Process Validation in Manufacturing of Biopharmaceuticals T H I R D E D I T I O N BIOTECHNOLOGY AND BIOPROCESSING SERIES Series Editor Anurag Rathore 1. Membrane Separations in Biotechnology, edited by W. Courtney McGregor 2. Commercial Production of Monoclonal Antibodies: A Guide for ScaleUp, edited by Sally S. Seaver 3. Handbook on Anaerobic Fermentations, edited by Larry E. Erickson and Daniel YeeChak Fung 4. Fermentation Process Development of Industrial Organisms, edited by Justin O. Neway 5. Yeast: Biotechnology and Biocatalysis, edited by Hubert Verachtert and René De Mot 6 . Sensors in Bioprocess Control, edited by John V. Twork and Alexander M. Yacynych 7. Fundamentals of Protein Biotechnology, edited by Stanley Stein 8. Yeast Strain Selection, edited by Chandra J. Panchal 9. Separation Processes in Biotechnology, edited by Juan A. Asenjo 10. LargeScale Mammalian Cell Culture Technology, edited by Anthony S. Lubiniecki 11. Extractive Bioconversions, edited by Bo Mattiasson and Olle Holst 12. Purification and Analysis of Recombinant Proteins, edited by Ramnath Seetharam and Satish K. Sharma 13. Drug Biotechnology Regulation: Scientific Basis and Practices, edited by Yuanyuan H. Chiu and John L. Gueriguian 14. Protein Immobilization: Fundamentals and Applications, edited by Richard F. Taylor 15. Biosensor Principles and Applications, edited by Löíefc J. Blum and Pierre R. Coulet 16. Industrial Application of Immobilized Biocatalysts, edited by Atsuo Tanaka, Tetsuya Tosa, and Takeshi Kobayashi 17. Insect Cell Culture Engineering, edited by Mattheus F. A. Goosen, Andrew J. Daugulis, and Peter Faulkner 18. Protein Purification Process Engineering, edited by Roger G. Harrison 19. Recombinant Microbes for Industrial and Agricultural Applications, edited by Yoshikatsu Murooka and Tadayuki Imanaka 20. Cell Adhesion: Fundamentals and Biotechnological Applications, edited by Martin A. Hjortso and Joseph W. Roos 21. Bioreactor System Design, edited by Juan A. Asenjo and José C. Merchuk 22. Gene Expression in Recombinant Microorganisms, edited by Alan Smith 23. Interfacial Phenomena and Bioproducts, edited by John L. Brash and Peter W. Wojciechowski 24. Metabolic Engineering, edited by Sang Yup Lee and Eleftherios T. Papoutsakis
Process Validation in Manufacturing of Biopharmaceuticals THIRD EDITION BIOTECHNOLOGY AND BIOPROCESSING SERIES Series Editor Anurag Rathore Membrane Separations in Biotechnology, edited by W Courtney McGregor Commercial Production of Monoclonal Antibodies: A Guide for Scale-Up, edited by Sally S Seaver Handbook on Anaerobic Fermentations, edited by Larry E Erickson and Daniel Yee-Chak Fung Fermentation Process Development of Industrial Organisms, edited by Justin O Neway Yeast: Biotechnology and Biocatalysis, edited by Hubert Verachtert and René De Mot Sensors in Bioprocess Control, edited by John V Twork and Alexander M Yacynych Fundamentals of Protein Biotechnology, edited by Stanley Stein Yeast Strain Selection, edited by Chandra J Panchal Separation Processes in Biotechnology, edited by Juan A Asenjo 10 Large-Scale Mammalian Cell Culture Technology, edited by Anthony S Lubiniecki 11 Extractive Bioconversions, edited by Bo Mattiasson and Olle Holst 12 Purification and Analysis of Recombinant Proteins, edited by Ramnath Seetharam and Satish K Sharma 13 Drug Biotechnology Regulation: Scientific Basis and Practices, edited by Yuan-yuan H Chiu and John L Gueriguian 14 Protein Immobilization: Fundamentals and Applications, edited by Richard F Taylor 15 Biosensor Principles and Applications, edited by Lưíefc J Blum and Pierre R Coulet 16 Industrial Application of Immobilized Biocatalysts, edited by Atsuo Tanaka, Tetsuya Tosa, and Takeshi Kobayashi 17 Insect Cell Culture Engineering, edited by Mattheus F A Goosen, Andrew J Daugulis, and Peter Faulkner 18 Protein Purification Process Engineering, edited by Roger G Harrison 19 Recombinant Microbes for Industrial and Agricultural Applications, edited by Yoshikatsu Murooka and Tadayuki Imanaka 20 Cell Adhesion: Fundamentals and Biotechnological Applications, edited by Martin A Hjortso and Joseph W Roos 21 Bioreactor System Design, edited by Juan A Asenjo and José C Merchuk 22 Gene Expression in Recombinant Microorganisms, edited by Alan Smith 23 Interfacial Phenomena and Bioproducts, edited by John L Brash and Peter W Wojciechowski 24 Metabolic Engineering, edited by Sang Yup Lee and Eleftherios T Papoutsakis 25 Biopharmaceutical Process Validation, edited by Gail Sofer and Dane W Zabriskie 26 Membrane Separations in Biotechnology: Second Edition, Revised and Expanded, edited by William K Wang 27 Isolation and Purification of Proteins, edited by Rajni Hatti-Kaul and Bo Mattiasson 28 Biotransformation and Bioprocesses, Mukesh Doble, Anil Kumar Kruthiventi, and Vilas Gajanan Gaikar 29 Process Validation in Manufacturing of Biopharmaceuticals: Guidelines, Current Practices, and Industrial Case Studies, edited by Anurag Singh Rathore and Gail Sofer 30 Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, edited by Sadettin S Ozturk and Wei-Shou Hu 31 Process Scale Bioseparations for the Biopharmaceutical Industry, edited by Abhinav A Shukla, Mark R Etzel, and Shishir Gadam 32 Processs Synthesis for Fuel Ethanol Production, C A Cardona, Ó J Sánchez, and L F Gutiérrez 33 PAT Applied in Biopharmaceutical Process Development And Manufacturing: An Enabling Tool for Quality-by-Design, edited by Cenk Undey, Duncan Low, Jose C Menezes, and Mel Koch 34 Stem Cells and Revascularization Therapies, edited by Hyunjoon Kong, Andrew J Putnam, and Lawrence B Schook 35 Process Validation in Manufacturing of Biopharmaceuticals, Third Edition, edited by Anurag Rathore and Gail Sofer BIOTECHNOLOGY AND BIOPROCESSING SERIES VOLUME 35 Process Validation in Manufacturing of Biopharmaceuticals THIRD EDITION EDITED BY ANURAG S RATHORE GAIL SOFER Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2012 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S Government works Version Date: 20120404 International Standard Book Number-13: 978-1-4398-5094-7 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint Except as permitted under U.S Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers For permission to photocopy or use material electronically from this work, please access www.copyright com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400 