).

26 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445120_BP0614_026.pgs 05.24.2014 03:41 ADV Peer-reviewed: antibody Production

Antibody N-glycosylated forms were analyzed the concentrated feed media was increased by PNGase F digestion, reductive amination from 835 mOsm/kg of 1X medium to 2432 labeling, followed by separation using ultra- mOsm/kg for 2.50X medium. To test if a sur- performance liquid chromatography (UPLC). factant can increase the solubility of medium In a qPCR array, 3 × 106 cells of day 9 components without increasing the medium and day 11 from each treatment were pH, 2.50X concentrated feed media with vari- used to extract messenger ribonucleic acid ous percentages of PS80 or higher levels of (mRNA) following the protocol of RNeasy P188 were made. The data show that these Mini Kit (Qiagen). 400 ng of total RNA for each sample was reverse transcribed into comple- mentary deoxyribonucleic acid 2014 2016 BULK Chemicals for Scale-Up & Production (cDNA) using RT2 First Strand Kit (Qiagen). Four experimental cocktail solutions composed of cDNA synthesis reaction and SABiosciences RT2 qPCR mas- ter Mix (Qiagen) were dispensed into 4 × 96 wells (10 µL/well) in 384-well PCR unfolded protein response array (Qiagen). The array was analyzed by ViiA 7 real-time PCR system (Life Technologies). The raw CT val- ues were further analyzed by free web-based RT2 profiler PCR array data analysis version 3.5 (Qiagen). The ratios of gene levels between day 9 and day 11 for each treatment were reported. The gene functions were obtained from the website of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/gene). results Concentrated feed medium M1 test in shake flasks. In the concentrated feed medium study, the original concentration is designated as 1X. Also Available from The 1X medium was used as a con- Fisher Scientiﬁc & VWR International trol. Feed media with concentra- and other Domestic and International Distributors tions of 1X, 1.30X, 1.60X, 1.75X, 1.90X, and 2.50X were prepared by Scan adding 10 N NaOH to increase the with your • Active Pharmaceutical Ingredients Smartphone • US DEA Controlled Substances pH and dissolve all of the compo- to order a • Cosmetic Ingredients nents into the solutions. The catalog • Excipients higher the feed concentration, the Scan • Dietary Supplements more the base needed for solubil- with your • FCC & Food Grade Chemicals ity, resulting in an increase of pH Smartphone • Custom Synthesis Services from pH 8.58 for 1X medium to to order a pH 10.41 for 2.50X medium, as 800.772.8786catalog SpectrumChemical.com shown in Table I. The osmolality of

June 2014 www.biopharminternational.com BioPharm International 27

magentablackcyanyellow ES445119_BP0614_027.pgs 05.24.2014 03:41 ADV Peer-reviewed: antibody Production

Figure 1: Concentrated M1 feed media test in shake fasks. (a) The titer on day 14 and specifc productivities of cell line A. (b) The titer on day 14 and specifc productivity of cell line B. The cultures were fed with 1X M1 or concentrated media as indicated. The titer and specifc productivity were normalized to those fed with the 1X M1 medium. The error bars were from the standard deviation of duplicate fasks. PS80 is Polysorbate 80; P188 is Poloxamer 188.

(a) 1.6 Normalized productivity Normalized specifc productivity

1.2

0.8 productivity

0.4 Normalized productivity/specifc

0.0 1x 1.30x 1.60x 1.75x 1.90x 2.50x 2.50x 2.50x 2.50x 2.50x Control +0.01% +0.05% +0.5% +2.5% PS80 PS80 P188 P188 (b)

2.4 Normalized productivity

2.0 Normalized specifc productivity

1.6

1.2

productivity 0.8

0.4 Normalized productivity/specifc 0.0 1x Control 2.50x + 0.5%P188

media required less NaOH compared to the The concentrated feed media described 2.50X concentrated medium, resulting in a previously were used in the fed-batch culture lower final medium pH and lower osmolality of cell line A. The feed volumes are shown Table I Table I (see ). in . Nutrients were added to the cell ALL FIGURES ARE COURTESY OF THE AUTHORS

28 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445125_BP0614_028.pgs 05.24.2014 03:41 ADV Peer-reviewed: antibody Production

Figure 2: Antibody production in bioreactors using concentrated feed medium of M1 or M2. (a) – (c) The culture of cell line A was grown in 7-L bioreactors and fed with 1X M1 or 2.5 fold concentrated M1 supplemented with 0.5% Poloxamer 188 (P188) as indicated in Table I. The productivity was normalized to the titer on day 14 fed with the 1X M1 medium. (d) The culture of cell line C was grown in 7-L or 5000-L bioreactors and fed with 1X M2 or concentrated M2 as indicated in Table I. The productivity was normalized to the titer on day 14 fed with the 1X M2 medium. The error bars were from the standard deviation of duplicate bioreactors.

(a) (b)

250 100 1x Control /ml

5 200 2.50x+0.5% P188 80

150 60

100 40 Viability %

50 1x Control

Viable cell density x10 20 2.50x+0.5% P188

0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Time (day) Time (day) (c) (d) 1.4 1.4 1x Control 1.2 1x (7 L) 1.2 2.50x+0.5% P188 1.33x (7 L) 1.0 1.0 1.33x (5000 L) 0.8 0.8

0.6 0.6

0.4 0.4 Normalized productivity 0.2 Normalized productivity 0.2 0.0 0.0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Time (day) Time (day)

culture for the concentrated feed media at the 2.50X + 0.5% P188 feed medium have the same quantities for the 1X feed medium. been observed as these are closer to the 1X The productivities on the last day of the control condition (data not shown). The process (day 14) showed that the 1.60X 2.50X + 0.5% P188 feed medium also led medium led to 12% and 20% increase of to 42% and 116% increase in the final titer the titer and specific productivity respec- and specific productivity of cell line B tively, which is significantly higher than respectively (see Figure 1b). the 1X medium control (p < 0.05). The 2.50X Antibody production in 7-L bioreactors medium had the lowest productivity (see using concentrated feed media of M1. To test Figure 1a), suggesting that there was an upper the scalability as well as to remove the nega- concentration limit of feed media prepared tive effects from the high pH feed media, 7-L by raising the pH, which was due to the bioreactors were used to run cell line A pro- lower cell viability at higher cell culture pH cess with pH control. The profiles of viable and osmolality (data not shown). Among cell density and viability are shown in Figure the surfactants evaluated, the addition of 2a and 2b. The 2.50X + 0.5% P188 medium 0.5% (w/v) P188 in 2.50X medium increased was tested in parallel to the 1X medium the titer and specific productivity by 15% that was used as a control. The viability for and 25%, respectively, while the feed volume the control decreased from day 8, whereas could be reduced by 2.5-fold. More favorable the concentrated feed maintained higher culture pH and osmolality profiles from viability for a longer period of time. The

June 2014 www.biopharminternational.com BioPharm International 29

magentablackcyanyellow ES445124_BP0614_029.pgs 05.24.2014 03:41 ADV Peer-reviewed: antibody Production

Figure 3: Product quality of antibody produced in 7-L bioreactors using concentrated feed medium of M1. (a) Charge variant distribution. (b) N-glycan profles. The culture of cell line A was fed with 1X M1 or 2.5-fold concentrated M1 supplemented with 0.5% Poloxamer 188 (P188) as indicated in Table I. Protein quality was assayed using the samples from day 14. The error bars in the experiments were from the standard deviation of duplicate bioreactors.

