Gene Therapies Push Viral Vector Production

Home , ApiJect Systems, Bioproduction, Viral vector

Volume 33 Number 5

BioPharm International BioPharm INTERNATIONAL

BioMay 2020 PharmThe Science & Business of Biopharmaceuticals MAY 2020 www.biopharminternational.com

GENE THERAPIES PUSH VIRAL VECTOR PRODUCTION

UPSTREAM PROCESSING SYNTHETIC BIOLOGY

COVID-19 I Modern Manufacturing I Development Efforts Efforts I Development Manufacturing I Modern COVID-19 DOWNSTREAM PROCESSING FLUID HANDLING CONSIDERATIONS

MANUFACTURING CLOSED FILLING SYSTEMS

ANALYTICS BIOASSAY DEVELOPMENT

QUALITY/REGULATIONS REMOTE AUTOMATION PRACTICES

OUTSOURCING CONTINUOUS BIOPROCESSING

PEER-REVIEW RESEARCH IR SPECTROSCOPY FOR BIOCHARACTERIZATION Volume 33 Number 5 33 Number Volume Whether it’s a vaccine, recombinant protein, or gene therapy, Avantor ® can help transform how these therapies are brought to market, combining our unique workflow expertise with advanced biologic products engineered to satisfy the highest quality standards and regulatory requirements.

We help enable breakthroughs in life-changing biologics. avantorsciences.com/moves-biopharma-forward INTERNATIONAL

BioPharmThe Science & Business of Biopharmaceuticals

ADVERTISING EDITORIAL Publisher Mike Tracey [email protected] Editorial Director Rita Peters [email protected] National Sales Manager Scott Vail [email protected] Senior Editor Agnes M. Shanley [email protected] Managing Editor Susan Haigney [email protected] European Sales Manager Linda Hewitt [email protected] European Editor Felicity Thomas [email protected] European Senior Sales Executive Science Editor Feliza Mirasol [email protected] Stephen Cleland [email protected] Manufacturing Editor Jennifer Markarian [email protected] VP/Managing Director, Pharm/Science Group Dave Esola Assistant Editor Lauren Lavelle [email protected] [email protected] Senior Art Director Marie Maresco C.A.S.T. Data and List Information Graphic Designer Maria Reyes Michael Kushner [email protected] EDITORIAL ADVISORY BOARD AUDIENCE DEVELOPMENT BioPharm International’s Editorial Advisory Board comprises distinguished specialists involved in the biologic manufacture of therapeutic drugs, Audience Development Christine Shappell [email protected] diagnostics, and vaccines. Members serve as a sounding board for the editors and advise them on biotechnology trends, identify potential MJH LIFE SCIENCESTM authors, and review manuscripts submitted for publication. Chairman and Founder Mike Hennessy, Sr K. A. Ajit-Simh Hanns-Christian Mahler Vice Chairman Jack Lepping President, Shiba Associates Head Drug Product Services Lonza AG President and CEO Mike Hennessy, Jr Madhavan Buddha Chief Financial Officer Neil Glasser, CPA/CFE Freelance Consultant Jerold Martin Independent Consultant Executive Vice President, Operations Tom Tolvé Rory Budihandojo Executive Vice President, Global Medical Affairs and Director, Quality and EHS Audit Hans-Peter Meyer Boehringer-Ingelheim Lecturer, University of Applied Sciences Corporate Development Joe Petroziello and Arts Western Switzerland, Senior Vice President, Audience Generation and Edward G. Calamai Institute of Life Technologies Managing Partner Product Fulfillment Joy Puzzo Pharmaceutical Manufacturing K. John Morrow Senior Vice President, Content Silas Inman and Compliance Associates, LLC President, Newport Biotech Senior Vice President, I.T. & Enterprise Systems John Moricone Suggy S. Chrai David Radspinner Vice President, Human Resources & Administration Shari Lundenberg President and CEO GE Healthcare The Chrai Associates Vice President, Business Intelligence Chris Hennessy Tom Ransohoff Leonard J. Goren Vice-President and Senior Consultant Executive Creative Director, Creative Services Jeff Brown Global Leader, Human Identity BioProcess Technology Consultants Division, GE Healthcare Uwe Gottschalk Anurag Rathore Vice-President, Biotech CMC Consultant Chief Technology Officer, Faculty Member, Indian Institute of Pharma/Biotech Technology Lonza AG © 2020 MultiMedia Pharma Sciences LLC All rights reserved. No part of this Susan J. Schniepp publication may be reproduced or transmitted in any form or by any means, electronic Fiona M. Greer Executive Vice President of or mechanical including by photocopy, recording, or information storage and retrieval Global Director, Post-Approval Pharma without permission in writing from the publisher. Authorization to photocopy items BioPharma Services Development and Distinguished Fellow for internal/educational or personal use, or the internal/educational or personal use of SGS Life Science Services Regulatory Compliance Associates, Inc. specific clients is granted by MultiMedia Pharma Sciences LLC for libraries and other Rajesh K. Gupta users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA Tim Schofield 01923, 978-750-8400 fax 978-646-8700 or visit http://www.copyright.com online. Vaccinnologist and Microbiologist Consultant For uses beyond those listed above, please direct your written request to Permission Dept. Alexa Rockenstein, [email protected]. Reprints: Contact Michael Denny Kraichely CMC Sciences, LLC Tracey, [email protected]. Associate Director Johnson & Johnson Paula Shadle MultiMedia Pharma Sciences LLC provides certain customer contact data (such Principal Consultant, as customers’ names, addresses, phone numbers, and e-mail addresses) to third parties Stephan O. Krause Shadle Consulting who wish to promote relevant products, services, and other opportunities that may be of Director of QA Technology interest to you. If you do not want MultiMedia Pharma Sciences LLC to make your contact AstraZeneca Biologics information available to third parties for marketing purposes, simply email mmhinfo@ Alexander F. Sito mjhlifesciences.com and a customer service representative will assist you in removing Steven S. Kuwahara President, your name from MultiMedia Pharma Sciences LLC lists. Principal Consultant BioValidation GXP BioTechnology LLC BioPharm International does not verify any claims or other information appearing in Michiel E. Ultee any of the advertisements contained in the publication, and cannot take responsibility for Eric S. Langer Principal any losses or other damages incurred by readers in reliance of such content. President and Managing Partner Ulteemit BioConsulting BioPharm International welcomes unsolicited articles, manuscripts, photographs, BioPlan Associates, Inc. illustrations, and other materials but cannot be held responsible for their safekeeping or return. Thomas J. Vanden Boom Howard L. Levine VP, Biosimilars Pharmaceutical Sciences To subscribe, email [email protected] President Pfizer BioProcess Technology Consultants Hank Liu Krish Venkat Head of Quality Control Managing Partner Sanofi Pasteur Anven Research Herb Lutz Steven Walfish Principal Consulting Engineer Principal Scientific Liaison Merck Millipore USP Table of Contents Volume 33 Number 5

BioPharm International integrates the science and business of biopharmaceutical research, development, and manufacturing. We provide practical, peer-reviewed technical solutions to enable biopharmaceutical professionals to perform their jobs more effectively.

COVER STORY 12 Gene Therapies Push Viral Vector Production Viral vectors show promise as a delivery mechanism for gene therapy, but which virus types are commercially viable? Cover Design by Maria Reyes. Images: Kateryna_Kon - Stock.adobe.com

FEATURES COLUMNS AND DEPARTMENTS

UPSTREAM PROCESSING PEER-REVIEW RESEARCH FROM THE EDITOR Biopharmaceutical Manufacturing Shaping IR Spectroscopy into Will moving at “warp speed” to develop and the Power of Synthetic Biology a Powerful Tool for Biopharma a vaccine impact efficacy or safety? David McElroy Characterizations Rita Peters ��6 Synthetic biology has advanced the scope Dipanwita Batabyal, Libo Wang, and scale with which biologically derived Jeffrey Zonderman, and Mats Wikström REGULATORY BEAT therapeutics can be developed. ��22 The authors present experimental Policy makers seek to ensure supplies studies showcasing the performance of of new therapies and to limit shortages. DOWNSTREAM PROCESSING microfluidic modulation spectroscopy in Jill Wechsler ��8 Fluid Handling Considerations the secondary structure characterization Jennifer Markarian of biopharmaceutical products ��42 CELL THERAPIES PIPELINE ��10 Pumps and other components meet the demands of single-use systems QUALITY/REGULATIONS PRODUCT SPOTLIGHT ��56 in biopharmaceutical downstream Good Automation Practices processing. ��28 for Remote Operations AD INDEX ��57 Lauren Lavelle MANUFACTURING Having remote operations in place ASK THE EXPERT Straight Talk on is crucial to maintaining good Having a better understanding Closed Aseptic Systems automation practices. ��48 about compliance will be of James Agalloco benefit when looking for a job or The industry’s aversion to risk has OUTSOURCING for furthering one’s career. led to its treating closed aseptic Biomanufacturing: Demand for Siegfried Schmitt ��58 processing systems as miniature Continuous Bioprocessing Increasing cleanrooms, resulting in redundant Eric S. Langer and expensive practices. ��32 But are innovations sufficient to increase SPONSORED CONTENT adoption? CMOs are demanding better ANALYTICS continuous bioprocessing options. �� 51 BIOPHARMA INSIGHTS Building Better Bioassays Challenges in Bulk Drug Cynthia A. Challener Substance Management Next-generation therapeutics and Michael Eder regulatory requirements create demand Closing the gap between for complex, fit-for-purpose tests. ��37 downstream and fill–finish. ��20

BioPharm International is selectively abstracted or indexed in: • Biological Sciences Database (Cambridge Scientific Abstracts) • Biotechnology and Bioengineering Database (Cambridge Scientific Abstracts) • Biotechnology Citation Index (ISI/Thomson Scientific) • Chemical Abstracts (CAS) • Science Citation Index Expanded (ISI/Thomson Scientific) • Web of Science (ISI/Thomson Scientific) BioPharm International ISSN 1542-166X (print); ISSN 1939-1862 (digital) is published monthly by MultiMedia Healthcare LLC 2 Clarke Drive, Suite 100, Cranbury, NJ 08512. Subscription rates: $82.95 for one year in the United States and Possessions; $112.35 for one year in Canada and Mexico; all other countries $159.60 for one year. Periodicals postage paid at Trenton, NJ 08650 and additional mail- ing offices. Postmaster Please send address changes to BioPharm International, PO Box 457, Cranbury, NJ 08512-0457, USA. PUBLICATIONS MAIL AGREEMENT NO. 40612608, Return Undeliverable Canadian Addresses to: IMEX Global Solutions, P. O. Box 25542, London, ON N6C 6B2, CANADA. Canadian GST number: R-124213133RT001. Printed in U.S.A.