CCC is a not-for-profit organization that provides licenses and registration for a variety of users For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com Contents Preface .ix Editors xiii Contributors xv Chapter Guidelines to Process Validation Gail Sofer Chapter Commentary on the US Food and Drug Administration’s 2011 “Guidance for Industry, Process Validation General Principles and Practices” 11 Hal Baseman Chapter Applications of Failure Modes and Effects Analysis to Biotechnology Manufacturing Processes 51 Robert J Seely and John Haury Chapter Process Characterization 63 James E Seely Chapter Scaled-Down Models for Purification Processes: Approaches and Applications 89 Ranga Godavarti, Jon Petrone, Jeff Robinson, Richard Wright, Brian D Kelley, and Glen R Bolton Chapter Adventitious Agents: Concerns and Testing for Biopharmaceuticals 141 Raymond W Nims, Esther Presente, Gail Sofer, Carolyn Phillips, and Audrey Chang Chapter Lifespan Studies for Chromatography and Filtration Media 159 Anurag S Rathore and Gail Sofer Chapter Validation of a Filtration Step 185 Jennifer Campbell vii viii Contents Chapter Analytical Test Methods for Well-Characterized Biological and Biotechnological Products 235 Nadine Ritter and John McEntire Chapter 10 Facility Design Issues: A Regulatory Perspective 269 Susan Vargo and Nancy Kavanaugh Chapter 11 Validation of Computerized Systems 313 Monica J Cahilly Chapter 12 Process Validation with a CMO 355 Susan Dana Jones, Sheila G Magil, and Gail Sofer Chapter 13 Risk Management and Validation 375 James Agalloco Chapter 14 Process Validation in Membrane Chromatography 385 Suma Ray and Miyako Hirai Chapter 15 Leveraging Multivariate Analysis Tools to Qualify ScaledDown Models 411 John Pieracci and Helena Yusuf-Makagiansar Chapter 16 Process Validation of a Multivalent Bacterial Vaccine: A Novel Matrix Approach 441 Narahari S Pujar, Marshall G. Gayton, Wayne K. Herber, Chitrananda Abeygunawardana, Michael L. Dekleva, P. K. Yegneswaran, and Ann L Lee Chapter 17 Validation of the Zevalin® Purification Process: A Case Study 455 Lynn Conley, John McPherson, and Jörg Thömmes Chapter 18 Viral Clearance Validation: A Case Study 491 Michael Rubino, Mark Bailey, Jeffrey C Baker, Jeri Ann Boose, Lorraine Metzka, Valerie Moore, Michelle Quertinmont, and William Wiler Preface This updated edition of Process Validation in Manufacturing of Biopharmaceuticals provides insights into current guidelines and expectations Current practices are discussed and illustrated with industrial case studies Chapter presents an overview of the new validation paradigm of process development, process qualification, and continuous monitoring—an approach that is intended to provide even greater assurance that a process will perform consistently to produce a drug substance with its requisite critical quality attributes (CQAs) Some of the current process validation concerns are presented Chapter provides background and industry commentary on the final version of the Food and Drug Administration (FDA) 2011 “Guidance for Industry, Process Validation General Principles and Practices,” commonly referred to as the Process Validation Guidance or PVG, which was issued in final form on January 24, 2011 Understanding the background and intent of the PVG should make it easier to develop plans that are aligned with the recommendations presented in the PVG and result in more effective process validation approaches In Chapter 3, the use of a risk assessment method (failure modes and effect analysis [FMEA]) is presented as a means to prioritize process parameters for further process characterization prior to validation FMEA provides a logical approach that can aid in establishing critical parameters and ensure process robustness Specific examples on the use of FMEA will aid readers in establishing this method in their own organization Process characterization is a prerequisite for process validation In Chapter 4, a description of how to carry out thorough and consistent process characterization is presented Precharacterization studies, which are used to help define the scope of the actual experimental characterization work, are also discussed The discussions on timing of process characterization, needed resources, and a stepwise approach provide valuable insights The importance of scale down in process characterization is also addressed Accurately scaling down to mimic manufacturing processes is essential in several aspects of process validation Chapter provides further guidance and strategies for scaling down unit operations, including chromatography, chemical modification reactions, ultrafiltration, and microfiltration In addition to general scale-down principles and parameters, the authors address specific problems and present some examples Prior to establishing a process that can be validated, it is essential to consider potential risks from adventitious agents, which include viruses, bacteria, fungi, mycoplasma, and transmissible spongiform encephalopathies The potential sources of these agents and testing programs for them are described in an updated Chapter Current examples of contamination events in biopharmaceutical manufacturing are presented Bioburden assessment and sterility issues are also addressed, and a summary table describes adventitious agents, recommended tests, and stages at which to perform testing ix x Preface In Chapter 7, the lifespan of both chromatography and filtration media is addressed There are discussions on the various factors that influence lifespan, and experimental approaches for validation The use of small-scale models for validation is discussed The application of concurrent validation to provide lifespan data, an approach that is gaining more acceptance, is discussed in this chapter Chapter begins with an overview of filtration validation and a discussion of validation that can be performed in scaled-down studies as well as those aspects that require manufacturing scale Next is a section on the validation of sterilizing grade filters Subsequent sections address validation of filters used for clarification and virus removal filters Details of tangential flow filter validation are presented Also included are descriptions of specific validation issues in clarification of bacterial cell harvest and lysate clarification, mammalian cell clarification, and protein