(a)

70 Acidic peaks Main peak 60 Basic peaks 50

Charge variant (%) Charge variant 20

0 1x Control 2.50+0.5%P188

(b) 60 GO-GN 50 GO Man5 GO-GN GO Man5 G1-GN G1a, G1b G2

40 N-acetylglucosamine Galactose Mannose G1-GN G1a 30 G1b G2

N-glycan (%) 20

0 1x Control 2.50+0.5%P188

productivity profiles shown in Figure 2c significantly higher (p < 0.05) specific pro- indicate that use of the concentrated feed ductivity than the control (data not shown). medium improved the productivity by 33% These results suggest that productivity can towards the end of the process. The use of be improved when feed volume is reduced, the concentrated feed medium also led to a which is consistent with the shake-flask stud-

30 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445121_BP0614_030.pgs 05.24.2014 03:41 ADV Peer-reviewed: antibody Production

Table II. Gene level changes from day 9 to day 11 during the process of feeding with 1X M1 control medium and 2.50X + 0.5% P188 M1 medium. Fold change from day 9 to Gene Protein Function day 11

1X Control 2.50X + 0.5% P188

Hspa5 HSP70 family Protein folding chaperone -3.15 -1.06 Hsp90b1 HSP90B1 Protein folding chaperone -2.20 -1.07 Calr3 Calreticulin 3 ER Ca2+-binding chaperone -1.83 1.68 Calr Calreticulin ER Ca2+-binding chaperone -1.74 -1.27 Pdia3 PDIA3 Protein disulfide isomerase family -1.66 -1.14 Nucleotide exchange factor for unfolded Sil1 Nucleotide exchange factor SIL1 -1.59 1.23 proteins Homocysteine-responsive ER-resident Ubiquitin-like domain protein, ER associated Herpud1 -1.51 1.36 ubiquitin-like domain member 1 protein degradation LOC100763514 Unknown N/a -2.29 1.73 LOC100763648 Unknown N/a -1.50 1.07 LOC100762174 Unknown N/a -2.09 1.29 Ero1 ERO1L ER redox homeostasis -1.32 1.53 Prevention protein from aggregation; Hspa1L Heat shock 70 kDa protein 1L -1.18 1.66 assistance of protein folding Srebf1 Sterol regulatory element-binding protein 1 Regulation of sterol biosynthesis 1.54 1.00 Insig1 Insulin-induced gene 1 protein Regulation of cholesterol biosynthesis 1.58 1.09 Scap SREBP cleavage activating protein Regulation of sterol biosynthesis 1.59 -1.15 Htra3 HtrA serine peptidase 3 Serine peptidase 4.31 2.07 LOC100754863 Unknown N/a 1.53 -1.03 Note: Blue is downregulated; orange is upregulated; grey is non-differentiated expression. ER is endoplasmic reticulum. P188 is Poloxamer 188. N/a is not available.

ies. The product qualities, including charge the same as that of the 1X M2 control. Data heterogeneity and N-glycan profiling, were from a 5000-L manufacturing bioreactor run not affected by the increase of P188 con- performed using 1.33X M2 feed medium are centration in the feed (see Figure 3). There overlaid in Figure 2d. The productivity of were no changes in antibody monomer per- 1.33X M2 was 11% higher than that of 1X centages and protein purity with addition of M2 at both development and 5000-L manu- P188 (data not shown). facturing scales. Antibody production in 5000-L bioreactor Gene regulation by feeding with concen- using concentrated feed media of M2. The trated media. To understand the rationale concept of concentrated media was applied for improving antibody specific produc- in the development of a feed medium for tivities in cultures fed with concentrated the process of cell line C. A feed medium feed media (see Figure 1), an unfolded pro- was developed by concentrating the original tein response qPCR array was chosen in feed medium to 7.5 fold, designated as M2 this study. The array included 84 genes for (see Table I). From M2, a 10-fold increase of protein expression, folding, and secretion. the original feed medium was achieved by Gene level changes from day 9 to day 11 in adjusting pH to between 8.8 and 9.5, leading the protein-production phase were inves- to 1.33X M2 feed medium. The two media tigated in this assay, using two samples were tested in parallel using 7-L bioreac- prepared on both days from 1X M1 feed tors. The feeding volume of 1.33X M2 was medium treatment and two samples from reduced by 33% so that the total amount of 2.50X + 0.5% P188 treatment. Gene lev- each component added to the bioreactor was els that changed more than 1.5-fold from

June 2014 www.biopharminternational.com BioPharm International 31

magentablackcyanyellow ES445126_BP0614_031.pgs 05.24.2014 03:41 ADV Peer-reviewed: antibody Production

these treatments are listed in Table II. The media could be obtained by raising pH, there downregulated genes in the control samples was an optimal pH of feed media in which (highlighted in blue) include heat shock cell growth and antibody production were protein family genes (Hspa5, Hsp90b1), not adversely affected. The addition of a Ca2+-binding chaperones (Calr3, Calr), pro- surfactant makes it possible to prepare more tein disulfide isomerase family gene (Pdia3), concentrated feed media while maintaining nucleotide exchange factor for unfolded the pH. In this study, 2.50X concentrated proteins (Sil1), ubiquitin-like domain medium supplemented with 0.5% P188 led protein for endoplasmic reticulum (ER)- to a 10–50% increase in titers while feeding associated degradation (Herpud1), and three as low as 40% of the original feed volume. unknown genes. Instead of downregulation, The surfactant addition to the feed did not these genes were either upregulated or non- have any impact on measured protein qual- differentially expressed in cells fed with ity attributes. 2.50X + 0.5% P188 (see Table II). Highlighted Medium storage stability is a general con- in gray in Table II, Ero1 encoding an ER cern for manufacturing and development oxidoreductase and Hspa1L encoding an ER using concentrated feed media. In this study, chaperone were non-differentially expressed the 2.50X + 0.5% P188 M1 and 1.33X M2 in cells fed with the 1X medium, but up- media have a shelf life of 60 days at 2–4 oC regulated in cells fed with 2.50X + 0.5% and 26 days at ambient temperature respec- P188. These data suggest that the 2.50X + tively (data not shown). There was no precip- 0.5% P188 medium could maintain and itation observed and antibody titers were not elevate levels of genes for chaperones and affected. These data show that concentrating folding enzymes that ensure protein quality feed medium either by raising pH or add- control in the secretory pathway. This finding surfactant is an effective and applicable ing is consistent with the higher cell viabil- approach in simplifying the manufacturing ity and higher productivity observed in the process and improving productivity. concentrated medium. Among the up-regulated genes of the acknowledgeMents 1X M1 medium treatment (highlighted in The authors would like to thank Gautam orange in Table II), Srebf1, Insig1, and Scap Nayar, Joel Goldstein, and Frank Ritacco for are genes for sterol biosynthesis (8). These their review and comments on this manu- genes were non-differentially expressed in script. The authors would also like to thank cells fed with 2.50X + 0.5% P188. P188, a sur- Amanda Bell, Wenkui Lan, and Ya Fu for factant consisting of hydrophobic and hydro- their support in protein purification and pro- philic blocks, has been reported to reduce tein quality analysis. cell death (9, 10). It may play a role similar to lipids, which have hydrophobic tails and references hydrophilic heads that confer cell protec- 1. B. Kelley, mAbs. 1 (5) 443-452 (2009). tion from stress. In addition to three sterol 2. F. Li et al., mAbs. 2 (5) 466-479 (2010). synthesis genes, Htra3, a serine peptidase 3. The Merck Index (Merck & Co. Inc., Whitehouse Station, NJ, 13th ed., 2001). gene, was upregulated more than four-fold 4. P. Hossler et al., Biotechnol. Prog. 29 (4) 1023- from day 9 to day 11 in the 1X M1 medium, 1033 (2013). while only about two-fold in the concen- 5. S.S. Bhadoriya et al., Biochem. Pharmacol. 2 (2) trated medium. Given that serine peptidase 1000113 (2013) doi:10.4172/2167-0501.1000113. contributes to protein degradation, a higher 6. K.H. Bhandari et al., Biol. Pharm. Bull. 30 (6) level of this protease may be one of the rea- 1171-1176 (2007). sons for the lower antibody levels observed 7. J. Sruti et al., Indian J. Pharm. Sci. 75 (1) 67-75 in the 1X M1 medium compared to that in (2013). the 2.50X + 0.5% P188 medium. 8. R. McPherson and A. Gauthier, Biochem. Cell Biol. 82 (1) 201-211 (2004). discussion 9. D.M. Phillips and R.C. Haut, J. Orthop. Res. 22 (5) In this study, different strategies of prepar- 1135-1142 (2004). ing concentrated feed media were compared. 10. G. Serbest et al., FASEB J. 20 (2) 308-310 Although higher concentrations of feed (2006). ◆