4 BioPharm International May 2020 www.biopharminternational.com

From the Editor

How Fast Is Too Fast?

he rapid spread of the novel coronavirus is a reminder of the interconnected nature of global populations and economies. A few other realities also should be obvious. T First, no single individual, company, or country can solve the pandemic crisis alone. Second, while getting a vaccine to patients quickly is crucial, decisions about quality and efficacy should be based on science, not political or public pressure. The competition for vital medical resources in the early days of the pandemic illustrated how uncontrolled or misguided competition and anxiety can quickly overwhelm response to an emergency. Fortunately, collaborative efforts are shaping the development of treatments and vaccines. For example, the World Health Organization (WHO) is coordinating global efforts for vaccine development and clinical trials for treatment options. Rita Peters is In the United States, the Accelerating COVID-19 Therapeutic Interventions and the editorial director of Vaccines partnership is a public-private effort to develop a framework to prioritize vac- BioPharm International. cine and drug candidates, streamline clinical trials, coordinate regulatory processes, and leverage assets to respond to the COVID-19 and future pandemics. More than a dozen biopharmaceutical companies have joined the National Institutes of Health (NIH), Foundation for the NIH, Health and Human Services Office of the Assistant Secretary Will moving at for Preparedness and Response, FDA, Centers for Disease Control and Prevention, and the European Medicines Agency in an effort to advance the most promising vaccine and “warp speed” therapeutic candidates (1). While groups are collaborating on development and clinical trial phases; the need for a to develop coordinated manufacturing strategy cannot be overlooked. For an industry used to development timelines counted in years, the COVID-19 vac- a vaccine impact cine and therapy development process is moving very fast. Sponsors of the leading vaccine candidates now in early clinical trials are expressing confidence that the product can reach efficacy or safety? patients by the end of 2020, months or years earlier than predicted by experts just one week ago. To achieve that goal, manufacturing capacity for unproven vaccines must be created now, a risky prospect for the companies developing the capacity and for patients. Drug companies and contract manufacturers are accepting the challenge; governments and tax- payers may bear the financial risk. In April 2020, AstraZeneca and the University of Oxford announced a development and manufacturing partnership. Johnson & Johnson announced manufacturing agree- ments with Catalent and Emergent BioSolutions for its vaccine candidate, as did Lonza with Moderna for its mRNA vaccine. Both Johnson & Johnson and Moderna have received funding from the Biomedical Advanced Research and Development Authority. PRUDENT PATIENCE In late April, media outlets reported a Trump Administration plan—dubbed “Operation Warp Speed”—to have 300 million doses of coronavirus vaccine available by the end of 2020 (2). Details for this program—including which vaccines would be manufactured— were not available at press time. After weeks of stay-at-home orders, social distancing, closed restaurants, high unem- ployment numbers, and bad economic news, optimism about the early arrival of a vaccine, as well as treatments such as Gilead Sciences’ remdesivir, offers some good news and excitement. With anxiety for a resolution to the pandemic driving vaccine development activity, however, it is crucial that science-based information—from the global bio/pharma experts—should drive development, approval, and manufacturing decisions. REFERENCES 1. NIH, “NIH to Launch Public-Private Partnership to Speed COVID-19 Vaccine and Treatment Options,” Press Release, April 17, 2020. 2. J. Jacobs and D. Armstrong, “Trump’s ‘Operation Warp Speed’ Aims to Rush Coronavirus Vaccine,” Bloomberg.com, April 29, 2020. ◆

6 BioPharm International May 2020 www.biopharminternational.com Coming soon from USP Biologics Monoclonal Antibodies helping to drive the biologics revolution

Part of our commitment to Currently Available: biologics is keeping up with our USP Monoclonal IgG System Suitability RS customers’ needs. Catalog #: 1445550

That’s why we’re expanding our family of Vial contains: Monoclonal Antibody Reference Standards 2 mg of IgG lyophilized (mAb RS) by adding 3 new options that are part of Monoclonal Antibody the same IgG1 subclass, each with unique quality attributes. These Reference Standards can be Coming Soon: used for many non-compendial purposes that a laboratory may need, including: USP mAb 001 RS, Monoclonal IgG1 • As an independent control material for USP mAb 002 RS, Monoclonal IgG1 method development, training, and method transfer USP mAb 003 RS, Monoclonal IgG1 • As an internal assay control Coming soon from USP Biologics: • For standardization of physico-chemical testing, such as intact mass, charge 3 new IgG1 monoclonal antibody heterogeneity, size variants, purity and Reference Standards glycan analyses

When you choose USP Biologics, you’re choosing quality and reliability that can only come from our rigorous and collaborative testing and development process.

Explore all 4 of our mAb RS and our full biologics catalog at usp.org/biologics/reference-standards/igg To contact someone directly about USP Reference Standards please email [email protected] Regulatory Beat

Modern Drug Manufacturing Key to COVID-19 Response Policy makers seek to ensure supplies of new therapies and to limit shortages.

oncerns about access to medicines and diagnostics critical to containing the Potential difficulties in C coronavirus pandemic (COVID-19) and treating infected patients have broadened sup- ensuring sufficient supplies port for advanced pharmaceutical manufacturing and test methods. FDA officials have long of newly discovered therapies pressed industry to adopt continuous manufacturing and online testing methods able to scale up quality production quickly and efficiently. and anticipated vaccines The current pandemic has boosted support for such initiatives, as global health organizations have moved to center stage. and US policy makers recognize the importance of being able to produce millions of doses of test a variety of drugs. Gates further empha- any promising new treatments. sized that the federal government should provide While the lack of personal protective gear and suppport for building production facilities now, respirators has dominated the headlines since as making such investments before knowing the pandemic outbreak, potential difficulties in that a product will be used is excessively risky for ensuring sufficient supplies of newly discovered manufacturers. therapies and anticipated vaccines have moved Funds to advance efficient drug manufacturing to center stage. China’s prominence in produc- systems to ensure access to vital therapies and ing key APIs for antibiotics and many common prevent shortages due to supply disruptions from medicines, plus a recent move by the Indian gov- China and elsewhere were included in the ini- ernment to halt the export of multiple drugs and tial $8.3-billion emergency coronavirus funding drug ingredients, heightened concerns about package enacted March 5, 2020. FDA gained $61 the United States’ dependence on global sup- million to support the development of new med- ply chains. Rumors that an existing ical countermeasures and vaccines, of advanced malaria treatment might be effective manufacturing for medical products, and for mon- against COVID-19, for example, created itoring medical product supply chains (1). The a run on the drug, leading to notable subsequent $2-trillion coronavirus aid and relief shortages at some manufacturers. package approved March 27, 2020 similarly pro- These concerns have spurred calls vided an added $80 million to FDA to advance the for greater US investment in high- development and approval of medical counter- tech biopharma production sys- measures and vaccines (2). The legislation also tems. In announcing the COVID-19 directed FDA to use some of the funds to fur- Therapeutics Accelerator in early ther the adoption of advanced manufacturing Jill Wechsler is March to advance treatments for the systems for medical products and to monitor BioPharm International’s pandemic, the Bill and Melinda Gates supply chains for potential threats to access to Washington editor, Foundation cited the need to quickly medicines and APIs imported from abroad. To

[email protected]. build up manufacturing capacity to help FDA anticipate looming supply problems, W.Scott McGill - Stock.adobe.com