concentration and diafiltration Cleaning validation for reusable membranes is also discussed It has been said that without assays, you have nothing In an updated and current Chapter 9, analytical test methods are discussed with a special focus on well-characterized biological and biotechnological products Appropriate methods for testing raw materials and in-process samples during the various manufacturing steps are addressed The authors also discuss process analytical technology (PAT), which is being driven by the FDA as a means to better control processes Another section of this chapter presents methods used for product characterization, release, and stability testing Also included are the ever-problematic potency assay and strategies for choosing a quality control testing scheme Other topics discussed are the use of assays for demonstrating comparability, assay validation, dealing with out-of-specification (OOS) results, and assay revalidation In Chapter 10, the reader is provided with a regulatory perspective on facility design and validation issues Written by two former FDA employees, this chapter provides details on the regulatory requirements and the information that should be provided in a license application Also presented are the requirements for cell inoculum suites and areas intended for fermentation/harvest, purification, and bulk filtration In addition, support areas, such as those used for preparation of media and buffers, and the use of closed systems to reduce environmental classifications are discussed There are extensive sections on utilities, cleaning, and environmental monitoring Multiproduct facility issues are addressed In the section on facility inspections, the authors provide insight into the current focus of inspections Chapter 11 has been updated and discusses the importance of taking a risk-based approach toward computerized system compliance and how it adds value to the product and process that is commensurate with cost It is concluded that a sound computer system validation (CSV) program encourages the introduction of new and exciting technologies with the ultimate promise of safer, more effective, and more affordable medicines Today, many firms are dependent on contract manufacturing organizations (CMOs) to perform process validation Chapter 12 presents strategies for a team approach to ensure process validation is carried out according to current expectations A table that delineates the responsibilities of the sponsor and CMO provides a practical tool The application of risk management in validation is described in Chapter 13 488 Process Validation in Manufacturing of Biopharmaceuticals FTIR Spectrum of HCl pH Residue 4000 3500 3000 2500 2000 1500 Wavenumber (cm–1) 1000 FTIR Reference Spectrum of Silica 4000 3500 3000 2000 1500 2500 Wavenumber (cm–1) 1000 FIGURE 17.14 FTIR spectra of HCl pH extraction buffer residue and silica references standard consistency These parameters are continually reevaluated as further manufacturing experience is gained This reevaluation will allow revision of the action limits and acceptance criteria of future validations as a larger data set is obtained The Zevalin purification process has been shown to effectively and consistently produce a product that meets its predetermined specifications and quality attributes The validation of the purification process was accomplished by using a combination of full- and small-scale studies that encompassed a broad range of activities Full-scale process validation studies demonstrated the consistency and reliability of the purification process under normal operating conditions Small-scale process validation studies demonstrated the capability of the process at extreme operating conditions The small-scale models for the chromatography and nanofiltration steps were qualified as being representative of the manufacturing process prior to or concurrent with each validation Small-scale studies demonstrated that the process is capable of removing and inactivating a broad range of viruses as well as other impurities and contaminants to safe levels The small-scale process validation studies complemented the full-scale studies by providing additional understanding of the purification process In the current development of newer processes, process validation issues are taken into consideration much earlier in development of the purification process by use of Validation of the Zevalin® Purification Process 489 a design of experiment methodology Design of experiment (DOE) is a statistically based methodology in which several operational parameters can be evaluated at the same time using two levels, one at high level and one at low level Significant operational parameters and their interactions affecting a process step can be identified, and a model predicting the response or output of a performance parameter can be developed The design of experiment can be set up to provide a high degree of statistical confidence in the predicted outcome of performance parameters The DOE can be used to find and predict the range of operational parameters that will provide the optimal performance parameters in terms of yield and purity The 95% prediction interval from the model developed by the DOE can be used to define acceptance criteria in process validation Characterization studies evaluating both the high and low maximum process operating ranges at small scale or pilot scale are an acceptable means to verify the DOE model such that it can be used to set acceptance criteria for full-scale validations REFERENCES International Conference on Harmonization (ICH) “Good Manufacturing Practice for Active Pharmaceutical Ingredients, Q7A, Step 4,” November 10, 2000 U.S Food and Drug Administration “Guideline on General Principles of Process Validation,” May 1987 U.S Food and Drug Administration “Manufacturing, Processing or Holding Active Pharmaceutical Ingredients,” March 1998 Seely, R., M Tomusiak, and R Kuhn In Biopharmaceutical Process Validation Edited by G Sofer and D Zabriske New York: Marcel Deker, 2000, 130 Gardner, A., T Smith, R Gerber, and D Zabriskie “Worst Case Approach to Validating Operation Ranges.” In Validation of Bio-pharmaceuticals Manufacturing Processes, ACS Symp. Ser No 698 Edited by B Kelly and A Ramelmeir Washington, DC: ACS Books, 69–79 Smith, T., E Wilson, R Scott, J Misczak, J Bodek, and