32 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES445122_BP0614_032.pgs 05.24.2014 03:41 ADV MAb Manufacturing: Where are we headed, cost or capabilities?

ON-DEMAND WEBCAST

www.biopharminternational.com/MAb

EVENT OVErVIEW: Investing money to reduce costs is a good business practice. Investing money to increase value and expand capabilities is better. In monoclonal antibody (MAb) production, investments in improved capabilities through smartly integrated process designs can turn each dollar spent on capability improvements into 5 or 20 revenue dollars...or more.

View this one hour webcast and learn how you can improve your capabilities: Presenter: Günter Jagschies n Upstream: high MAb titers, while reducing time for cell culture Strategic Customer Relations operations GE Healthcare Life Sciences n Downstream: capacity of capture and polishing chromatography steps

n Integration: combine technologies into a highly fexible facility, with small footprint and minimized fxed cost

Sponsored by register today for this FrEE WEBCAST.

Presented by

For questions, contact Sara Barschdorf at [email protected]

magentablackcyanyellow ES446149_BP0614_033_FP.pgs 05.28.2014 03:13 ADV Downstream Processing Biopharma Moves to integrated, single-Use, Downstream Processing

Cynthia A. Challener

Suppliers see challenges to the adoption of single-use technologies for downstream

processing as s ge a m I y t opportunities. t e G / RF ch r a e s o t o F

hile single-use systems Desire for integration have been widely adopted Higher cell densities in biopharmaceu- Wfor upstream biopharma- tical manufacturing have led to both ceutical processing, imple- higher titers and higher impurities, both mentation in downstream operations has of which are increasingly challenging been limited to date. A desire to better downstream processing, according to integrate all processes, as well as the clear Willem Kools, head of field market- demonstration of the upstream perfor- ing and the biomanufacturing sciences mance and benefits of single-use technol- network with EMD Millipore. “Better ogy, are combining to drive interest in process economics can be obtained if the single-use approach for downstream process optimization occurs on both applications. Suppliers of single-use sys- the upstream and downstream sides,” he tems have made progress in some areas, notes. On the upstream side, improving such as tangential flow filtration (TFF), the output from cell culture by improv- and are working to address the need for ing expression of the protein of interest other single-use purification technolo- and reducing the impurity challenges gies. At the same time, they are building will put less stringency on purifica- their product portfolios to assist biologics tion, ultimately reducing the number manufacturers with the integration of of unit operations required, simplify- production processes. ing processes, improving productivity

34 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES444957_BP0614_034.pgs 05.24.2014 02:14 ADV Downstream Processing

and efficiency, and driving the Derek Masser, sales director with batch operations.” New concepts cost of goods lower, according to ASI–Life Sciences, also notes the for continuous manufacturing Christine Gebski, product man- continued emphasis on alleviat- are indeed evolving to support ager for bioproduction at Thermo ing the bottleneck that is inher- higher throughput downstream Fisher Scientific. For example, ent in downstream processes rather and increase integration, accord- Kools points to new technologies than integrating upstream and ing to Millie Ullah, senior product that enable initiation of the puri- downstream processes. “In par- manager for single-use technolo- fication process in the bioreac- ticular,” he says, “we see efforts gies with Thermo Fisher Scientific. tor itself, bringing upstream and focused on the application of sin- She also notes that companies are downstream process development gle-use technologies for continu- developing approaches for reduc- teams closer together. ous processes as opposed to strict ing the number of processing

A Sampling of New Tools for Downstream Processing

Several recent developments are leading to an increase Additional column sizes are also becoming available, in adoption of single-use technology in downstream and some vendors are now willing to work with any processing, and more progress can be made, but it is resin type, which is increasing the flexibility component important for providers to offer the right technologies at for chromatography, according to Christine Gebski, the right scale, according to Helene Pora, vice-president product manager for bioproduction at Thermo Fisher of single-use systems with Pall Life Sciences. “As a whole, Scientific. She adds that customers are also mapping suppliers are developing single-use technologies that are the total time spent on the actual supportive (non- continuous in nature, and these advances have helped producing) workflow associated with column packing and the adoption of single-use technologies in downstream testing and are realizing the potential efficiency gains applications,” Derek Masser, sales director with ASI–Life associated with moving to pre-packed columns. Thermo Sciences adds. Fisher has developed high-performance resins that “In reality, however, areas such as tangential flow increase efficiency, throughput, productivity, and costs filtration (TFF) and chromatography have not seen a great for downstream processing and pre-packed, limited- deal of development in terms of single-use, ready-to-use, use columns for more rapid validation and pilot scale-up integrated models,” notes Pora. “There is still a lot to prove studies and early-phase clinical manufacturing. regarding reliability, particularly at large scale, although ASI has introduced a disposable heat exchanger for Pall has made the first steps with the introduction of use in continuous downstream processes where jacketed ready-to-use gamma irradiated TFF modules and a single- systems are currently employed. The exchanger can be use platform that offers cross-functional downstream easily set up and integrated and, in addition to providing solutions for running depth filtration, virus filtration, virus more efficient and continuous heat transfer for accurate inactivation, and polishing applications with membrane temperature control, it also helps reduce bioburden chromatography.” concerns, according to Masser. EMD Millipore also offers fully-automated single- Masser also notes that recent advances in single-use use TFF and chromatography systems that have been sensing technology have made it possible to provide designed for both operational flexibility and scalability, individualized sensing capabilities that are beneficial according to Willem Kools, head of field marketing and the for downstream manufacturing because the sensors biomanufacturing sciences network. “The modular system are individualized to the process. In addition, new can be used for both TFF and chromatography with a bioprocessing films with reduced extractable and leachable minimum number of component changes, reducing both profiles are also helping biopharmaceutical companies capital investment and product development times, and as a meet regulatory requirements, according to Millie Ullah, result, has attracted attention from contract manufacturers senior product manager for single-use technologies with and biopharma companies installing pilot-plant operations,” Thermo Fisher Scientific. Kools says. EMD Millipore has also introduced new high- In 2013, Pall expanded its product offerings in performance single-use process consumables, including purchasing Medistad Holding, SoloHill Engineering, and clarification filters and flocculants for handling cell removal the LifeSciences business of ATMI, while Thermo Fisher and initial purification, as well as pre-packed columns. acquired Life Technologies.