8 BioPharm International May 2020 www.biopharminternational.com Regulatory Beat

Congress instructed manufacturers (HHS) to develop countermea- manufacturing scale-up capabili- to send FDA information on where sures and vaccines in response to ties. At a “virtual summit” in March interruptions in supply affect APIs the pandemic. This includes build- sponsored by the Biotechnology as well as drugs and extended such ing the Rapid Aseptic Packaging Innovation Organization (BIO), requirements to medical device of Injectable Drugs (RAPID) con- industry leaders cited the challenge makers during this public health sortium with a network of up to in needing to expand manufactur- emergency. To further address these eight domestic facilities to rapidly ing capabilities before knowing they issues, Congress provided $1.5 mil- fill and finish millions of prefilled have a viable product, and the fast lion for the National Academies of syringes for delivering vaccines launch of clinical trials for candidate Sciences, Medicine, and Engineering and therapies for COVID-19 (3). vaccines aggravates those difficulties. (NASEM) to prepare a study on ways An innovative syringe developed On many fronts, industry is ris- to strengthen the manufacturing by ApiJect Systems utilizes existing ing to the challenge, with pharma supply chain for drugs and devices to blow-fill-seal technology plus an companies partnering with smaller avoid shortages. interlocking needle hub to provide biotechs and federal agencies that low-cost, easy-to-use injectables for offer innovative drug and vaccine MILLIONS FOR MANUFACTURING the Strategic National Stockpile. candidates for established firms Added support for establishing to test and produce. In March, advanced biopharma manufactur- Johnson & Johnson announced ing facilities is found in sections Industry is rising to that a $1-billion partnership of its of the pandemic relief legislation, Janssen unit with BARDA planned which provides some $30 bil- the challenge, with to rapidly scale up vaccine man- lion for federal health agencies to ufacturing capacity to be able develop countermeasures and vac- pharma companies to supply over one billion doses cines, plus platform technologies of vaccine globally (5). Such fast to advance US production of new expansion of production capacity therapeutics, diagnostics, vaccines, partnering with will be needed for clinical trials and medical supplies. Congress slated to begin this fall and then specified that a portion of $3.5 bil- smaller biotechs and to provide emergency use access to lion for the Biomedical Advanced any promising product. Research and Development federal agencies. Authority (BARDA) should be used REFERENCES to construct or renovate US-based Similarly, additional funding for 1 . H.R. 6074, 116th Congress, Public Law 116–123—Mar. 6, 2020, Coronavirus next-generation manufacturing the National Institute of Standards Preparedness and Response facilities. Similar directions are and Technology (NIST) supports pro- Supplemental Appropriations Act, included in providing added funds grams to accelerate production of 2020. 2. Amendment H.R. 748 “An Act to amend for the Defense Research Advanced critical materials, build additional the Internal Revenue Code of 1986 to Projects Agency (DARPA) in the production facilities, ensure supply Repeal the Excise Tax on High Cost Department of Defense, building chains for vital ingredients, develop Employer-Sponsored Health Coverage,” on its research programs related and train manufacturing workers, US Senate, March 21, 2020. 3. HHS, “HHS Announces New Public- to advancing biopharma manufac- and return to the US the manufac- Private Partnership to Develop U.S.- ture. Among other things, these ture of critical conventional drugs Based, High-Speed Emergency Drug initiatives have supported the (4). NIST has worked with biotech Packaging Solutions,” Press Release, March 18, 2020. development of RNA and DNA vac- firms for several years to address 4. NIST, “NIST Funding Manufacturing cines to fight infectious diseases challenges in developing more effi- Institutes to Support Pandemic such as Chikungunya and Ebola cient and reliable ways to produce Response,” Press Release, March 31, and to overcome manufacturing high quality cell and gene therapies. 2020. 5. J&J, “Johnson & Johnson Announces a challenges to faster scale-up. These and other projects Lead Vaccine Candidate for COVID-19; The pandemic legislation also stand to assist manufacturers Landmark New Partnership with U.S. provides $27 billion for the Public on accelerated timelines for test- Department of Health & Human Services; and Commitment to Supply Health & Social Services Emergency ing promising COVID-19 ther- One Billion Vaccines Worldwide for Fund managed by the Secretary apies and vaccines looking to Emergency Pandemic Use,” Press of Health and Human Services establish systems for fast, reliable Release, March 30, 2020. ◆

www.biopharminternational.com May 2020 BioPharm International 9 Cell Therapies Pipeline

Researchers Tap Scorpion Venom Daiichi Sankyo Seeks Approval for for CAR T Cell Therapy Lymphoma Car T Cell Therapy Scientists from City of Hope, a not-for-profit clinical research On March 30, 2020, Daiichi Sankyo Company submitted a new center, developed and tested the first chimeric antigen drug application (NDA) to Japan’s Ministry of Health, Labor receptor (CAR) T-cell therapy using chlorotoxin (CLTX), an and Welfare (MHLW) for the chimeric antigen receptor (CAR) element in scorpion venom, to encourage T cells to pinpoint T cell therapy, axicabtagene ciloleucel, for adult patients with brain tumor cells (1). relapsed/refractory diffuse large B-cell lymphoma and related Throughout the study, researchers found that, when using lymphomas (1). tumor cells in resection samples from a group of patients The NDA was submitted after the cell therapy received with glioblastoma (GBM) to compare CLTX attachment with positive results from a global trial and a Phase II study targeted antigens, the CLTX attached to a large number conducted in Japan. The trials focused on individuals with of tumors and cells in the GBM patients. The researchers four types of relapsed/refractory B-cell lymphomas, including concluded that the CLTX–CAR T cells selected and killed large diffuse large B cell lymphoma (DLBCL), primary mediastinal populations of GBM cells in cell-based assays and in animal B-cell lymphoma (PMBCL), transformed follicular lymphoma models while disregarding non-tumors and toxicity. (TFL), and high-grade B cell lymphoma. The Phase II study “Our chlorotoxin-incorporating CAR expands the met its primary endpoint for objective response rate. In 2018, populations of solid tumors potentially targeted by CAR T axicabtagene ciloleucel received Orphan Drug Designation cell therapy, which is particularly needed for patients with from the MHLW for the treatment of DLBCL, PMBCL, TFL, and cancers that are difficult to treat, such as glioblastoma,” high-grade B-cell lymphoma. said Christine Brown, PhD, City of Hope’s Heritage Provider The cell therapy targets CD19, a cell membrane protein, while Network professor in Immunotherapy and deputy director of using the patient’s immune system to fend off B-cell lymphoma the T Cell Therapeutics Research Laboratory, in a press release. on its own. Daiichi Sankyo received exclusive development, “This is a completely new targeting strategy for CAR T therapy manufacturing, and commercialization rights for the cell with CARs incorporating a recognition structure different from therapy in Japan from Kite, a Gilead company, in 2017. Kite other CARs.” received approval for axicabtagene ciloleucel under the brand “Much like a scorpion uses toxin components of its venom name Yescarta in the United States in October 2017 (2) and to target and kill its prey, we’re using chlorotoxin to direct the in the European Union in August 2018 (3). Yescarta had $456 T cells to target the tumor cells with the added advantage that million in 2019 sales (4). the CLTX–CAR T cells are mobile and actively surveilling the “We are pleased to confirm submission of the NDA for brain looking for appropriate targets,” added Michael Barish, axicabtagene ciloleucel following positive topline results from PhD, City of Hope professor and chair of the Department of the [Phase II] bridging study in Japan,” said Wataru Takasaki, Developmental and Stem Cell Biology, in the press release. PhD, corporate officer, head of Oncology Function and head of “We are not actually injecting a toxin but exploiting CLTX’s the R&D Division in Japan, Daiichi Sankyo, in a company press binding properties in the design of the CAR. The idea was to release. “We will continue to work with regulatory authorities to develop a CAR that would target T cells to a wider variety of develop this important new cell therapy for eligible patients in GBM tumor cells than the other antibody-based CARs.” Japan who need additional treatment options for relapsed or FDA recently granted the cell therapy first-in-human clinical refractory DLBCL and related lymphomas.” trial privileges and the study team is currently screening potential GBM patients for participation.

Reference References 1. City of Hope, “From Scorpion to Immunotherapy: City of 1. Daiichi Sankyo Company, “Daiichi Sankyo Submits Hope Scientists Repurpose Nature’s Toxin for First-Of- Application for CAR T Therapy Axicabtagene Ciloleucel for Its Kind Car T Cell Therapy To Treat Brain Tumors,” Press Treatment of Patients with Certain Relapsed/Refractory Release, March 4, 2020. ◆ B-cell Lymphomas in Japan,” Press Release, March 30, 2020. 2. FDA, “FDA Approves CAR-T Cell Therapy to Treat Adults with Certain Types of Large B-Cell Lymphoma,” Press Release, Oct. 18, 2017. 3. Gilead Sciences, “Yescarta (axicabtagene ciloleucel) Receives European Marketing Authorization for the Treatment of Relapsed or Refractory DLBCL and PMBCL, After Two or More Lines of Systemic Therapy,” Press Release, Aug. 27, 2018. ◆

10 BioPharm International May 2020 www.biopharminternational.com Boost your Host Cell Protein Analytics Help ensure clinical success

Antibody Affinity Extraction™ and Mass Spectrometry Services from Cygnus HCP experts. When it comes to outsourcing your critical Host Cell Protein analytical projects, it is essential to work with a dependable partner that has pioneered advanced orthogonal methods and HCP analysis solutions you can trust. Avoid delays in clinical trials due to an unforeseen need to optimize the purification process, adverse patient reactions, poor drug shelf life and reduced drug efficacy. Save time and resources on your journey to a quality drug substance.

• Assure that your HCP ELISA is fit for purpose of process monitoring and product lot release • Enrich and identify hitchhiker HCPs and improve MS resolution • Optimize your purification process to remove problematic HCPs • Provide comprehensive HCP characterization data to regulatory agencies

With you all the way.

cygnustechnologies.com

Cygnus_Ad_AAE-MS_8x10.75-B.indd 1 4/17/20 11:20 AM Cover Story: Gene Therapies

Gene Therapies Push Viral Vector Production Viral vectors show promise as a delivery mechanism for gene therapy, but which virus types are commercially viable?

FELIZA MIRASOL

everal virus types have been studied for use as viral vectors therapy. Compared to adenovirus and AAV, retrovirus-based vec- in gene therapy, including adenovirus, lentivirus, retrovirus, tors show relatively low transduction efficiency in-vivo, but their S and adeno-associated virus (AAV). Of these, AAVs have advantage lies in their ability to integrate stably and confer long- gained much attention as a gene delivery vehicle and are being term transgene expression in dividing tissues. Retroviruses and explored for their commerical viability. The commercial viability lentiviruses, therefore, are most suitable for ex-vivo gene transfer, of other virus types also continues to be explored, particularly for Chaudhary states. treatments that require a larger payload, transient expression, or Each viral vector system is characterized by an inherent set insertion of a gene of interest in the genome. of properties that affect its suitability for gene therapy or other The potential opportunity of a vector depends on the type specific applications, says Florence Vicaire, global gene ther- of gene therapy being developed, says Gaurav Chaudhary, apy business development leader, Cytiva (formerly part of GE CEO of Roots Analysis, a business research and consult- Healthcare). “There is no one-fits-all multipurpose viral vector ing firm. In general, there are two types of gene therapies, appropriate for all applications; each of the vectors has its own namely ex-vivo and in-vivo, Chaudhary points out. “In advantages, limitations, and range of applications,” Vicaire says. ex-vivo, cells are modified outside the patient’s body and the corrected version is transplanted back into the patient. THE VIRAL-VECTOR MECHANISM Opposite of ex-vivo is what we call in-vivo, where cells are Viral vector systems are gutted viruses where the packaging sig- treated inside the patient’s body. The corrected copy of the nals (e.g., the genetic signals that target the wrapping into the genes is transferred into the body of the patient.” viral particles) are fused to the genes of interest (the payload), In both cases, cells may be treated either with a viral or non-vi- and the remaining necessary elements are placed on separate ral vector carrying the corrected copy of the gene. Generally, AAV genetic elements, often three or more, says Carsten Carstens, and adenoviruses are primarily used for creating in-vivo gene senior scientist, R&D, Agilent Technologies. Kateryna_Kon/Stock.Adobe.com

12 BioPharm International May 2020 www.biopharminternational.com Take control of your bioprocess Cedex Bio and Bio HT Analyzers

Quantify IgG in Real Time • Accuracy over a wide range (10 mg/L to 8 g/L*) • Customizable to your human IgG molecule • No sample fi ltration or pretreatment required Results Comparable to HPLC IgG titer monitored on two Cedex • Also available: Human Ig Fab and Bio HT Analyzers and HPLC over Mouse IgG Assays 18 days. Variation coeffi cient <6% to Protein A HPLC. *Additional on-board dilution capability Data on fi le at Roche.