D Zabriskie “Establishment of Operating Ranges in a Purification Process for a Monoclonal Antibody.” In Validation of Biopharmaceuticals Manufacturing Processes, ACS Symp. Ser No 698 Edited by B Kelly and A Ramelmeir Washington, DC: ACS Books, 80–92 International Conference on Harmonization (ICH) “Guideline Q6A Step 4, Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products,” March 1999 International Conference on Harmonization (ICH) “Q5A Step 4, Consensus Guideline, Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin,” (CPMP/ICH/295/95), 1995 Darling, A “Validation of Biopharmaceutical Purification Process for Viral Clearance Evaluation.” Mol Biotechnol., May 2002, 57–83 10 Center for Biologics Evaluation and Research “Points to Consider in the Manufacturing and Testing of Monoclonal Antibody Products for Human Use.” Rockville, MD: US Food and Drug Administration, 1997 11 World Health Organization “Acceptability of Cell Substances for Production of Biologicals.” World Health Organization Tech Report Ser 747, 1987 12 Griffiths, E “WHO Expert Committee on Biological Standardization: Highlights of the Meeting of October 1996.” Biologicals 25 (1997): 359–362 490 Process Validation in Manufacturing of Biopharmaceuticals 13 Sofer, G., and L Hagel Handbook of Process Chromatography: A Guide to Optimization Scale-Up and Validation San Diego, CA: Academic Press, 1997, 159 14 Gagnon, P Purification Tools for Monoclonal Antibodies Tucson, AZ: Validated Biosystems, 1996, 174–175 15 “Technical Report No 26, Sterilizing Filtration of Liquids.” PDA J Pharm Sci Technol., 2008, 52 (suppl.) 16 Stone, T., V Goel, and J Loszcak Methodology for Analysis of Filter Extractables: A Model Solvent Approach.” Pharm Technol 18 (1994): 116–130 17 Reif, O., P Solkner, and J Rupp. “Analysis and Evaluation of Filter Cartridge Extractables for Validation in Pharmaceutical Downstream Processing.” Pharm Technol 50 (1996): 399–410 18 Weitzmen, C “The Use of Model Solvents for Evaluating Extractables from Filters Used to Process Pharmaceutical Products.” Pharm Technol 10 (1997): 72–99 18 A Case Study Viral Clearance Validation Michael Rubino, Mark Bailey, Jeffrey C Baker, Jeri Ann Boose, Lorraine Metzka, Valerie Moore, Michelle Quertinmont, and William Wiler CONTENTS 18.1 Strategy and Planning 491 18.2 Logistical Considerations 493 18.3 Protocol Development and Execution 494 18.3.1 Calculation of Reduction Factors 495 18.4 Results 496 18.4.1 Cytotoxicity, Viral Interference, and Frozen Viability 496 18.4.2 Laboratory-Scale Process Evaluation 496 18.4.3 Viral Clearance per Individual Process Steps 497 18.4.3.1 Viral Inactivation Step 497 18.4.3.2 Chromatography Step 497 18.4.3.3 Heat 497 18.4.3.4 Nanofiltration 497 18.4.3.5 Chromatography Step 500 18.4.4 General Comments on Viral Clearance 501 18.5 Conclusion 501 Acknowledgments 502 References 502 18.1 STRATEGY AND PLANNING The development and planning of a viral clearance study, as stipulated in the guidance documents, is related to the potential for viruses to enter the production system from either the cell line or other sources such as raw materials [1–6] The International Conference on Harmonization (ICH) Q5A, in fact, ranks cell lines based on the presence of viral particles or viruses [7] This stratification of the cell line will then determine the viral clearance that needs to be demonstrated Other documents written by the Food and Drug Administration (FDA) or the European authorities provide general guidance and in some cases details on the design and implementation of viral clearance studies The activities surrounding the planning and designing for the viral clearance study for a mammalian cell-derived protein included use of the guidance documents 491 492 Process Validation in Manufacturing of Biopharmaceuticals as a source for the design Two primary considerations were taken into account in the planning stages First, the expression system for production of the protein is a human-derived cell line Second, the production and purification of the protein in this study required the use of animal-sourced materials An analysis of the cell line provided information needed to determine the impact on the viral clearance studies Although the cell line had not been reported in the scientific literature to contain retroviral particles or any evidence of retrovirus infection, the cell line prior to and subsequent to the production of a good manufacturing practice (GMP) master cell bank was tested for retroviruses, retroviral particles, and other viruses No evidence of viral infection or expression of viral particles was detected Based on this information, the ICH Q5A makes the cell line a Case A The ICH Q5A then suggests that model viruses be used for viral clearance studies Prior to the initiation of any studies, a viral clearance strategy was formulated in consultation with outside experts This included an external viral safety consultant knowledgeable in regulatory issues within and outside the United States A biosafety contract laboratory was also chosen This laboratory had excellent capability and had sufficient expertise in regulatory affairs to assist in the design of all aspects of the study Discussion with internal and external experts in the design of the marketing application studies started more than two years prior to the proposed submission date Another key element of the study design was the identification of purification steps to be evaluated in the viral clearance studies Clearance studies had already been conducted on the purification process This previous data provided insight into the level of clearance that could be expected from the steps evaluated There are two steps in the process dedicated to viral inactivation and viral removal Additional steps with viral clearance possibilities included two chromatography processes and one step in which there was an increase in temperature of the process solution to 40°C After identifying the process and purification steps to be evaluated, it was necessary to decide which viruses to use No specific viruses had to be included because no particles were identified in the cell line Consideration of which viruses to use was related to identifying viruses that could potentially grow in the production cell line and the use of bovine-sourced materials in production The literature was a source of information