June 2014 www.biopharminternational.com BioPharm International 35

magentablackcyanyellow ES444960_BP0614_035.pgs 05.24.2014 02:14 ADV Downstream Processing

steps and linking steps together to approach and working with stan- used for downstream manufactur- improve process efficiencies. dardized single-use components and ing to more continuous approaches a wide variety of vendors to offer requires the development of rev- CollaBoration customers more choice and connec- olutionary technologies, such as anD CUstoMization tivity, according to Ullah. “Suppliers continuous chromatography sys- Increased levels of integration can need to develop and commercialize tems. On the smaller scale, Masser be achieved by focusing closely high-value, effective product and also notes that scrutiny by FDA on developing complete solutions workflow solutions,” asserts Gebski. and internal quality control with the right components for any In the end, Pora believes that it becomes more intense the closer particular application and dem- is all about looking at the bigger a process step is to the final thera- onstrating that they will work as picture and seeing how the pieces peutic, and as a result there have expected, according to Helene Pora, fit together to create quality solu- historically been concerns regard- vice-president of single-use systems tions. “Without quality and product ing the evaluation of plastics in with Pall Life Sciences. As is still knowledge, the level of integration terms of extractables and leach- done for upstream applications, it does not matter,” she states. ables. “These concerns have, how- is necessary for suppliers to collabo- ever, been successfully addressed rate with customers on the integra- a laCk of single-Use by single-use suppliers through tion of downstream processes and teChnologies validation studies,” he stresses. the implementation of single-use The greatest barrier for adoption technology. There is, however, a of single-use technology for down- Drivers for aDoPtion greater need for customer collabo- stream applications has been a lack The success that biopharma- ration and personalized integration of adequate technology, particularly ceutical manufacturers have had for downstream processes, accord- in the sensor area, that provides with single-use technologies for ing to Masser. “Downstream pro- for a robust automation approach, upstream applications is providing cessing is very customer-centric. according to Pora. “Not all down- significant impetus for their appli- Biopharmaceutical manufactur- stream process steps are available in cation to downstream operations. ers have their own unique ways single use,” agrees Ullah. For those “Validation studies have proven of manufacturing their products, technologies that are available, she that single-use technologies work, particularly when close to the final adds, scalability, performance, and and as a result, companies are therapeutic; therefore, being able cost are typical limitations. more inclined to trust them,” Pora to custom-configure single-use sys- Chromatography is the last puri- comments. In addition, she notes tems for each process helps increase fication unit operation to move that the industry is changing and the level of integration. In addi- to disposability and has seen only requires more flexibility than any tion, collaborating with customers limited use, according to Gebski. traditional set up can offer. “Single- enables integration of single-use She notes that the adoption of use technologies are, quite simply, equipment that is unique to their pre-packed, limited-use chroma- more agile and can be implemented processes, can fit in their tight tography in the industry has been much more quickly; the flexibility cleanroom spaces, and can monitor particularly slow, even though the they offer is certainly a key factor the results that are relevant to their value proposition for early clinical behind the growing willingness of products.” phase manufacturing is clear (time people to give them a try.” Biopharma suppliers can also savings, where time can be spent Furthermore, according to Masser, help to increase levels of integration on more value-added activities). “It the continued integration and and bring biological drugs to market is possible that customers do not extensive use of plastics in upstream faster by offering more insight on perceive column packing as a pinch manufacturing has meant that the how various unit operations affect point (non-value-add point) in their evaluation and study of plastics has each other, according to Kools. manufacturing process (i.e., the been extensive as well, and the com- “Coupling application knowledge problem is not big enough) or per- fort level achieved with upstream with system design and introduc- ceive a decrease in flexibility with systems is now being transferred to tion of high-performance technolo- respect to defining the required col- downstream applications. “The level gies will lead to the increased levels umn geometries,” she suggests. of bioburden reduction and elimi- of integration for which the indus- From the larger perspective, nation that single-use technology try is looking.” Integration is sup- Masser points out that the con- provides is another huge motivator ported at Thermo Fisher through version of the traditional batch for its adoption in downstream pro- the use of an open architecture manufacturing processes currently cessing,” he says. ◆

36 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES444952_BP0614_036.pgs 05.24.2014 02:13 ADV Fast, Robust LC and LC-MS Workﬂows for the Comprehensive Characterization of Bio-Therapeutic Proteins

ON-DEMAND WEBCAST Register free at http://www.biopharminternational.com/fast

EVENT OVERVIEW: Key Learning Objectives: Over the past decade, there have been a growing number of mAb ■ How to chose an appropriate solid core column dependent candidates entering the clinical pipeline. This results in a large on the application increase on the demand for analytical characterization. This seminar ■ How solid core morphology will discuss new advances in analytical method development with can be optimized for small and analytical run times below 10 minutes for all routine methods large molecule analysis

with intelligent, integrated chromatography workfows. Orbitrap ■ How solid core technology can technology has been established as the most powerful mass extend column lifetimes spectrometry technology for protein characterization. Procedures Who Should Attend: for incorporating this technology into a complete workfow for ■ Biopharmaceutical research biopharma analysis will be presented. and development chemists and laboratory managers

■ Biopharmaceutical QC chemists and lab managers

■ Protein characterization chemists Presenter Moderator Dr. Ken Cook Mike Tracey ■ Fermentation production EU Bio-separations Group Publisher analysts Support Expert BioPharm International Thermo Fisher Scientifc ■ Biotherapeutic protein clone selection chemists

Presented by Sponsored by

For questions, contact Kristen Moore at [email protected]

magentablackcyanyellow ES446557_BP0614_037_FP.pgs 05.28.2014 21:00 ADV Lot-release testing Assuring equivalency of Alternative Lot-release test methods Cynthia A. Challener

New test methods can provide improved quality and s e g efficiency, but a m I y t t e G they must be / c s odi t o h

validated to P / s r a e

demonstrate P A t r e ob equivalency. R

he biopharmaceutical indus- ing can offer numerous advantages in try is advancing at an unprec- this situation, but there are challenges Tedented rate as new cells to assuring equivalence to standard and viruses are being used methods. to generate biological therapeutics in greater yields with shorter produc- Numerous beNefits tion cycles and more efficacious prop- New test methods can improve qual- erties, according to Audrey Chang, ity and promote efficiency, according executive director of global services at to Niall Dinwoodie, global coordinator BioReliance. of analytical testing in the Biologics “New cell substrates (e.g., trans- Testing Solutions business of Charles formed cell lines, insect cells), new River Laboratories. “New methods or product types (e.g., gene and cell improved versions of traditional meth- therapy, tissue engineering), new pro- ods allow quality control laboratories duction process, and the discovery/ to provide a greater level of information detection of new contaminants have about the finished product, whether contributed to an elevated complex- through improved detection limits, dis- ity of product testing to evaluate criminating power, or quantification,” Cynthia A. Challener is a contributing safety and efficacy,” Chang explains. he observes. “In addition, newer tech- editor to BioPharm International. Newer techniques for lot-release test- niques tend to be more automated,

38 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES446016_BP0614_038.pgs 05.28.2014 02:11 ADV Effective Viral Contamination Testing Programs for Biologics Product Manufacturing ON-DEMAND WEBCAST