Reliable Photometric Technology • High accuracy and precision • Broad range and sensitivity Broad and Expandable Menu • Glucose, Lactate, Ammonia, Glutamate, Glutamine, IgG, Pyruvate, Ala-Gln, Acetate, Phosphate +17 more

For more information, visit go.roche.com/cedex-info or contact [email protected]

For use in quality control/manufacturing process only. Glutamine, Ala-Gln, Iron, LDH: For quality control/manufacturing of IVD/ medical devices/pharmaceutical products only. CEDEX is a trademark of Roche. © 2020 Roche. MC-US-06401-0420

MC-US-06401_CedexBio_BioPharm_AD.indd 1 3/30/2020 11:02:04 AM Cover Story: Gene Therapies

“The objective here is to prevent for- wide target range. The upside of pseudo- Most packaging systems will result in mation of a replication-competent viral typing is a tailored target range. The infectious particles that will not, or at least particle,” Carstens emphasizes. “The downside is that some modifications of infrequently, integrate into the genome, non-payload elements are generally the envelopes lead to destabilization of and, thus, infection results in transient referred to as the packaging system. If the viral particle and that pseudotyping expression such as the adenoviral vectors. performed properly, packaging results requires engineering of the packaging sys- “The virus can integrate into the genome only in infectious particles carrying the tem that is often not accessible to most at a very specific site (e.g., the ‘lysogenic’ payload.” customers,” Carstens states. form). However, this requires modi- The primary objective of these viral Lentiviral vectors, meanwhile, are a fication to the packaging system,” says vector systems is to achieve the high- subgroup of retroviral vectors. Lentiviral Carstens. Upsides to AAV include the est possible titers without forming vectors have displaced traditional retro- relative safety of these vectors (versus len- replication-competent variants. “For lentiviral vectors because lentiviral vectors do tiviral), relatively low immune responses, viral packaging, there are iterative cycles of not require dividing host cells for integra- and reasonably broad tropism. Some of safety improvements. Agilent vectors, for tion, Carstens further explains. This char- the downsides include a limited payload, example, are suitable and compatible with acteristic broadens the utility of lentiviral difficulty achieving high titers and func- generation-four packaging systems (i.e., vectors compared to traditional retrovi- tional titers, and difficulty in pseudotyping, the latest version of packaging systems).” ral systems. Upsides to using a lentiviral Carstens reports. For all practical purposes, a viral vec- vector include the ability of the system tor is a genetic element wrapped in its to carry a relatively large payload and THE PROMISE OF AAV own transfection reagent, which makes genetic elements that become active only Of the different virus types that have been transfection titrations possible (e.g., dos- after integration. This makes it relatively explored for gene therapy, AAV is the age responses) and highly effective. “In an straightforward to achieve desired titers most commonly used because it offers R&D setting, this becomes preferable if and to achieve a single integration ele- certain advantages that make it a promis- the same reagent is transfected (for exam- ment, Carstens emphasizes. Downsides ing vector compared to other vector types. ple, CAS9 delivery vectors) since all the include the fact that it is more difficult Chaudhary points out some of the advan- work is in the generation of the viral stock, to achieve large titers. In addition, each tages that AAV offers: not in the infection,” Carstens says. integration event constitutes a mutation • AAV possesses characteristics that Carstens points out that the greatest in its own right, which becomes a safety allow efficient manipulation of the limitation of viral vector systems is their concern when it comes to gene therapy, vector (as required). tropism, that is, the type of cells that a Carstens notes. • AAV possesses the ability to be virus will naturally infect. Infectivity, he In comparison, adenoviral vectors do easily purified, as they are not readily explains, is usually dictated by the enve- not integrate into the host genome and degraded by shear forces, enzymes, or lope proteins present in a virus and the are thus a transient delivery system. “They solvents. cellular surface components to which were once thought to have potential in • AAV exhibits reduced risk of adverse those proteins bind. “Depending on the gene therapy approaches, but there are inflammatory reactions because of its application, the ideal vector either has potentially strong immune responses non-pathogenic nature and because it an extremely broad tropism or extremely when used in vivo, which is why they has less immunogenic properties. narrow if the objective is gene therapy of are typically not used in gene therapy • AAV allows for the delivery of genetic a very specific cell type (in this case this is approaches anymore,” Carstens states. sequences of up to approximately 4 kb. a safety precaution),” Carstens elucidates. Finally, AAV is a “defective” virus that • AAV exhibits reduced risk of ectopic Some vectors, such as adenoviruses, is not replication competent by itself. integration of the therapeutic DNA. have a fairly broad tropism, and the target “[AAV] is famous as being the only “Adenovirus (AV) was the first viral range is often modified by engineering known vertebrate virus to form a lysogen, vector investigated for gene therapy use; part of the envelope proteins. Carstens meaning it integrates into the genome however, after the death of a patient cautions, however, that biomanufactur- in a dormant state from which it can in 1999 due to immunogenic reaction ers need to be aware that such modifi- be rescued by superinfection with cer- against AV (1), researches on gene ther- cations are properties of the packaging tain viruses, most notably the adenovirus apy using AV were put to a halt, and system, not the viral vector. “The process (hence its name) or by some forms of other vectors were investigated, includ- of changing the tropism of the vectors is chemical treatments (butyric acid and ing AAV, especially due to AAV’s lower referred to as pseudotyping. For instance, others),” notes Carstens. The natural immunogenicity. The latest approvals of Agilent has retroviral and lentiviral vector defectiveness of AAV makes it an inher- gene therapies using AAV (e.g., Luxturna, systems that are pseudotyped to achieve a ently safe delivery system. Zolgensma) have led to a surge in AAV

14 BioPharm International May 2020 www.biopharminternational.com An ever-changing journey needs an agile partnership

The dynamic nature of drug development and your world, means you need an adaptable partner. By joining forces with you, we provide a highly ﬂ exible, expert service covering contract development and manufacturing services for mAbs and viral vectors, product characterization, biosafety testing and toxicology testing services. So whatever direction your project needs to take, we’ll help guide you there.

To find out more, visit: SigmaAldrich.com/BioReliancePartner

MilliporeSigma, the vibrant M and BioReliance are trademarks of Merck KGaA, Darmstadt, Germany or its aﬃ liates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.

The Life Science Business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the US and Canada. Cover Story: Gene Therapies

use as it has proven safe and effective,” MANUFACTURING “For this, we also should not forget adds Vicaire. BOTTLENECK Industry 4.0 and the use of data analytics Based on recent approvals and the ben- Manufacturers, meanwhile, are contend- and machine learning within biopharma efits of AAV, including its low immuno- ing with the challenges of manufacturing (such as multivariate data analysis). This genicity, the fact that it does not cause any viral vectors. Among the main manufac- will influence the production of AAV disease in humans—something other viral turing challenges today for AAV sero- and other viral vectors. Learning how vectors have the potential to do—its abil- types is the use of the adherent human process parameters in the upstream pro- ity to confer long-term gene expression in embryonic kidney-293 (HEK-293) cell cess influence the purification process is vivo, its potential to infect replicative as line. The current AAV manufacturing fundamental for a good quality-by-de- well as non-replicative cells, and its poten- process using the HEK-293 cell line can sign strategy. Furthermore, it will be tial to transduce a wide range of tissues, only be increased by a “scale-out” method, interesting to see the developments in the AAV is currently considered the best viral notes Touw. This is carried out by imple- intensification of monoclonal antibody vector for in-vivo gene therapy, and is also menting more hyperstacks, or cell stacks, (mAb) production processes. Lessons considered as a potential delivery vehicle for production. “Scale-out of these sys- learned and technologies developed here for other applications, such as gene editing, tems result in more laboratory handlings could have the same benefits for the pro- asserts Vicaire. and, subsequently, a higher failure rate duction of viral vectors,” says Touw. By now, a wide variety of AAV due to contaminations,” Touw says. “After production, vectors need to be serotypes have been studied, adds “This can be prevented by implement- recovered from large volumes of cell Kai Touw, technical lead at Batavia ing a novel fixed-bed bioreactor sys- lysate or medium which includes: the Biosciences, a Netherlands-based tem for adherent cell production like harvesting of the producer cells, cell contract development and manufac- the iCELLis (Pall) or scale-X system lysis procedures to release AAV vector, turing organization (CDMO). Some (Univercells),” Touw continues. “Batavia the clarification and removal of cellular of the serotypes have been success- managed to collaborate with the sup- impurities, vector separation and purifi- ful preclinical and clinical followed by pliers to optimize these systems and cation, and vector formulation and sterile regulatory approval in both Europe make them more suitable for process filtration,” adds Chaudhary. and the United States. “This makes development and clinical manufactur- Most downstream approaches to viral it a popular starting point for gene ing, specifically for viral vectors, leading vector separation and purification are therapy, although many serotypes con- to lower failure rates from significantly based around traditional laboratory pro- tinue to struggle with manufacturing less contaminations and, therefore, lower cesses that are not scalable or suitable challenges, such as low production cost of goods.” for clinical-grade manufacture because yields and therefore high cost of goods,” With a better understanding of the the viral vector field is still an emerging Touw says. upstream process for the various AAV sector in biomanufacturing, Chaudhary “Two distinct advantages of AAV vec- vectors, productivity can be increased, but also cautions. Despite the fact that the tors is the safety profile at large doses and this could give rise to new challenges outcome of such conventional purifi- the long-term expression (durability) of downstream, such as changes in the cation approaches would result in pure a gene of interest. This means they can impurity profile of the cell harvest, Touw product, the lengthy processing time in be used to treat genetic disorders, such as adds. “Because each serotype has its own manufacturing and the complexity of spinal muscular atrophy and hemophilia,” challenges, it’s not possible to simply add such a process results in a more expen- Chris Murphy, vice-president and general in available plug-and-play technologies,” sive downstream purification procedure manager, Viral Vector Services, Thermo he states. and ultimately, cumulative yield losses. Fisher Scientific, further adds. More recent innovations, such as novel “Therefore,” Chaudhary explains, “the “Most packaging systems with AAV membranes and monoliths, are suitable lack of scalable platform technologies for have been engineered so that it does not as a capture step, Touw notes, while at purification has proven to be a bottle- integrate into the host genome, and, as the same time lowering buffer consump- neck in downstream processing. In addi- a result minimizes any potential non-in- tions. Other innovations, such as affinity tion, it is important to maintain AAV tended mutations, versus lentiviral vectors, ligands for specific AAV serotypes are vector biological activity when removing which are only active after integration. being developed for use with these tech- impurities and contaminates present in a The relatively low immune response com- nologies to further enhance the resolution feedstock that originates from host cells pared with adenoviral vectors makes of the capture step. A major challenge or culture media. Next to this, the vast AAV a more favorable approach,” concurs remains, however, and that is to control majority of described AAV purification David Weiss, product manager, Genome the amount of empty and hence non-in- processes rely on the specific serotype Engineering, Agilent Technologies. fectious particles during manufacturing. produced. This requires the design of a