on the viruses to which the cells were susceptible In addition, a study was conducted in which the cell line was challenged with a subset of bovine viruses to determine its susceptibility Because the range of viruses that grew in the cell was varied, viruses were chosen that represented a range of biochemical and morphological types The following six viruses were used in the initial viral clearance study: xenotropic murine leukemia virus (MuLV), bovine viral diarrhea virus (BVDV), adenovirus, pseudorabies virus (PRV), poliovirus, and minute mouse virus (MMV) BioReliance provided the virus and a certified titer for each study These model viruses were chosen for the following reasons: MuLV was previously used to support clinical trials and is a model for any potential retrovirus contamination of the cell line or the process BVDV was previously used in viral clearance studies to support clinical trials and was used again in the current study BVDV is a common contaminant of Viral Clearance Validation 493 bovine serum, and therefore there is a potential for the cell line to be exposed to this virus Adenovirus serotype (Ad-2) was used in the spiking studies because the human cell line is susceptible to them Pseudorabies virus (PRV) was chosen because it is a herpes virus, a family of viruses that can grow in the cell line It also completes the spectrum of viruses used since it is a nonenveloped DNA virus Poliovirus is a small (30 nm) RNA virus belonging to the paramyxovirus family Poliovirus is resistant to many environmental conditions, and because it is so robust, the scientific literature has reported that it is commonly used in other viral clearance evaluations Minute mouse virus is a parvovirus, the smallest family of mammalian viruses MMV is nonenveloped and from 20 to 25 nm in size MMV is a ubiquitous parvovirus and has previously caused contaminations of CHO cell bioreactor runs Because the cell line will support growth of MMV, it is important to evaluate the clearance of this virus The objectives of a viral clearance study were (1) to demonstrate that the cell culture and purification processes are capable of removing or inactivating viruses, (2) to determine the clearance of each process step and the clearance for the entire process, (3) to demonstrate the kinetics of inactivation in those process steps in which inactivation is the primary method of clearance, and (4) to demonstrate that the column chromatography regeneration solutions and processes can inactivate model viruses At the end of the strategy and planning phase, it was important to have good communication with local management, with internal regulatory and quality control personnel, and among the scientific staff Included in this communication was an understanding of the goals or acceptance criteria by which the studies would be evaluated It was key to the success of the studies that the strategy was strong and well developed prior to initiating the actual work 18.2 LOGISTICAL CONSIDERATIONS BioReliance of Rockville, Maryland, was selected as the biosafety contract laboratory with whom the viral clearance studies would be performed Once legal and quality contractual agreements were completed, the technical and quality staff conducted an audit of BioReliance Communication between the parties was essential in designing the specifics of the study The viral spiking studies of the scaled-down process occurred at Lilly in a biosafety level (BSL-2) laboratory for the chromatography and nanofiltration steps Samples from these steps during the studies were then frozen and shipped to BioReliance at a later date for testing Analysis of any inactivation step was done on-site at the BioReliance facilities A written agreement of the study design had to be in place with BioReliance, which prepared a statement of work (SOW) The SOW is a detailed protocol of the study and includes a list of the model viruses and a list of the samples BioReliance would receive for testing Prior to starting any clearance study, the purification scientific staff had to design and validate scaled-down models of the process steps The scaled-down process 494 Process Validation in Manufacturing of Biopharmaceuticals steps needed to be comparable to the full-scale commercial process Regulatory guidance documents provided information helpful to the design of the scale steps A table was prepared listing the process parameters such as column height and flow rate and the actual conditions used for commercial and laboratory scale, which were similar Whenever process parameters could not be duplicated at the different scales, viral clearance studies were conducted under the worst-case conditions Once the laboratory-scale models were designed, protocols were written to outline the objectives of the studies, outline conditions that would be used, and the acceptance criteria for each step. Management reviewed and signed the protocols All clearance studies were done at least in duplicate, preferably on different days For chromatography steps, the viral clearance was evaluated using columns packed with virgin resin and columns packed with resin that had been regenerated the maximum number of times that will be allowed in production before the resin is replaced This measures the viral clearance robustness of the step relative to resin age At least three separate process steps were evaluated for each virus, but they were not always the same three steps used in the evaluation of the other viruses As per the guidelines, process solutions used in the viral clearance studies were obtained from the full-scale process Process solutions were ordered from both the clinical trial pilot plant and from the commercial production facility For chromatography runs, sufficient process solution had to be ordered to support at least twice the number of planned viral runs plus sufficient amounts for preliminary runs Prior to each chromatography run, the purification scientist conducted at least two or three preliminary runs without virus One run included a spike with the viral suspension media used by BioReliance Process solutions were also obtained from the commercial-scale or pilot plant and were submitted for cytotoxicity, viral interference, and frozen viability studies The purpose of the cytotoxicity study was to determine whether the process solutions in the viral spiking studies