EVENT OVERVIEW: Presenters: Viruses pose a threat at all stages of the biopharmaceutical manufacturing process from raw materials, to cell lines and Archie Lovatt cell culture, through bulk harvests and biomanufacturing. Scientifc Operations Director SGS Vitrology Biopharmaceutical manufacturers need comprehensive

virus testing programs to ensure product safety through- Euan W. Milne out the various manufacturing stages. Electron Microscopist In this webcast, experts will describe strategies and best SGS Life Science Services practices for developing and implementing efective virus testing programs, including a review of regulatory guid- Ruth Paul Director, Paul Regulatory ance. In addition, participants will learn about test methods Consulting Services for virus detection and quantitation during various biopharmaceutical manufacturing phases. A range of testing Moderator: technologies, including in vitro adventitious virus assays, PCR, ELISA, plaque assays, and electron microscopy, as well Rita Peters as sampling methods, will be discussed. Editorial Director BioPharm International The panelists will provide an analysis of the advantages and limitations of each method, as well as methods that are most suitable for specifc situations. Sponsored by

Key Learning Objectives:

n Develop a comprehensive plan for efective testing for viral contaminants n Review current testing methods and revise current methods to ensure product safety Presented by n Understand pros and cons for various methods for detecting viruses

Who Should Attend:

n Product development scientists n Process Development scientists For questions, n Manufacturing engineers contact Sara Barschdorf at n QA/QC and regulatory personnel [email protected]

magentablackcyanyellow ES446131_BP0614_039_FP.pgs 05.28.2014 03:12 ADV Lot-release testing

allowing faster sample throughput such as polymerase chain reac- relating the conclusions gen- in larger numbers.” tion (PCR) for mycoplasma, next- erated from the old and new The use of newer analytical generation sequencing for virus methods when there are appar- techniques can also help mitigate screening, and digital PCR for ent differences between the data risks, according to Chang. “The residual DNA quantification,” she sets, according to Chang. “A good cost and safety impact of con- comments. Charles River is also example is the use of PCR, where tamination has led the industry to seeing growing interest in rapid the presence of the nucleic acid is embrace quality-by-design princi- microbiology methods for sterility used as a substitute to monitor the ples with respect to building qual- testing of autologous or allogeneic presence of live/infectious organ- ity into a product and process. As cell therapies where cells need to isms. Frequently, the term ‘false a result, biopharmaceutical com- be infused into patients before tra- positive’ is used when a positive panies are starting off with knowl- ditional sterility results would be result is seen with a PCR assay and edge of the risks involved and how a negative result is obtained with best to mitigate those risks, which the corresponding cell culture includes the use of the most effec- the use of newer assay (e.g., mycoplasma PCR and tive analytical methods,” she says. mycoplasma cell culture assays). In analytical techniques actuality, the PCR result was not a VArious choices false positive; the presence of the The adoption of alternative meth- DNA produced the PCR positive ods into a regulatory program can help mitigate risks. result as the assay was intended.” can be based on several factors, The alternative method may also such as the desire to reduce/ available, according to Dinwoodie. identify new or increased levels of eliminate the need for animal He also believes that PCR coupled known impurities, according to testing, shorten the turnaround with mass spectroscopy (Plex-ID) Dinwoodie. “If batches used for times for testing, and/or provide and massively parallel sequenc- safety and clinical assessments are a more comprehensive safety ing may provide advantages over still available for testing and can profile through either increased current techniques for detecting be shown to contain similar levels, sensitivity or specificity, accord- adventitious agents in cell sub- the impact of these impurities can ing to Chang. Some alternative strates and biological products. be more readily assessed.” In addi- test methods are in fact improve- “New techniques for determining tion, in the case of quantitative ments over existing techniques sub-visible particulates that enable assays, he notes that the alterna- rather than true alternatives and the detection and evaluation of tive methods may be concordant rely on the same properties of the particles of much smaller sizes are (i.e., show similar dose-response molecules to perform the analy- also providing conformational curves) but not truly equivalent. ses, according to Dinwoodie. He data that goes beyond traditional “Understanding the relationship points to capillary electropho- light obscuration methods,” adds between data sets derived from retic methods as substitutes for Dinwoodie. the two assays is critical, particu- gels to obtain greater resolution of larly in the case of potency assays,” closely related proteins within the compLicAted process Dinwoodie stresses. product and provide more robust To prove equivalence statisti- Another key challenge for quantification of these compo- cally, a large data set is needed. Chang is correlating the limit of nents. In addition, he suggests the “Generating a data set to show detection (LOD) of the two assays. use of ultra high-pressure liquid equivalence requires testing “Again, using the mycoplasma chromatography (UHPLC) meth- multiple samples that cover all example, the PCR LOD is reported ods instead of high-performance potential degradation routes and as genomic copies (GC), whereas liquid chromatography (HPLC) impurity profiles. Therefore, in the cell-based method is reported to reduce run times and increase order to show equivalence of a in colony-forming units (CFUs). sample throughput as an example. new method, testing must be con- The lack of an industry standard New technological methods for ducted on a bank of retain sam- mycoplasma stock can make it dif- rapid detection, identification, ples from historical production ficult to compare results because and quantification of adventitious runs and new stress and stability the GC/CFU ratio can vary agents have also emerged, accord- samples,” Dinwoodie states. depending on the species, storage, ing to Chang. “Many of these Beyond this issue, companies handling, and processing param- techniques are molecular-based, often face the challenge of cor- eters,” she explains.

40 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES446017_BP0614_040.pgs 05.28.2014 02:11 ADV Lot-release testing

pLAN ANd documeNt pitfALLs to AVoid “The best way to show equivalence is through It is crucial to include degraded or historical produc- statistical comparison of the results for the same tion samples in the equivalence test when changing a samples tested under standard and alternative method, according to Dinwoodie. In addition, if the methods; the greater the range of samples, the alternative is to be used from the start of the develop- more convincing the statistical argument,” asserts ment program, the validation of the method must be Dinwoodie. He adds that it is important to recog- considered. Chang adds that it is important to assess nize that the process will be a long one, and that an alternative assay’s readiness for use in a release alternative methods should be introduced to sta- program, which requires planning and accounts for bility programs when there is typically sufficient performing the validation and documentation of the sample available and they can be run alongside the information. “Companies need to ensure that resources current method to provide an initial assessment of are dedicated to a validation quality program to ensure equivalence. that the performance characteristics of the alternative If an alternative method has been used from method meet the requirement for the intended applica- the start of product development, it is possible to tion. In addition, for all methods, but particularly for justify using that method rather than the standard new/alternative methods, plans need to be in place to approach, according to Dinwoodie, but ultimately monitor assay performance over time,” she states. it is necessary to provide data to show that the Finally, Dinwoodie recommends that regula- method is fit for purpose. “In general, though, tra- tory authorities be consulted whether considering ditional techniques are commonly used in the early an alternative for an existing method used for an stages of product development and newer, more approved product or when developing the specification for a new product at the investigational new if an alternative method has been drug stage. ◆ used from the start of product development, it is possible to justify using that method rather than the standard approach.

sensitive, technologies are introduced as the product moves through clinical trials; this approach • Lot traceability gives time to run the stability studies and gather T data on multiple batches before the final submis- • ype III DMF sion,” he notes. The key message from Chang is the need to have • EP well-executed validation strategies. “Good science n requires well-planned, well-executed, well-docu- • Passivatio mented assays that generate meaningful interpretation of the results. The overall validation approach needs to be risk-based, with the level of validation required for each step established. The goal is to define the risk and validation strategies employed, as well as outline the steps in the validation lifecycle. If this approach is adopted, industry/regulators Contact sales at will be encouraged to move away from old tech- 215-957-9333 niques and embrace the next generation of novel platforms that are far more sensitive and rapid,” www.eaglestainless.com she asserts.