16 BioPharm International May 2020 www.biopharminternational.com Cover Story: Gene Therapies

new process for each different serotype.” ugation is employed,” agrees Murphy. stream recovery (currently at best 30%, Chaudhary further explains that the “Thermo Fisher has developed down- new nanofiber technologies coupled presence of empty capsids presents yet stream processes using scalable technol- with next generation of ligands could another challenge associated with AAV ogy (e.g., column chromatography) that significantly improve recovery),” Vicaire vector purification. can handle large volumes of intermedi- further explains. The gene therapy manufacturing pro- ate while separating out impurities. This At Cytiva, the company offers a struc- cess is an overall exhaustive and complex eliminates the downstream bottleneck tural solution to increasing viral vec- task, presenting manufacturing challenges often making upstream production the tor manufacturing capacity through its such as the following: yield limiting step in the process,” he KUBio box, a modular biomanufactur- • Sterility and avoidance of states. Another manufacturing challenge ing environment. The company has tai- adventitious agents. It is difficult in viral vector production, Murphy points lored its KUBio product line to include a to maintain a complete sterile out, is the development and qualification standardized bioproduction environment environment using open systems of robust analytical methods to measure designed specifically for viral vector pro- that are extensively used in the product quality. duction. In addition, Cytiva has developed manufacturing units. “At the present time, there is cur- a technical solution that involves new • Live viral vector. Gene therapies rently a shortage of vector supply driven fiber-based purification products (Fibro involving the use of a live viral vector by the rapid growth of gene therapies technology) that increases downstream need extra care during handling and both in vivo and ex vivo,” adds Vicaire. recovery levels, according to Vicaire. manufacturing processes to maintain The shortage in viral vector supply is also the potency of the virus. Improper under pressure from inefficient manufac- EXPLORING NON-VIRAL handling leads to loss of viral potency. turing process and the need for specific GENE DELIVERY VEHICLES • Raw materials. Raw materials, such facilities with increased biosafety level Viruses, however, are not the only type as growth factors and cytokines, (BSL-2), which are currently lacking, of delivery vehicle being explored for that are required for manufacturing Vicaire points out. gene therapy. One area that is showing of gene therapy products are neither The solution to increasing viral vec- promise is with gene editing of cells easily available nor affordable. In tor capacity involves both structural and that can then be re-administered to the addition, the growth factors need technical issues. Vicaire states that a com- patient to correct a genetic mutation, to be continuously and manually bination of structural evolution on the says Murphy. He highlights the fact added/replenished in the medium. CDMO side and development of new that some innovators are using guide This cumbersome process leads to an technical solutions on equipment suppli- RNA with an endonuclease (e.g., Cas9) increase in the chances of errors. ers’ side can solve production challenges. to modify cells ex vivo and make the • Cell expansion environment. “We must bring robust, scalable, indus- edits. “I expect we’ll see some products Growing a large number of cells trialized manufacturing processes as fast approved in the future using this tech- is a challenging task due to lack of as possible (less manual and more auto- nology,” he asserts. technologies to monitor the cell- mated processes) and GMP solutions for While there is a significant amount expansion phase. Moreover, some both small-scale and large-scale produc- of work going into viral vectors to of the cells are sensitive to chemical tion,” Vicaire says. improve their tropism, rate of infec- transfection reagents. Furthermore, “The CDMO market is currently frag- tion, and adverse immunological because a cell-harvesting process mented with more new players offering to response, there is also much attention involves several steps, the manufacture vectors. However, the capac- on developing other gene therapy probability of committing mistakes ity is still not covering the current needs. mechanism options. “If we look beyond is much higher. Capacity increases should resolve the vector-based approaches, there are new • Cell preservation. Freezing the cells supply shortage, which will be followed CRISPR technologies that are mov- for preservation is one of the major by the consolidation of CMOs [contract ing into this space without some of challenges as only a limited number manufacturing organizations] with smaller the drawbacks of vector or virus-based of vessels are available for use in a players being acquired,” Vicaire adds. systems,” Weiss adds. “Two of these cryopreservation environment, “On the technical side we must approaches, Base editing and Prime directly impacting the cell viability improve titer upstream (high titer sta- editing, have the ability to introduce and quality. ble producer cell line and/or cell line small changes or corrections to the host “For certain manufacturing processes, and media optimization) and for AAV genome without introducing foreign downstream processing can be a bot- specifically, improve ratio of full versus DNA. Base editing has the potential to tleneck, for example, when ultracentrif- empty capsids; as well as improve down- specifically change single nucleotides

www.biopharminternational.com May 2020 BioPharm International 17 Cover Story: Gene Therapies

in a defined pattern (G–A, C–T, T–C, to the target cells with, for example, ible promoters are widely preferred as A–G) depending on the type of Cas9/ mammalian cell-line produced engi- gene delivery tools, owing to the fact deaminase complex being used. Prime neered exosomes, start to show their that these vectors offer the flexibility to editing, meanwhile, involves the use of capabilities,” Touw interjects. “As with activate/deactivate gene expression as a reverse transcriptase complexed to a all new technologies in biopharma, required,” Chaudhary explains. Cas9 nickase in combination with an there are several hurdles to take, but Various modified versions of plas- extended gRNA that contains an RNA we follow the developments in the mid DNA vectors have been developed, template used to confer the desired edit,” field closely and take along the lessons including minicircles and minivectors, Weiss explains. This process allows for learned from other molecules.” that are being used as vectors, Chaudhary a defined change to be made to a small “Plasmid vectors, meanwhile, are one further adds. “Apart from plasmids there region of the host genome without of the most common non-viral gene are liposomes, lipoplexes, polyplexes, and significant off-target and unintended delivery tools that are used to insert oligonucleotides [that can be used] as mutations, he adds. transgenes into target cells,” Chaudhary non-viral modes of gene delivery.” “Each of these CRISPR-based tech- states. “Plasmids can be easily modi- Vicaire also notes that using non-vi- nologies open up new avenues to make fied to deliver a therapeutic gene and ral vehicles offers a safety benefit in targeted changes to a host genome optimize its expression in a host cell. that there is less immunotoxicity com- with less of a potential immunologic The incorporation of a multiple cloning pared to viral vectors and potentially response. There are certainly drawbacks site into a plasmid enables the insertion lower cost as well as ease of produc- with these approaches as well, and there of a gene of interest with the help of tion, such as for synthetic or mechanical is more work that needs to be done in restriction enzymes. The circular DNA approaches. Challenges to using non-vi- this area, but it is a truly exciting devel- is nicked at the multiple cloning site ral approaches, however, include poorer opment,” Weiss emphasizes. and, after the incorporation of the gene efficacy, the potential of shorter dura- “With gene therapies, both with of interest, the nicks are annealed via a tion of the gene therapy expression (i.e., episomal expression and gene integra- ligation step. The gene present in these repeated dosing may be required), and tion, the main purpose is to have a vectors is generally flanked by a pro- poor targeted delivery because there long-lasting effect. At this moment moter sequence and a transcription ter- may be less tissue specificity. viral vectors are the most promising minator sequence to facilitate proper technology. That being said, other expression after it is incorporated into REFERENCE technologies such mRNAs delivered the host genome. Plasmids with induc- 1. S. Lehrman, Nature 401, 517–518 (1999). ◆

More on gene therapy-related manufacturing and development

For more on gene therapy and gene therapy-related manufacturing and development, read these articles on BioPharmInternational.com:

• MilliporeSigma Adds Viral Vector and Gene Therapy Capacity www.BioPharmInternational.com/milliporesigma-adds-viral-vector-and-gene-therapy-capacity-0 • MicrofluidX Raises Funds to Develop Novel Cell Bioprocessing Technology www.BioPharmInternational.com/microfluidx-raises-funds-develop-novel-cell-bioprocessing-technology • Orgenesis, ExcellaBio Announce Breakthrough Manufacturing Process for Bioxomes www.BioPharmInternational.com/orgenesis-excellabio-announce-breakthrough-manufacturing-process-bioxomes • Univercells Launches CDMO to Support Cell and Gene Therapy Developers www.BioPharmInternational.com/univercells-launches-cdmo-support-cell-and-gene-therapy-developers • Amicus Opens New Gene Therapy Center of Excellence www.BioPharmInternational.com/amicus-opens-new-gene-therapy-center-excellence • Navigating GMPs for Gene Therapies www.BioPharmInternational.com/navigating-gmps-gene-therapies • Sourcing Success in Cell and Gene Therapy Development www.BioPharmInternational.com/sourcing-success-cell-and-gene-therapy-development-1 • Using the “Cubic Effect” to Drive Cell and Gene Therapy Commercialization www.BioPharmInternational.com/using-cubic-effect-drive-cell-and-gene-therapy-commercialization-0