were toxic to the cell lines used to quantitate the viruses The viral interference study also determined whether the process solutions would inactivate the viruses or interfere with their recovery from spiked solutions The frozen viability studies were conducted to determine whether the model viruses were stable in process solutions when frozen at –80°C The frozen viability studies were conducted over a period of a few weeks Virus was spiked into the different dilutions of process solutions and then frozen Samples were removed to determine whether the virus was stable at –80°C for that length of time Frozen viability studies determined the length of time samples could be stored before being tested The samples must be diluted with cell culture media at the dilution determined in the cytotoxicity studies After being diluted, the samples can then be frozen for testing at a later date 18.3 PROTOCOL DEVELOPMENT AND EXECUTION The actual viral spiking studies could be initiated only after all the preliminary planning had been completed, the protocols at both BioReliance and Lilly were approved, and results had been received for cytotoxicity, viral interference, and frozen viability For chromatography processes, a control run in the BSL-2 laboratory was first performed with only process solution Next, a run with process solution plus the Viral Clearance Validation 495 viral suspension media was performed This sequence was important because some components of the suspension media, such as bovine serum albumin, could interfere with the chromatography Either the purification scientific staff set up the chromatography runs in a BSL-2 laboratory at Lilly or Lilly personnel went to BioReliance to aid in the conduct of the batch process analysis The overall approach included first spiking a process solution with virus of a known titer, then processing the solution using the laboratory-scale process step, and finally determining the amount of virus remaining in the product stream An overall (global) reduction factor was calculated for each virus The final reduction factor was the cumulative sum of the clearance seen for each step for that virus Small fractions were collected on the chromatography run and pooled to generate the samples submitted for viral testing As samples were collected from the batch process or from the chromatography run, they were diluted in cell culture medium supplied by BioReliance The dilution used was the one identified in the cytotoxicity/ viral interference/frozen viability studies Aliquots were prepared and immediately frozen at –80°C After all samples had been collected, they were shipped to BioReliance in a dry-ice shipper BioReliance was notified in advance that samples were being shipped so that they would expect the shipment and make arrangements to start testing immediately For studies that were conducted at BioReliance, samples were tested immediately For chromatography, the viral clearance was determined taking into account the amount of virus remaining in the chromatographic protein peak or mainstream It was necessary to decide on the criteria to be used to define the peak before the chromatography study was initiated Main peak collection for viral clearance is usually slightly broader for laboratory studies than for those at full scale The broader peak collected in the laboratory represents a worst-case analysis for viral clearance Besides the main peak fraction, other parts of the chromatography run—from the postcolumn loading flow-through to the fractions prior to and after the peak—should be collected In addition, for chromatography, it was especially important to maintain accurate records on the volumes involved for the fractions that are obtained The calculation of log reduction requires knowledge of the volume of process solution used in the studies In the studies described here, Eli Lilly provided those volumes to BioReliance It was also necessary to provide data showing that the laboratory model was run in a manner similar to the full-scale systems For batch processes, this appears to be straightforward Solutions can be placed in containers and stirred using a stir bar over a magnetic stirrer For the chromatography systems, demonstration of comparability is more complex The rule is to keep the same contact time at laboratory scale and at full scale The height of the resin bed needs to be similar or worst-case for laboratory scale Other process parameters were kept the same between the two scales 18.3.1 Calculation of Reduction Factors At the conclusion of the study, BioReliance calculated the reduction factors for each individual study The virus reduction factor is the log10 of the ratio of the input virus load to the output virus load The reduction factor can be calculated as follows If 496 Process Validation in Manufacturing of Biopharmaceuticals V(i) and T(i) represent the input volume (ml) and virus titer, respectively, and if V(o) and T(o) represent the output volume and output titer, respectively, then V(i)T(i) is the input virus load and V(o)T(o) is the output virus load The virus reduction R is given by the following formula: R = log10 V (i )T (i ) V (o)T (o) 18.4 RESULTS 18.4.1 Cytotoxicity, Viral Interference, and Frozen Viability The results of these studies are listed in Table 18.1 In some cases, the dilution needed to quench the reaction was on the order of 2–3 logs 18.4.2 Laboratory-Scale Process Evaluation The process steps were scaled to a size that would allow them to be performed in a biosafety facility where viral spiking studies can be conducted All process solutions used in the laboratory studies came from the GMP large-scale process at either the pilot plant or the commercial facility All buffers and resins were also from the GMP run or a comparable process The resin to be evaluated for the end of use was obtained from scaled-down runs at the commercial plant TABLE 18.1 Test Article Dilutions or Quench Dilutions as Determined by the Cytotoxicity, Viral Interference, or Frozen Viability Studies Viruses Used in Studies Step Sample Chromatography Load Flow-through Prepeak Peak Postpeak Load Load Flow-through Prepeak Peak Postpeak Load – no TX100 Load + TX100 Posttreatment Nanofilter Chromatography Viral inactivation Heat MuLV BVDV PRV Ad-2 Poliovirus 1:300 1:300 1:3 1:3 1:3 1:100 1:100 1:10 1:100 1:300 1:3 Undilute 1:100 1:100 