June 2014 www.biopharminternational.com BioPharm International 41

magentablackcyanyellow ES444953_BP0614_041.pgs 05.24.2014 02:13 ADV synthetic peptides control strategies for synthetic therapeutic peptide apis part iii: Manufacturing process considerations

Ivo Eggen, Brian Gregg, he United States Pharmacopeia and solid-phase synthesis methods, Harold Rode, (USP) Therapeutic Peptides the scope of synthetic peptide chemis- Aleksander Swietlow, TExpert Panel was formed in try has been further expanded in the Michael Verlander, and 2013 at the direction of the past two decades by the development Anita Szajek Monographs-Biologics & Biotechnology of native chemical ligation techniques, Expert Committee to evaluate quality which allow the coupling of unpro- attributes for synthetic peptides based tected peptide fragments to give even USP’s on currently available regulatory guid- larger assemblies (5). ance and expectations. This series of Therapeutic three articles by the panel explores the Manufacturing of current manufacturing and regulatory synthetic peptides Peptides Expert landscape and provides a comprehen- The chemical manufacturing of pep- sive overview of quality attributes to tides generally involves the following Panel discusses be considered for successful synthetic sequence of operations: peptide APIs from development through • Assembly of the protected peptide manufacturing manufacturing to lot release. The first sequence article (1) covered analytical character- • Removal of the semi-permanent processes and ization methods, lot-release tests and protecting groups points to consider for synthetic peptide • Modifications such as disulfide impurity control API manufacturers entering the market. bond formation and fragment cou- The second article (2) focused on raw plings for synthetic materials used in the chemical synthesis • Purification of the crude peptide of peptides. This last article in the series by preparative chromatography fol- peptide APIs. is devoted to manufacturing processes lowed by salt exchange and impurity control for synthetic pep- • Isolation of the final, purified peptide APIs. tide. In the 1950s, pioneers in the field, Assembly of the protected peptide sequence. such as Bodanszky and Du Vigneaud, The assembly of the peptide backbone produced the first bioactive peptides involves a series of cycles including a by purely synthetic methods in solu- coupling and a deprotection step. tion (3). Synthetic peptide chemistry During the coupling step, an acti- Ivo Eggen is section lead global technical received a big boost in 1963, when vated AA is coupled, usually in molar operations api chemistry at aspen pharma, Bruce Merrifield developed the method excess to ensure complete conversion, Brian Gregg is chief operating officer for synthesis on a solid support (solid- to the N-terminal amino acid of the at Bachem americas, Harold Rode is a phase peptide synthesis [SPPS]) (4). growing peptide chain. This AA is pro- consultant at rode Biologics consulting, Nowadays, solid-phase techniques and tected by a temporary protecting group Aleksander Swietlow is principal materials have evolved to the point that on its Nα-amino function and, if the scientist at amgen, Michael Verlander sequences exceeding 100 amino acids side chain of the AA is reactive under is technical advisor at polypeptide (AAs) in length have become feasible. the coupling conditions, it may also group, and Anita Szajek is principal Besides various hybrid techniques based be protected by a semi-permanent pro- scientific liaison at the us pharmacopeia. on extractive methods and solution- tecting group. Following coupling, the

42 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES444959_BP0614_042.pgs 05.24.2014 02:15 ADV More than an event

like Dolly was more than a sheep

Valuable education, partnering, global networking, exhibits and entertainment makes BIO 2014 much more than an event. The BIO International Convention regularly attracts 15,000 of the most powerful biotech and pharma players from 60+ countries, and every year we work to improve the experience. This year is no exception, with more networking, insight and opportunities delivering value to you and your business long after the event ends. Join us in San Diego and discover where BIO 2014 can take you.

magentablackcyanyellow ES446135_BP0614_043_FP.pgs 05.28.2014 03:12 ADV synthetic peptides

Nα-amino function of the AA is of the semi-permanent protect- ability of the selected purification selectively deprotected, leaving ing groups, with some approaches process to purge the impurities. side chain protecting groups on the employing orthogonal thiol pro- Following purification, a salt growing peptide intact and liberat- tecting groups to selectively effect exchange step is typically imple- ing the N-terminus of the grow- the desired connectivity, partic- mented to remove salts originating peptide for further elongation. ularly when multiple disulfide ing from the buffers used during Removal of excess AA derivative is bonds are required. purification to convert the peptide essential to prevent impurity for- Fragment couplings, as well as to the desired counterion and set mation. The same principles apply other modifications of specific pH. This salt exchange step may be to both classical solution-phase functions on the peptide sequence, achieved by an additional prepara- synthesis and solid-phase synthe- usually involve the selective depro- tive chromatography step or by use sis. However, in the latter approach, tection of the functional groups to of ion exchange resins. the growing peptide chain is be modified. Several types of pro- Isolation of the purified peptide. anchored at the C-terminus tecting groups for the various AA The isolation of the peptide API to a solid support, which allows functional groups have been devel- usually occurs through lyophili- removal of excess amino acid deriv- oped, which are either orthogonal zation of the concentrated aque- atives and coupling reagents by to the normal protecting schemes ous peptide solution following washing and filtration. or display a higher sensitivity purification and salt exchange. Removal of the semi-permanent pro- towards acidolysis. Alternatively, precipitation may tecting groups. Following assembly Purification of the crude peptide by be used as a more economical of the protected peptide sequence, preparative chromatography and salt process, although this may not the semi-permanent protect- exchange. Protected peptide frag- be feasible for all sequences and ing groups are generally removed ments have a low propensity to is likely to involve significant by acidolysis and the peptide is crystallize and, contrary to other development and engineering simultaneously cleaved from the classes of compounds, impurities to ensure that the API meets the resin support in the case of SPPS. formed in the course of peptide desired solid-state properties and Carbocations originating from synthesis usually accumulate up is fit for use in the drug product. the cleaved protecting groups are to the stage of the crude peptide. Although not yet widely applied generated under the harsh condi- This is particularly the case for in peptide manufacturing, other tions of acidolysis, requiring the peptides assembled on a solid sup- techniques, such as spray drying, use of scavengers to minimize port. The crude peptide is, there- may be used. impurities resulting from the fore, unlikely to meet the purity modification of the sensitive pep- specifications set for the API and types of iMpurities tide chain. In orthogonal protect- must be purified. Purification in synthetic peptides ing schemes involving only one typically involves preparative Several types of impurities may acidolytic deprotection step at chromatography, which may be encountered in synthetic pep- the end of the backbone assem- comprise sequential purification tides, which either originate from bly (Z and Fmoc chemistry), the steps that are based on differ- the raw materials, from the manu- integrity of the peptide sequence ent retention principles, such as facturing process, or are formed is better preserved than in a pro- ion exchange or reversed phase. by degradation during the manu- tecting scheme, which involves Purification processes developed facturing process or during stor- acidolysis in every cycle of the for peptide APIs typically result age (Figure 1). The various types of peptide assembly, followed by a from an extensive initial screen- manufacturing process impurities, harsh acidolysis after the backbone ing of various purification meth- together with their origins, are pre- assembly (Boc chemistry). ods on a laboratory scale and rely sented in Table I, while typical deg- Modifications such as disulfide bond on a meticulously developed and radation impurities are presented formation and fragment couplings. A validated in-process analytical in Table II. Identification methods peptide may contain one or more high-performance liquid chroma- and detectability are indicated in generally intramolecular and well- tography (HPLC) method, which both tables. defined disulfide bonds between is able to discern actual impuri- Deletion sequences. Deletion Cysteine residues. Formation of ties generated by the synthesis sequences lack one or more amino disulfide bonds may be achieved process. This requires an in-depth acid residues. These sequences during assembly of the peptide knowledge of the impurity pro- originate either from incomplete backbone, or after the removal file and an understanding of the coupling or from incomplete

44 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES444956_BP0614_044.pgs 05.24.2014 02:14 ADV synthetic peptides

Figure 1: Peptide manufacturing flow.