18 BioPharm International May 2020 www.biopharminternational.com STB-AD.qxp:Layout 1 3/1/18 10:04 AM Page 1 Highly-Polished Sanitary Stainless Steel Vessels FOR THE PHARMACEUTICAL INDUSTRY

SPOTLIGHT ON THE STB SERIES > Type III DMF > Certifications Available - 316L MOC Lot Traceability - 10 Ra Surface Finish - USP Class VI PSIL Gaskets

STB SERIES CTL SERIES (1mL TO 750mL) (10L TO 300L)

BTB SERIES CTH SERIES (0.5L TO 30L) (4L TO 500L)

EagleStainless.com | 215.957.9333 ISO 9001 Certified Biopharma Insights SPONSORED CONTENT THOUGHT-LEADERSHIP FEATURE

• Protect: A Robust Storage and Challenges in Bulk Drug Shipping (RoSS) shell protects every single-use bag available through BULK.STREAM. Its Substance Management sophisticated construction offers many advantages (www.susupport. Closing the gap between downstream com/en/bulk-stream/ross). and fill–finish. • Fill/Drain: RoSS.FILL is a flexible, fully automated platform for the inal liquid drug substance pro- In addition, numerous challenges allocation and distribution of bulk duction and fill–finish activi- and complexities must be over- drug substance in single-use bags in F ties rarely occur at the same come throughout the supply chain RoSS shells (www.susupport.com/ site. Nearly all pharmaceutical and to comply with good distribution en/bulk-stream/ross-fill). biotechnology companies outsource practices as well as governmental • Freeze/Thaw: RoSS.pFTU a portion of their fill–finish needs and other regulations (3, 4). is a plate-based freeze and thaw to contract manufacturing organiza- platform that offers safe and tions (CMOs) to help them address HOW TO STREAMLINE THE consistent freezing of high-quality unexpected demand, satisfy second BIOPHARM SUPPLY CHAIN substances in various scales (www. source policies, or better match scale BULK.STREAM® from Single susupport.com/en/bulk-stream/ with need (1). Thus, the shipping of Use Support closes the gap between freeze-thaw). liquid drug substances worldwide is downstream and fill–finish activi- • Store/Ship/Track & Trace: RoSS. often inevitable as well as filled with ties with a new, secure management SHIP is a highly robust, stackable, challenges and complexities. process for liquids (Figure 1). Using and coolable device that holds up single-use bags and plate-freezing, to 20 RoSS trays for storage and COMMON SUPPLY BULK.STREAM® guarantees fast shipping of frozen substances in a CHAIN CHALLENGES and safe protection, filling, freez- protected manner (www.susupport. Proteins or monoclonal antibod- ing, storing, shipping, and thawing com/en/bulk-stream/ross-ship). ies with highly complex struc- of high-quality substances from one tures require cold chain handling. set of hands to another. The single- HOW TO OVERCOME Insufficient freezing of these sub- use bag agnostic technologies increase CRYOCONCENTRATION stances not only renders them inef- patient safety and minimize the risk Freezing drug substance is essential fective, but also harmful and possibly of biocontamination and product loss for storing and shipping, but the pro- even life-threatening to patients (2). toward 0%. BULK.STREAM® also cess stresses the protein and can lead Many sets of hands are involved enables the storage and shipment of to denaturation. Cryoconcentration in packaging, lifting, hauling, and high-quality substances in a faster and leads to factors that induce protein delivering products thus creating more secure manner, and at a lower denaturation. For example, the high great potential for human errors. loading volume than to date. protein concentration itself causes Figure 1. Single Use Support’s BULK.STREAM closes the gap between downstream and fill–finish.

20 May 2020 SPONSORED CONTENT Biopharma Insights THOUGHT-LEADERSHIP FEATURE

denaturation through protein Figure 2. Blast freezing versus plate freezing. aggregation (5, 6). The solubility of the dissolved buffer salts decreases as temperature decreases. As freezing proceeds, ice formation excludes solutes (including protein) from the growing ice crystal. Solutes migrate in front of the growing ice that consist of small finger- like projections called dendrites (7, 8). These dendrites trap some solutes as they grow, so solutes are found in all regions of the block instead of only at the last point to freeze. As the solution trapped between dendrites freezes, however, it continues to undergo cryoconcentration as the water component is removed and as ice and the composition changes. antibodies in the center of the BULK.STREAM, an end- Cryoconcentration can never bag. As demonstrated with blue to-end solution for all scales, be eliminated, but with “con- color agent in Figure 2, slow addresses these challenges by trolled freezing,” geometry and freezing leads to cryoconcen- freezing large volumes of expen- freezing rate are determining fac- tration. When comparing the sive drug substances in batches tors in practical systems of freez- freezing results of static freez- to facilitate the production of ing (9). Using a slow freezing rate ers vs. plate freezers, it is evident medicinal drugs based on real- leads to a high cryoconcentration that fast freezing achieves more time commercial or clinical with a comparable low ice surface. homogenous freezing and pre- requirements. Cleaning, ster- The low freezing rate causes the vents excessive cryoconcentration. ilization, and repeating docu- ice to form slowly enough for the mentation are not needed with proteins to be pushed along the HOW TO COPE single-use systems. Moreover, ice rather than being trapped by it. WITH SCALABLE the RoSS platforms represent Exposure to concentrated solutes MARKET DEMAND flexible, fast, and scalable solu- can result in a loss of a protein’s Small volumes of drug substance tions that help decrease the risk thermodynamic stability, eventu- are often produced for early-stage of contamination and increase ally causing aggregation. clinical phases. Traditionally, the patient safety. Freezing rates that are too use of single-use drug substance In general, awareness of the high lead to the opposite effect, bulk freezing containers has gap in bulk drug substance man- namely a dendritic ice surface. been the norm for the 50 mL to agement (between downstream The dendritic structure envelopes 1 L range. While this size offers and fill–finish) is steadily increas- proteins in low cryoconcentra- an acceptable operational fit for ing. Securing the highest possible tions, but compared with a plane dispensing drug substance for standard is not only important in ice front, the surface of the den- smaller-volume pharmaceutical terms of efficiency, but also for Michael Eder drites is larger. manufacturing processes, it is not safeguarding high-quality prod- is marketing manager ■ Studies have shown that plate ideal for larger-volume dispensing ucts for patients (11). at Single Use Support. freezing technology achieves processes. It is typical for commer- FOR REFERENCES, GO TO Biopharma Insights— the best results because the fast cial programs to generate several Thought Leadership freezing process hinders the hundred liters of drug substance THE ONLINE ARTICLE AT from Marketers/ accumulation of proteins and per batch, for instance (10). BIOPHARMINTERNATIONAL.COM Paid Program

May 2020 21 Upstream Processing

Biopharmaceutical Manufacturing and the Power of Synthetic Biology Synthetic biology has advanced the scope and scale with which biologically derived therapeutics can be developed.

DAVID McELROY

raditional small-molecule drug discovery—the main- PICK A HOST... stay of pharmaceutical R&D for more than a century Biotherapeutic production generally relies on inserting T and a half—has become increasingly challenging, as genetic elements encoding the peptide, protein, or antibody the “low hanging fruit” of universally effective therapeutics of interest into a host organism capable of producing it in have been slowly exhausted. The only real potential areas for large quantities. There are a wide range of potential cel- major growth in the small-molecule drug sector are now the lular hosts that can be employed for bioproduction—each discovery of new drug targets that come from furthering our with its own unique advantages and limitations. At present, understanding of disease and the development of precision Escherichia coli (E. coli) or Chinese hamster ovary (CHO) medicines targeting specific subpopulations of patients. cells are the “go to” systems for the majority of labs looking Fortunately for the industry, the rise of biotherapeutics to produce biological molecules that are ultimately intended is making up for this gap in traditional discovery pipelines, for clinical applications. However, more often than not, this providing greater specificity, new avenues of therapy, and is because they are the host organisms researchers are most the possibility of combating previously untreatable diseases. familiar with, rather than the optimal choice of expression Somewhat less fortunately, the commercial development system. This generally isn’t an issue in an upstream R&D and large-scale production of biological agents are far more setting, where only small quantities of the biotherapeutic complex than classical chemical syntheses. This means that are required, but can lead to yield and solubility issues when

many novel biotherapies never make the transition from * DAVID McELROY , PHD, [email protected], is chief the lab bench to the clinic, simply because the requisite business officer at Ingenza Ltd, UK. biological molecules can’t be produced in the purity or *To whom correspondence should be addressed. quantity required. ELNUR/STOCK.ADOBE.COM

22 BioPharm International May 2020 www.biopharminternational.com Upstream Processing

pda.org/EU/Bio2020

attempting to scale up production for preclinical and clinical studies, or for manufacturing. This is a real stumbling block for many potential biotherapeutics, as the resources required to scale up production in an inefficient or unsuitable host 2020 PDA EUROPE can make it economically unviable.