1:3 Undilute Undilute 1:10 1:10 1:10 1:3 1:100 1:3 Undilute 1:100 1:100 Undilute Undilute Undilute Undilute Undilute Undilute Undilute Undilute Undilute Undilute 1:300 1:300 1:10 Undilute Undilute 1:100 1:100 1:10 1:10 1:100 1:3 Not done 1:100 1:100 1:30 1:3 1:3 Undilute Undilute Undilute Undilute Undilute Undilute Not done 1:100 1:100 1:300 1:10 1:100 Undilute Undilute 1:100 Undilute Undilute Viral Clearance Validation 497 Some problems occurred with the chromatography runs, and the runs then had to be repeated These problems were related to the columns plugging and chromatographic runs that did not meet the process step acceptance criteria In addition, samples sometimes did not get properly diluted as specified by the cytotoxicity data and studies had to be repeated 18.4.3 Viral Clearance per Individual Process Steps A summary of the log reduction values obtained for the panel of viruses and the individual process steps are shown in Table 18.2 Further details for the input log and output log values for the individual viruses are shown in Tables 18.3–18.8 These tables provide the output load after volume and dilution corrections have been made Therefore, the log reduction is simply the initial load minus the output load As can be seen, depending on the virus, the titers ranged from just over logs/ml for PRV to over to 10 logs for adenovirus In most cases, the standard deviation reported was under 0.5 log/ml 18.4.3.1 Viral Inactivation Step Three lipid-enveloped viruses were evaluated in this step including MuLV, BVDV, and PRV Complete inactivation was achieved with the BVDV and PRV virus Only 2.91 logs of reduction were obtained for MuLV, a result that could not be explained In later studies, higher clearance was noted for this virus Other viruses in the panel were not assessed because they did not have lipid envelopes 18.4.3.2 Chromatography Step For calculations of log reduction, the output load for the chromatographic peak was used and subtracted from the initial load For chromatography step 1, log reduction values could not be obtained for lipid-enveloped viruses because the viral inactivant from the previous step was still present in the feed to the chromatography step. For Ad-2, poliovirus, and MMV, the clearances were 2.51, 3.04, and 4.57 logs of clearance, respectively 18.4.3.3 Heat One process step used mild heat (40°C) Although this temperature is lower than levels published to be effective, it was decided to evaluate this step on the panel of viruses Only with MuLV was there any significant log reduction (2.22 logs) 18.4.3.4 Nanofiltration This step, which was included as a second dedicated viral clearance step, was very effective across the panel Complete clearance to levels of detection for the assay was reported for all viruses except the two smallest Complete clearance of MMV (20 nm) and poliovirus (30 nm) was not achieved, but 3.09 and 2.96 logs of clearance were obtained 498 TABLE 18.2 Reduction of Viruses in Various Process Steps Virus Genome Envelope Size Chromatography Number Mild Heat Nanofiltration Chromatography Number Global Reduction Factor a BVDV Ad-2 PRV PolioVirus MVM RNA env 50–70 nm 4.99 4.84 (>4.92) Virucidal activity: no value DNA non-env 70–90 nm Not done DNA env 80–120 nm >5.75 >5.53 (>5.65) Virucidal activity: no value RNA non-env 30 nm Not done DNA non-env 20 nm Not done 2.62 4.08 4.79 (4.57) Not done 2.86 3.24 (3.09) Not done 2.33 2.08 (2.22) >3.29 >2.16 (>3.02) 2.81 3.63 (3.39) 11.5 2.80 0.09 0.16 1.21 (2.51) –0.16 0.47 0.49 0.60 3.26 (3.04) 0.05 0.42 >4.16 >4.05 (>4.11) 1.99 1.64 (1.84) 10.8 >3.17 >4.04 (>3.79) 4.66 4.00 (4.44) 10.8 >5.14 >4.97 (>5.06) 1.44 1.36 (1.40) 12.1 2.92 2.99 (2.96) 2.11 2.64 (2.45) 8.4 7.68 Numbers in the boxes from top to bottom for viral inactivation, heat, and nanofiltration are the log reduction for Run 1, Run 2, and the average For the chromatography steps, the numbers from top to bottom are the log reductions for new resin, used resin, and the average of the two Process Validation in Manufacturing of Biopharmaceuticals Viral Inactivation MuLV RNA env 100 nm 2.93a 2.89a (2.91)a Not done 499 Viral Clearance Validation TABLE 18.3 Reduction Factors for MuLV Process Step Viral inactivation—Run Viral inactivation—Run Viral inactivation—average Heat—Run Heat—Run Heat—Run 2, retest Heat—average Chromatography 2—Run (new resin) Chromatography 2—Run (used resin) Chromatography 2—average Nanofiltration—Run Nanofiltration—Run Nanofiltration—average Initial Load (log10 TCID50) Output Load (log10 TCID50) 8.19 ± 0.43 8.32 ± 0.48 5.26 ± 0.53 5.43 ± 0.46 9.20 ± 0.36 7.70 ± 0.40 8.20 ± 0.36 6.87 ± 0.43 8.00 ± 0.44 6.12 ± 0.32 7.95 ± 0.35 8.20 ± 0.43 5.14 ± 0.60 ≤4.57 8.43 ± 0.44 7.30 ± 0.37 ≤5.14 ≤5.14 Initial Load (log10PFU) Output Load (log10PFU) 7.67 ± 0.23 7.77 ± 0.07 7.58 ± 0.17 7.61 ± 0.06 7.94 ± 0.46 7.79 ± 0.09 ≤2.95 ≤2.95 8.69 ± 0.10 8.58 ± 0.14 ≤4.53 ≤4.53 8.33 ± 0.07 8.26 ± 0.10 8.26 ± 0.10 6.34 ± 0.19 8.08 ± 0.13 6.62 ± 0.22 0.09 ± 0.29 0.16 ± 0.09 0.13 ± 0.21 ≥4.99 ± 0.46 ≥4.84 ± 0.09 ≥4.92 ± 0.33 ≥4.16 ± 0.10 ≥4.05 ± 0.14 ≥4.11 ± 0.12 1.99 ± 0.20 0.18 ± 0.16 2.22 ± 0.24 8.26 ± 0.10 6.56 ± 0.03 1.64 ± 0.10 log10 Reduction 2.93 ± 0.68 2.89 ± 0.66 2.91 ± 0.67 2.33 ± 0.56 –0.30 ± 0.59 2.08 ± 0.48 2.22 ± 0.51 2.81 ± 0.69 ≥3.63 ± 0.43 ≥3.37 ± 0.57 ≥3.29 ± 0.44 ≥2.16 ± 0.37 ≥3.02 ± 0.40 TABLE 18.4 Reduction Factors for BVDV Process Step Heat—Run Heat—Run Heat—average Viral inactivation—Run Viral inactivation—Run Viral inactivation—average Nanofiltration—Run Nanofiltration—Run Nanofiltration—average Chromatography 2—Run (new resin) Chromatography 2—Run (used resin) Chromatography 2—Run (used resin), retest Chromatography 2—Run (used resin), resubmission (repeat) Chromatography 2—average log10 Reduction 1.84 ± 0.10 500 Process Validation in Manufacturing of Biopharmaceuticals TABLE 18.5 Reduction Factors for Adenovirus Process Step Heat—Run Heat—Run Heat—average Chromatography 1—Run Chromatography 1—Run Chromatography 1—average Chromatography (new resin) Chromatography (used resin) Chromatography 2—average Nanofiltration—Run Nanofiltration—Run Nanofiltration—average Initial Load (log10TCID50) Output Load (log10TCID50) 9.09 ± 0.43 9.22 ± 0.24 9.25 ± 0.40 8.75 ± 0.36 10.72 ± 0.37 10.74 ± 0.51 7.92 ± 0.40 9.53 ± 0.51 9.70 ± 0.49 9.20 ± 0.32 5.04 ± 0.41 5.20 ± 0.24 9.17 ± 0.36 8.32 ± 0.37 ≤5.13 ≤5.15 Initial Load (log10PFU) Output