OTHER MODIFICATIONS Disulfde Critical raw Backbone Fragment Acidolysis bond Purifcation Isolation API Stability materials assembly coupling (Amino acid derivatives) formation

Insertion Deletion Functional Functional Truncation Purging of Disulfde Functional sequences sequences group group sequences impurities modifcations group modifcations modifcations modifcations Diastereomers Insertion Diastereomers sequences Disulfde (Deamidation, Functional Substitution modifcations Acetylation) Truncation group sequences Disulfde sequences modifcations modifcations Diastereomers Functional group modifcations

Nα-deprotection steps, especially sequences contain one or more plings, which is often the case in around so-called “difficult “double” AA residues. The pres- SPPS. C-terminally truncated sequences” in SPPS, and hence ence of either Nα-unprotected AA sequences, on the other hand, may require careful in-process controls derivatives or dipeptides in the be generated when quenching is during backbone assembly. The starting AA derivatives leads to the part of the synthesis protocol to Kaiser colorimetric test (6), typi- formation of insertion sequences prevent insertion sequences. The cally applied in SPPS to monitor and can be controlled by setting structure of truncated sequences is completion of deprotection and appropriate specifications for usually sufficiently different from coupling reactions, is rapid and these materials. Alternatively, dur- the target sequence to allow for effi- straightforward but not always sen- ing peptide backbone assembly, cient removal during purification. sitive enough to determine quanti- incomplete removal of excess AA Diastereomers. Diastereomeric tative completion of coupling and derivative prior to the next depro- sequences contain one or more especially deprotection reactions. tection and coupling cycle will AA residues in the undesired chi- Hence, use of the Kaiser test may lead to the formation of insertion ral form. Diastereomers are usually not completely prevent the for- sequences. To prevent process- more difficult to remove during mation of deletion sequences. In related insertion sequences, fol- purification and typically present such instances, chromatographic lowing the coupling step, excess a greater separation challenge by analysis can provide quantitative activated AA derivative must analytical HPLC than other types results, but requires a soluble inter- be inactivated by quenching or of impurities. Their identification mediate or, in the case of SPPS, extraction prior to the subsequent usually depends on HPLC spiking requires cleavage of a resin sample. deprotection step, and the excess experiments of the peptide prod- Sequences lacking the C-terminal of inactivated AA derivative must uct with the synthesized diaste- residue may arise from incomplete be removed prior to the next cou- reomeric analogs. Such spiking coupling of the first AA residue to pling step. Removal of a specific experiments may also corroborate a solid-phase resin, when this step insertion sequence impurity dur- the suitability of the analytical is not followed by an efficient cap- ing purification is typically more release method(s). ping protocol for residual, active difficult when the inserted amino Diastereomers may originate anchoring sites. Removal of a spe- acid is relatively simple. from the presence of the optical cific deletion sequence impurity Truncation sequences. antipode in starting AA deriva- during purification is typically N-terminally truncated sequences tives, requiring the establish- more difficult when the missing may be generated when capping is ment of appropriate specification amino acid is relatively simple used as part of the synthesis proto- limits for these raw materials. (e.g., Glycine or Alanine). col to prevent deletion sequences Alternatively, diastereomers may

FIGURE 1 IS COURTESY OF THE AUTHORS Insertion sequences. Insertion that result from incomplete cou- be formed during peptide back-

June 2014 www.biopharminternational.com BioPharm International 45

magentablackcyanyellow ES444962_BP0614_045.pgs 05.24.2014 02:15 ADV synthetic peptides

Table I: Potential synthetic peptide process-related impurities. Substitution sequences. Impurities Origin of impurities Identifcation method HPLC detectability Substitution sequences occur when Deletion Synthesis LC-MS or LC-MS/MS + one or more AA residues have Raw material or been substituted by another AA Insertion LC-MS or LC-MS/MS + synthesis residue, the most common being ↔ Truncation Synthesis LC-MS + an Isoleucine Leucine substitution. Substitution sequences HPLC spiking with Raw material or Diastereomer synthesized diastereomeric +/- originate from the presence of synthesis analogs contaminants in the starting AA HPLC spiking with derivatives and can consequently Substitution Raw material synthesized analogs or - (Leu/Ile) be controlled by setting appro- isolation/AAA priate specifications for these Functional raw materials. The same purifica- group Synthesis or stability LC-MS or LC-MS/MS +/- modification tion and analytical HPLC chal- Disulfide lenges described for diastereomer Synthesis or stability LC-MS or LC-MS/MS ++ modification sequences may apply to substitution sequences, especially for the LC-MS is liquid chromatography-mass spectrometry. LC-MS/MS is liquid chromatography-tandem mass spectrometry. HPLC is high-performance liquid chromatography. AAA is amino acid analysis. Leu is Leucine. Ile is Isoleucine. Isoleucine ↔ Leucine substitution. Modifications of functional groups Table II: Potential peptide degradation impurities. and disulfide bonds. Several AA side Impurities Identifcation method HPLC detectability chains are susceptible to modifi- Deamidation of Gln/Asn/C- cation, either during synthesis or LC-MS or LC-MS/MS +/- terminus during storage. AAs may undergo Acetylation of amino rearrangements during coupling LC-MS or LC-MS/MS ++ functions (e.g., Asparagine, Aspartic acid, Disulfide modification LC-MS or LC-MS/MS ++ and Glutamine) or may be prone to degradation or electrophilic Gln is Glutamine. Asn is Asparagine. substitution during acidolysis (e.g., primary amides, Tryptophan, bone assembly through epimer- in the C-terminal position of the Tyrosine, and Methionine). ization. Several epimerization fragment being activated in the Alternatively, modifications of mechanisms are known that either fragment condensation. All other functional groups may arise from involve 5(4H)-oxazolone forma- residues in this position will lead incomplete removal of protecting tion during activation of carbox- to some degree of epimerization, groups. ylic functions (during coupling) or which can only be minimized by Impurities related to disulfide direct Cα-proton abstraction under applying special coupling proto- bond modification include reduced basic conditions. AA residues with cols. Furthermore, the esterifica- (linear) monomers, oxidized (par- a relatively acidic Cα-proton such tion of AA derivatives under basic allel and anti-parallel) dimers and as Cysteine and Histidine are espe- conditions, with the exception of higher polymers, isomers aris- cially sensitive to epimerization. Glycine and Proline, will result in ing from scrambling of disulfide Synthetic approaches have been some degree of epimerization, the bonds, and oxidized disulfides developed to minimize epimer- most frequent examples arising (thiosulfinates). The desired isomer ization, the most basic being that from esterification of the first AA is usually obtained under strictly the peptide backbone is assem- to a solid-phase resin. Finally, pep- controlled and optimized process bled from the C-terminus to the tides with a C-terminal Cysteine conditions. The handling of con- N-terminus by stepwise cou- ester are prone to epimerization centrated solution of the peptide pling of Nα-urethane protected during base treatment and, there- product following the purification AAs. Some AA derivatives, such fore, direct esterification to a process should be carefully con- as Cysteine and Histidine deriva- solid-phase resin for Fmoc synthe- trolled to avoid polymerization. tives, may require special coupling sis should be avoided. Numerous The most common degradation protocols. Concerning fragment coupling reagents have been devel- mechanisms include deamida- couplings, the only safe options oped over the years to promote fast tion of Asparagine, Glutamine and to prevent epimerization are the and epimerization-minimizing the C-terminal amide function, use of a Glycine or Proline residue coupling conditions. acetylation of amino functions