... ANY HOST Foreseeing and overcoming the various issues that can arise during the biomanufacture of novel drug products requires BioManu- a broad understanding of the various host organisms available, their strengths, and, crucially, their weaknesses. While there are no set rules to determine which host may be the facturing most effective for the production of a given biomolecule, a broad expertise and holistic understanding of each organism’s metabolism can help to avoid problems downstream in product development. Experience is crucial here, as is having access to an extensive toolbox of synthetic biology (synbio) and metabolic engineering technologies (Table I) designed to optimize gene expression and production of the target molecule. The major challenge for drug discovery groups— or even biotech companies—attempting to develop and exploit these technologies in-house is that most simply don’t have the breadth of knowledge necessary for success. They are, no doubt, experts in their specific technology or disease area, but the scale-up production of biotherapeutics requires a very different skill set, covering aspects of synthetic and molecular biology, fermentation, and chemistry as well as good manufacturing practice (GMP) manufacturing. The true power of synbio for biomanufacturing comes from the ability to combine commercially validated synbio tools (Figure 1) with other techniques and technologies to enhance the overall production system. For example, transcriptomics and proteomics—and even more tradi- Directly followed by the tional bioengineering techniques, such as ultraviolet (UV) PDA EUROPE PHARMACEUTICAL mutagenesis—can be used to identify further enhance- FREEZE DRYING TECHNOLOGY ments that improve the hosts’ overall production capabilities. Conference at the same venue! The expression capacity of the system can then be further increased through strategies such as ribosome engineering— to uplift overall gene expression—as well as the over-expression of regulatory genes, and the suppression or knockout of genes that may compete for resources, or could metabolize or 22-23 SEPTEMBER 2020 destabilize the final product. DUBLIN, IRELAND THE NEED FOR EXHIBITION: 22-23 SEPTEMBER A HOLISTIC APPROACH TRAINING: 24-25 SEPTEMBER The main challenge in biomanufacturing is that scaling up production of therapeutics requires not just an REGISTER BEFORE understanding of the individual elements involved in 26 JULY AND SAVE the process—cell culture maintenance and fermenta- UP TO €200! tion, genetic manipulation, codon optimization, directed evolution, metabolic engineering, transcriptomics, and proteomics, etc.—but also how all of these components

www.biopharminternational.com May 2020 BioPharm International 23

2020 BioManu_HP_vert_EU_85x241.indd 1 09.04.20 16:26 Upstream Processing

Table I. Summary of synthetic biology approaches to enhance biologics production. PCR is polymerase chain reaction. KOs are knock-outs. DSP is downstream processing.

Bioprocess steps Current limitations Synthetic biology benefits Restriction enzyme-based DNA cloning/ manipulation: “One-pot” combinatorial assembly of DNA: Gene design—cloning and • Slow and inefficient • Cheaper optimization of gene-expression construction • No rapid iteration designs by re-using all parts • Error prone due to PCR-derived DNA mutations • Better construct integrity by eliminating PCR errors

Limited range of naturally occurring expression Synthetic elements to control target gene elements: expression: Gene design—expression • Limits freedom to operate • Greater diversity of induction protocols available elements • Unstable • Better stability from non-repeating/orthogonal • Negatively impacts host gene expression expression elements

Trial and error empirical testing to optimize protein Superior protein stabilization using chaperones and stability: stability elements: Expression optimization • Highly unpredictable and costly • Better yield of target using co-expressed —protein stability • No systematic definition of criteria to enhance chaperone stability • Cheaper process from simplified target handling

Omics-driven exploitation of host factors impacting Traditional mutation/screening: target production: • Slow and unpredictable • Predictable genome editing to optimize gene • Damage to host from mutations expression • Difficult to define and combine beneficial traits • Faster and cheaper host bioengineering precision (e.g., protease KOs) Expression optimization —protein yield Direct selection and/or screening of high producing Trial and error testing: clones: • Slow, unpredictable • Faster identification of high yielding clones • Costly rebuilding of systems to be tested • Cheaper process optimization • Adaptable to apply to new targets

Precise gene targeting into the host genome: Random gene integration into the host genome: • Predictable integration at most favored genomic Expression optimization • Unpredictable outcomes locations —yield stability • Slow process to identify best expressing strains • Better expression and stability by re-use of optimal locations

Gene design “rules” and colorimetric reporters for best expressers: Trial and error testing: Analytical methods • Cheaper production by direct selection of well- • Slow and unpredictable process —expression optimization expressed soluble product • Costly rebuilding of systems to be tested • Better definition and translation of performance criteria to new targets

Large (>10,000 L) steel fermentation systems: Disposable (1 L–200 L), single use fermenters: • Expensive capital investment • Lower capital investment Upstream processing • Slow turnaround between runs • Cheaper manufacturing due to simpler process —fermentation • High resource consumption—water, energy, • Reduced environmental impact from less chemicals cleaning/validation

Proprietary target protein capture, release/ Large-scale, multi-column chromatography: maturation, and concentration: • Time-consuming and costly • Faster and cheaper purification with fewer DSP Downstream processing • Multiple cGMP process steps steps —protein purification • Separate operations for target purification and • Better purity through unique capture maturation • Adaptable to new targets • Safer handling of potent targets through simultaneous recovery/maturation

24 BioPharm International May 2020 www.biopharminternational.com Upstream Processing

Figure 1. Technology readiness level cascade for the commercial validation of synbio tools. DSP is downstream processing. cGMP is current good manufacturing practices. QMS is quality management system.

interact as part of a holistic bioman- no hard and fast rules on which vec- There are no shortcuts to overcoming ufacturing system. Worse still, opti- tor, insertion locus, individual strain, this issue, but experience can mean the mizing these bioproduction systems or even host organism may be best difference between success and failure. often requires the ability to screen suited to the production of a specific There are obviously numerous ways of hundreds or thousands of individual biological product, it is easy to see tackling any given challenge that may strains or mutants to identify the why so many biotherapeutics never arise during biomanufacturing scale-up, highest yielding candidates. With make it to the clinic. so working with an experienced partner

Changing the ART of analytical chromatography with μPACTM capLC columns:

Unrivalled separation power Micromachined reproducibility Flow rate exibility Plug-and-play connectivity 1 - 15 µl/min

Designed for increased robustness and throughput without losing sensitivity.

Discover our products on www.pharmauidics.com ALL FIGURES COURTESY OF THE AUTHOR.

www.biopharminternational.com May 2020 BioPharm International 25 Upstream Processing

Figure 2. A colorimetric screen for use in amine oxidase protein engineering assays must offer a quick and easy “yes/ and expression. This assay was developed and used by Ingenza to screen 105 individual E. coli transformants for target enzyme expression, with a one month no” assessment of every transformant, per round of protein evolution. HRP is horseradish peroxidase. and generally employ simple colorimetric or fluorimetric detection. The challenge here is that the common approach of using a generic reporter gene-linked expression assay—combining the target of interest with the expression of a reporter protein of some sort—is often not appropriate when optimizing biotherapeutic production, and so a predictive assay tailored to the individual target protein must be developed. This is often the rate-limiting step in the identification and selection of improved gene variants, which can significantly delay development, or even halt progress entirely if not deployed successfully. Working with a partner that has extensive experience in designing and performing this type of screening assay can dramatically accelerate timelines, generating lead candidates much sooner. Figure 2 shows an example of is often the fastest way to get to an effi- rapidly transfer production of a target a screening assay designed to improve cient and effective solution, without the biomolecule into a new host. For exam- transcription/translation of a novel cost and delays of following numerous ple, switching production from E. coli amine oxidase in E. coli. The assay, which dead ends. Outlined below are a num- to Pichia pastoris (P. pastoris) may elimi- was developed and validated in just a ber of issues commonly encountered nate solubility issues encountered in early few weeks, uses a colorimetric substrate during biotherapeutic development scale-up studies, allowing the high yield- (added to the culture media) to assess and scale up, with examples of vari- ing production and secretion of a high the expression of an enzyme involved in ous approaches to address them using purity product for GMP manufacturing. the in-vitro biocatalysis of an advanced different host organisms and synthetic pharmaceutical intermediate. Combined biology approaches. EXPRESSION SCREENING with design of experiment (DoE) LIMITATIONS approaches and an automated workflow, PROTEIN SOLUBILITY ISSUES Protein engineering for improved ther- this type of assay allows both the selec- One of the most common challenges apeutic function is often a numbers tion of high-yielding transformants and encountered when attempting to scale game; the more gene variants that can the determination of optimal expression up bioproduction is the solubility of the be screened, the higher the chance of conditions in a very short timeframe. target protein or peptide. The simplest, success. E. coli is often a good choice The development of efficient screen- and often fastest, way to overcome this is of host for this application—at least ing assays can also help to improve the to express your product in an alternative during the early development phase— product itself, by enabling larger studies host organism. However, this requires due to the plethora of bioengineering to be performed than would otherwise an understanding of which hosts might tools available for this host. However, be possible. High assay throughput pro- be appropriate, the tools to design, build, effective variant screening requires rapid, vides the opportunity for further product and test a suitable expression vector, and cost-effective assays that enable the qual- optimization, potentially in combina- the capacity to perform strain selection itative or semi-quantitative assessment tion with machine learning or artificial and growth optimization. While this may of target protein expression across large intelligence strategies, to identify poten- sound a daunting task for most labs, spe- numbers of individual transformants. tial enhancements or improvements cialist synthetic biotechnology companies Unlike the functional or kinetic assays in the design of the target biomole- routinely perform this type of project, and used in downstream characterization cule. For example, advanced screening have the knowledge and experience to studies, these variant library screening has been successfully combined with

26 BioPharm International May 2020 www.biopharminternational.com Upstream Processing

Figure 3. Synthetic biology and high throughput screening can be combined with machine learning to aid in the development of new biotherapeutics. FACS is fluorescence activated cell sorting. IP is intellectual property. DSP is downstream processing.