Load (log10PFU) 9.05 ± 0.10 8.63 ± 0.22 8.56 ± 0.12 8.03 ± 0.09 8.70 ± 0.17 8.51 ± 0.22 ≤2.95 ≤2.98 8.09 ± 0.20 7.90 ± 0.29 6.65 ± 0.25 6.54 ± 0.21 7.53 ± 0.24 7.73 ± 0.17 ≤2.56 ≤2.59 log10 Reduction –0.16 ± 0.59 0.47 ± 0.43 0.26 ± 0.52 2.80 ± 0.54 1.21 ± 0.72 2.51 ± 0.64 4.66 ± 0.64 4.00 ± 0.40 4.44 ± 0.53 ≥4.04 ± 0.36 ≥3.17 ± 0.37 ≥3.79 ± 0.37 TABLE 18.6 Reduction Factors for PRV Process Step Heat—Run Heat—Run Heat—average Viral inactivation—Run Viral inactivation—Run Viral inactivation—average Chromatography 2—Run Chromatography 2—Run Chromatography 2—average Nanofiltration—Run Nanofiltration—Run Nanofiltration—average log10 Reduction 0.49 ± 0.16 0.60 ± 0.24 0.55 ± 0.20 ≥5.75 ± 0.17 ≥5.53 ± 0.22 ≥5.65 ± 0.20 1.47 ± 0.32 1.36 ± 0.36 1.42 ± 0.34 ≥4.97 ± 0.24 ≥5.14 ± 0.17 ≥5.06 ± 0.21 18.4.3.5 Chromatography Step For calculation of log reduction, the output load from the chromatographic peak was subtracted from the input load Overall log reduction between viruses in the test panel varied greatly, from a low of 1.40 logs for PRV to a high of 4.44 logs for Ad-2 Samples from the BVDV study with used resin were retested after the first set of results yielded a difference of greater than logs To ensure that the second set of data was accurate, a backup sample was also submitted The log reduction value obtained was the average of the retest and the second submitted sample 501 Viral Clearance Validation TABLE 18.7 Reduction Factors for Poliovirus Process Step Heat—Run Heat—Run Heat—average Chromatography 1—Run (new resin) Chromatography 1—Run (used resin) Chromatography 1—average Chromatography 2—Run Chromatography 2—Run Chromatography 2—average Nanofiltration—Run Nanofiltration—Run Nanofiltration—average Initial Load (log10PFU) Output Load (log10PFU) 8.90 ± 0.29 7.81 ± 0.18 8.85 ± 0.33 7.39 ± 0.23 9.72 ± 0.06 10.17 ± 0.39 7.10 ± 0.21 6.91 ± 0.13 8.27 ± 0.20 9.23 ± 0.41 6.16 ± 0.19 6.59 ± 0.18 9.15 ± 0.07 9.24 ± 0.12 6.16 ± 0.23 6.32 ± 0.11 Initial Load (log10TCID50) Output Load (log10TCID50) 9.04 ± 0.35 9.29 ± 0.37 6.18 ± 0.40 6.05 ± 0.00 8.49 9.74 4.41 4.95 log10 Reduction 0.05 ± 0.44 0.42 ± 0.29 0.27 ± 0.37 2.62 ± 0.22 3.26 ± 0.41 3.04 ± 0.33 2.11 ± 0.28 2.64 ± 0.45 2.45 ± 0.37 2.99 ± 0.24 2.92 ± 0.16 2.96 ± 0.20 TABLE 18.8 Reduction Factors for MMV Process Step Nanofiltration—Run Nanofiltration—Run Nanofiltration—average Chromatography 2—Run (new resin) Chromatography 2—Run (old resin) Chromatography 2—average log10 Reduction 2.86 ± 0.53 3.24 ± 0.37 3.09 ± 0.46 4.08 4.79 4.57 18.4.4 General Comments on Viral Clearance In these studies, in most cases, there was less than a 1-log difference between the two independent runs, whether it was just a repeat of the same process step or, for chromatography, new and used resins were used It is customary to report overall or global log reduction values for each virus This is accomplished by adding the log clearance achieved at each step. Log reductions of 1.0 logs or less were not included The global reduction values from low to high were as follows: 7.68 logs for MMV, 8.4 logs for poliovirus, 10.8 logs for Ad-2, 10.8 logs for BVDV, 11.5 logs for MuLV, and 12.1 logs for PRV 18.5 CONCLUSION The studies demonstrated very good clearance of the model viruses by the process steps The viruses used represented a cross section of various morphological and 502 Process Validation in Manufacturing of Biopharmaceuticals biological types For each virus, at least one process step and in most cases two process steps resulted in more than logs of viral clearance There were at least three steps for each virus that provided viral clearance The two smallest viruses, MMV and poliovirus, exhibited the lowest clearance at over logs MuLV, a retrovirus, had 11 logs of clearance This may be lower than seen with product derived from CHO cells Since CHO cells have retroviral particles and this cell line does not, the clearance obtained for MuLV is more than sufficient The viral clearance study required almost years of activity from start to finish by personnel dedicated full-time to the project Good planning and communication, especially with the contract biosafety laboratory, were vital to the success of this study Studies of this nature require interaction among scientific staff in purification, production, quality control, and regulatory departments ACKNOWLEDGMENTS The author wishes to acknowledge the expert assistance of Eli Lilly and Company employee Mark Smith for his editorial comments Also, the author thanks Dr Carol Marcus-Sekura for her excellent advice REFERENCES Committee for Proprietary Medicinal Products (CPMP) “Note for Guidance: Validation of Virus Removal and Inactivation Procedures,” III/8115/89, 1991 Center for Biologics Evaluation and Research “Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use, Food and Drug Administration,” Bethesda, MD, 1997 Federal Health Office and Paul Erlich Institute Federal Office for Sera and Vaccines “Notice on the Registration of Drugs: Requirements for Validation Studies to Demonstrate the Virus Safety of Drugs Derived from Human Blood or Plasma.” Bundesanzeiger 84 (1994): 4742–4744 The European Agency for the Evaluation of Medicinal Products, Human Medicines Evaluation Unit—CPMP Biotechnology Working Party “Note for Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses,” CPMP/BWP/268/95 Final Version Center for Biologics Evaluation and Research “Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, Food and Drug Administration,” Bethesda, MD, 1997 International Commission on Harmonization “Topic Q5D, Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human and Animal Origin.” March 1998 International Commission on Harmonization “Topic Q5A, Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin.” October 1997 ... no cleaning validation for cleaning critical manufacturing areas Yet another was observed to have no cleaning Process Validation in Manufacturing of Biopharmaceuticals validation for laminar air... assessment of endotoxins or bioburden Another was cited for a cleaning validation report that was inadequate as it failed to include the testing of final rinse water for bioburden Cleaning: Cleaning validation. .. and inhibition of infectivity assays, scale-down accuracy, Process Validation in Manufacturing of Biopharmaceuticals effect of spike on process, cost of studies, and data interpretation In the