46 BioPharm International www.biopharminternational.com June 2014

magentablackcyanyellow ES444961_BP0614_046.pgs 05.24.2014 02:15 ADV synthetic peptides

by residual acetate, and disulfide cess, including identification of resulting from peptide backbone modification (i.e., polymerization). raw material attributes and process assembly, removal of protecting The identification of criti- parameters that affect API quality, groups, and modification of pep- cal process parameters, as well as an appropriate control strategy for tide functional groups. extensive stability studies, provide peptide-related impurities can be USP hopes that the work of the process and product understand- developed to achieve a manufac- Therapeutic Peptides Expert Panel ing, allowing for optimization of turing process that is both eco- and this series of articles will pro- the manufacturing process and nomically feasible and also able vide more consistent guidance to definition of hold-times and stor- to yield a peptide API meeting help support efforts to create a sus- age conditions, thereby contrib- predetermined quality attributes. tainable platform for the future of uting to the preservation of the An understanding of the ability of peptide-based drugs. integrity of the target peptide. the purification process to remove peptide impurities can be used references conclusion to define appropriate impurity 1. I. Eggen et al., BioPharm Intl. 27(3) Synthetic peptide-related impuri- specifications for AA derivatives (2014). ties may result from impurities in used as raw materials, as well as to 2. I. Eggen et al., BioPharm Intl. 27(4) AA derivatives used as raw materi- define and control critical process (2014). 3. Bodanszky, M. and du Vigneaud, V. J. als, from the manufacturing pro- parameters during manufactur- Am. Chem. Soc. 81 5688-5691 (1959). cess itself, and from degradation ing. Increased process knowledge 4. Merrifield, R.B. J. Am. Chem. Soc. 85 of the peptide during manufac- may provide an understanding of 2149-2154 (1963). turing or upon storage. Based on the ability of the purification pro- 5. P.E. Dawson et al., Science 266 776-778 a thorough understanding of the cess to tolerate variability in the (1994). peptide, its stability characteris- quality of raw materials as well as 6. E. Kaiser et al., Anal. Biochem. 34 595- tics and its manufacturing pro- variability in the crude peptide 598 (1970). ◆

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TOC Analyzer SEC-MALS Handheld Analyzer Increases Productivity Detector for UHPLC Improves Accuracy

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magentablackcyanyellow ES444981_BP0614_048.pgs 05.24.2014 02:15 ADV Regulatory Beat New Technology Showcase

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June 2014 www.biopharminternational.com BioPharm International 49

magentablackcyanyellow ES446126_BP0614_049.pgs 05.28.2014 02:59 ADV BIOLOGICS NEWS PIPELINE

Stem Cells From Human Body Fat Used to Fight Brain Cancer engineered bone from mice, then placed it in a microflu- Researchers from Johns Hopkins have stated that they idic device that steadily supplied nutrients and removed were able to extend the lives of mice with aggressive waste to mimic the circulation the tissue would experi- brain tumors by delivering biological treatments directly ence in the body. Marrow in the device remained healthy to the brains of the mice using stem cells derived from for up to one week, long enough to test the toxicity and human body fat. The researchers suggest that surgically effectiveness of a new drug. Similar to marrow from live removed glioblastomas (brain cancer cells) could be used mice, this engineered marrow was also susceptible to radi- in humans to find and destroy remaining cancer cells in ation, but an FDA-approved drug that protects irradiated difficult-to-reach areas of the brain. patients also protects the marrow on the chip. According The researchers used mesenchymal stem cells (MSCs) to the study, the device could be used to develop safe and harvested from human fat tissue in the mouse experi- effective strategies to prevent or treat radiation’s lethal ments. MSCs were modified to secrete bone morpho- effects on bone marrow without resorting to animal test- genetic protein 4 (BMP4), a small protein involved in ing. Bone marrow-on-a-chip could also be used to main- regulating embryonic development and known to have tain a cancer patient’s own marrow temporarily while some tumor suppression function. The researchers undergoing marrow-damaging treatments. The report is injected stem cells armed with BMP4 into the brains featured in the May 2014 online issue of Nature Methods. of mice that had already been given glioblastoma cells several weeks earlier. In a report published in Clinical NIH Study Reveals New Cancer Immunotherapy Method Cancer Research, the investigators say the mice treated Scientists at the National Cancer Institute (NCI) have with BMP4 had less tumor growth and spread, and developed a new method for using immunotherapy to their cancers were less aggressive and had fewer migra- specifically attack tumor cells that have mutations unique tory cancer cells compared to mice that didn’t get the to a patient’s cancer. The research showed that human treatment. In addition, the mice that received stem cells melanoma tumors often contain mutation-reactive with BMP4 survived significantly longer, living an aver- immune cells called tumor-infiltrating lymphocytes age of 76 days, as compared to 52 days in the untreated (TILs). The presence of these cells may help explain the mice. These findings build on research published in the effectiveness of adoptive cell therapy (ACT) and other March 2013 issue of PLOS ONE, which showed that har- forms of immunotherapy in the treatment of mela- vesting MSCs from fat was much less invasive and less noma. In this study, which appeared in the May 2014 expensive than harvesting from bone marrow. issue of Science, the researchers set out to determine whether TILs from patients with metastatic gastroin- Bone Marrow-on-a-Chip Developed to Test Drug Effects testinal cancers could recognize patient-specific muta- Researchers from Harvard’s Wyss Institute for tions. They analyzed TILs from a patient with bile duct Biologically Inspired Engineering have reproduced cancer that had metastasized to the lung and liver and the structure, functions, and cellular make-up of bone had not been responsive to standard chemotherapy. marrow, to create a device known as “bone marrow- The researchers used whole-exome sequencing, in on-a-chip.” The device could give scientists a tool to which the protein-coding regions of DNA are ana- test the effects of new drugs and toxic agents on whole lyzed to identify mutations that the patient’s immune bone marrow. The researchers packed dried bone pow- cells might recognize. They found that some of the der into an open, ring-shaped mold the size of a coin patient’s TILs recognized a mutation in a protein called battery, and implanted the mold under the skin of a ERBB2-interacting protein (ERBB2IP). The patient then mouse’s back. After eight weeks, the disk-shaped bone underwent adoptive cell transfer of 42.4 billion TILs, that had formed in the mold was surgically removed approximately 25% of which were ERBB2IP mutation- and examined with a CAT scanner. The scan showed reactive T lymphocytes, followed by treatment with four a honeycomb-like structure that looked identical to doses of the anticancer drug interleukin-2 to enhance natural trabecular bone. When the stained tissue was T-cell proliferation and function. Following transfer of examined under a microscope, the marrow was packed the TILs, the patient’s metastatic lung and liver tumors with blood cells, just like marrow from a living mouse. stabilized. When the patient’s disease progressed, they In addition, when the researchers sorted the bone mar- were retreated with ACT in which 95% of the transferred row cells by type, the mix of different types of blood cells were mutation-reactive T cells, and experienced and immune cells in the engineered bone marrow was tumor regression that was ongoing six months after the identical to that in a mouse thighbone. second T-cell infusion. These results suggest that a T-cell To sustain the engineered bone marrow outside of a response against a mutant protein can be harnessed to living animal, the researchers surgically removed the mediate regression of a metastatic epithelial cell cancer.

50 BioPharm International www.biopharminternational.com June 2014

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