machine learning to further enhance the so selecting a strain that excretes the example, many Bacillus species are spore potency of epidermicins, a new class of heterologous target protein will help to forming, which is an undesirable trait in antimicrobial peptides capable of rap- simplify downstream purification. a closely regulated GMP environment. idly killing potentially harmful bacte- It is also important to consider the The development of asporogenic strains ria, including drug-resistant species stability of the target protein within the has mitigated this issue, simplifying (Amprologix) (Figure 3) (1). expression system. For example, Bacillus culture management and, consequently, sp. have a number of protease enzymes reducing biomanufacturing costs. POST-TRANSLATIONAL that could cause the degradation of the EXPRESSION OPTIMIZATION target protein before it has even left the USING THE WHOLE TOOLBOX Looking beyond cell culture main- cell. Choosing knockout mutants that Advances in synthetic biology have tenance and the use of alternative lack the major proteases can therefore made it possible to coordinate the transcription/translation elements of increase overall yields by improving tar- expression of multiple genes to allow protein expression, there are a num- get protein stability. Another strategy the creation of novel biochemical path- ber of other factors to consider when to increase protein stability is to create ways. This allows the host organism to attempting to scale up bioproduction. a more favorable environment for your produce large quantities of a heterolo- Even for a well-characterized host such product. For example, co-expression of gous product that would normally be as Bacillus subtilis (B. subtilis)—which chaperonins has been successfully used outside of its metabolic “repertoire”. It is widely used in the production of in the production of recombinant factor is only by understanding each of these enzymes—there are various opportu- VIII in CHO cells by allowing high elements, while still considering the sys- nities to improve the overall bioman- levels of expression without leading to tem as a whole, that biomanufacturing ufacturing process in culture media misfolding or aggregation. can reach its true potential, and working composition, protein secretion, and Failure to consider these kinds of with a knowledgeable and experienced endogenous protease gene knockout. post-translational factors could sig- synbio partner is the best way to ensure Being able to extract the target protein nificantly reduce the overall yield, and successful scale up of novel biothera- from the bioreactor is obviously essen- therefore performance, of a bioman- peutics, on time and on budget. tial for efficient production, avoiding ufacturing system. Even beyond this, REFERENCE the need to destroy the culture to har- there are practical considerations when 1. Ingenza, “Amprologix Secures Funding vest the product. A number of secre- choosing the best expression system to Develop New Antibiotic with tion systems already exist for B. subtilis, or host for a target biotherapeutic. For Ingenza,” Press Release, Feb. 5, 2019. ◆

www.biopharminternational.com May 2020 BioPharm International 27 Downstream Processing

Fluid Handling Considerations Pumps and other components meet the demands of single- use systems in biopharmaceutical downstream processing.

JENNIFER MARKARIAN

he growing use of single-use systems and ongoing ciency, throughput, and product consistency while reducing development of continuous processing place new manufacturing costs. A typical example is chromatography. T demands on pumps and other fluid-handling compo- In batch mode, a column filled with resin runs through nents in downstream biopharmaceutical processing. Pumps the modes of equilibration, loading, washing, elution, and used with single-use systems include peristaltic pumps, in regeneration, one after the other. The continuous version is which the fluid flows through single-use tubing, and dia- called simulated moving bed chromatography. In this process, phragm pumps, which can also be single-use and move the several columns operate in parallel, with each of them in fluid using suction created by the diaphragm. BioPharm one of the aforementioned modes. Complex valve config- International spoke with Andreas Frerix, product manager urations are used to switch the column from one mode to for diaphragm pump manufacturer Quattroflow, a prod- the other. While this setup reduces the amount of expensive uct brand of PSG, a Dover Company, and Gregg Johnson, resin needed, it requires several pumps to run in parallel at global product manager for Masterflex and Masterflex high consistency over a long period of time. A high degree Ismatec peristaltic pumps at Cole-Parmer, about trends and of pump reliability and flow stability is needed because these best practices in fluid management. continuous processes can run for weeks rather than for only a day when in batch mode. TRENDS Johnson (Cole-Parmer): Both single-use and con- BioPharm: What trends do you see in downstream process- tinuous processing are growing trends because both offer ing and how do these relate to the requirements for the fluid unique advantages depending on the application need. pumping system? Single-use will be prominent in personalized cell/gene Frerix (Quattroflow): While by far most operations in therapy. Continuous processing will grow in large pro- downstream processing are in batch mode, there is a trend duction volume applications, such as virus or vaccine pro- to continuous bioprocessing because the industry is seeking duction, as operations search for opportunities to increase ways to advance their current technologies to improve effi- output and efficiency. innluga/Stock.Adobe.com

28 BioPharm International May 2020 www.biopharminternational.com Vaccines Illuminated: Biophysical Characterization, PAT, and Quality Control via Light-Scattering Techniques

Register for this free webcast at: LIVE WEBCAST: www.biopharminternational.com/bp_p/vaccines_illuminated Tuesday, May 19, 2020 11am EDT | 8am PDT | 4pm BST | 5pm CEST Event Overview Accurate preclinical characterization of vaccine candidates enables timely product and process development, leading to effective manufacturing, and quality control procedures. This webcast will highlight the role of light scattering techniques for characterizing a range of modalities, including DNA and mRNA Presenter vaccines, viruses and virus-like particles (VLPs), and protein- based vaccines. The role of light scattering in downstream process analytics and quality control also will be discussed.

Key Learning Objectives • How molar mass, size, titer, and composition of therapeutic and vaccine biomolecules are determined with size- exclusion chromatography coupled to multi-angle light scattering (SEC–MALS) Sophia Kenrick, Ph.D. Director of Analytical Services • How mRNA and lipid nanoparticle vaccines are Wyatt Technology characterized with field-flow fractionation coupled to MALS (FFF–-MALS) • How quality attributes of viruses and virus-like particles are quantified using MALS and dynamic light scattering (DLS) • How high-throughput formulation and stability studies of are carried out with plate-based DLS Moderator • How real-time MALS can monitor chromatographic purification of viruses and VLPs

Who Should Attend • Researchers involved in the biophysical characterization of novel vaccines • Downstream process development engineers responsible for Rita Peters implementing process analytical technology in Editorial Director vaccine production BioPharm International • Vaccine quality control managers • Regulators evaluating biophysical characterization for novel vaccines and therapeutics

Sponsored by Presented by

For questions or concerns, email [email protected] Downstream Processing

Peristaltic pumps are suitable in ation. Both concerns can be addressed both the single-use environment and “Single-use pumps by choosing the correct tubing mate- to provide, with a simple change of have the most rial for the application. For long life material, the long running life for with minimal failure risk, thermoplas- continuous processes. These pump benefit when used tic elastomer (TPE) formulations offer systems can address both areas of sin- in facilities with the capability of running for months at gle-use and continuous processing. a time in a ‘single-use’ capacity. When Continuous processes would poten- small to medium low particulates are needed, selection tially be looking for longer-lived tubing. of a smooth tubing surface signifi- Continuous processes and single-use capacities and cantly reduces spallation. The primary processes require pumps with mod- frequent product determinant when it comes to tub- ularity, easy maintenance, and dosing ing selection is chemical compatibility, accuracy and precision. Metering pump changes.” then any regulatory requirements, then technology featuring single-use wet- the life of the tubing. ted parts connected to a driving motor, —Andreas Frerix, Frerix (Quattroflow): There are such as the tubing in peristaltic pumps several trends affecting single-use and the pumphead in diaphragm Quattroflow components. While continuous man- pumps, satisfy the modularity and easy ufacturing requires lower flow rates maintenance requirements because the single-use components is growing in and longer operating times, we also parts that come into contact with the importance to mitigate risk and ease see a need for handling larger flow fluid are simply replaced. The accuracy scalability. Single-use components rates as the volumes of single-use bio- and precision of these systems is deter- are used in applications such as per- reactors are rising, which increases the mined by the sophistication of the drive, sonalized medicines and cell and demand for single-use equipment with and drive features such as precision gene therapy. The ability to eliminate larger diameters. Another trend where RPM [revolutions-per-minute] control, cross-contamination and decontam- single-use components will play an programmable interfaces, and analog ination processes drives the need for important role is personalized medi- signal control have become baseline. single-use components. cine/cell and gene therapies. Peristaltic technology is low-shear Single-use pumps are used from SINGLE-USE COMPONENTS and mechanically simple, consisting process development up to commercial BioPharm: What trends do you see of a single piece of material. However, production. The main benefits of sin- in single-use components for fluid some concerns with single-use tub- gle-use pumps are: handling? ing in peristaltic pumps, which are • Eliminating cleaning Johnson (Cole-Parmer): mentioned with some frequency by requirements, as all wetted Reliability and standardization of pump users, are tubing life and spall- components are replaced with a new single-use set after every Predictive maintenance offers improved efficiency. product run • Reducing time needed to complete Preventive maintenance uses a intelligence, such as machine- an installation and get the time-based schedule to perform learning algorithms that learn production facility up and running routine maintenance and prevent patterns of how equipment should • Reducing water and energy equipment breakdowns. Predic- be operating and then can identify consumption for cleaning and tive maintenance, however, is changes (e.g., in vibration or flow) need-based rather than only time- that may indicate a problem will sterilization based. It uses data from equip- occur. Equipment can also be • Decreasing risk of cross-product ment sensors so that, with insight compared to similar equipment contamination into when a problem might occur, in the same plant or in facilities • Allowing more operational process operators and engineers around the world, and these large flexibility with a modular approach can make better decisions about datasets improve accuracy of the • Reducing capital investment in how to intervene with the least dis- predictions (1). facilities and equipment. ruption to the process (1). Reference Looking at these benefits, sin- Predictive maintenance has been 1. J. Markarian, Pharm. Tech. 44 (1) gle-use pumps have the most benefit enabled by advances in artificial 36-40 (2020). when used in facilities with small to medium capacities and frequent prod-

30 BioPharm International May 2020 www.biopharminternational.com Downstream Processing

uct changes, such as clinical-trial manufacturing or contract RELIABILITY AND MONITORING manufacturing organizations (CMOs). BioPharm: Is remote monitoring/predictive maintenance Single-use diaphragm pumps have been used for many being used for biopharma pumps? years. For critical flow and/or pressure-controlled opera- Frerix (Quattroflow): While diaphragm pumps are tions like tangential-flow filtration, chromatography, virus reliable when used within the specified operating conditions, filtration, inline dilution, and many more, the advantages of it is possible to use sensors to detect leak-causing diaphragm diaphragm pumps are their low pulsation when using multi- damage and stop the process. This detection is especially ple diaphragms (such as quaternary diaphragm pumps); the helpful when multiple pumps are used in parallel in a fully capability of delivering the needed pressure, especially for automated manufacturing process. chromatography or tangential-flow filtration; the propor- Johnson (Cole-Parmer): We have customers asking us tional flow characteristics; and the shear-sensitive handling now if we have solutions for remote monitoring/predictive of biological products. maintenance. We have a tool for remote monitoring and control in place that provides operations the ability to ‘see’ “Reliability and standardization what is happening with the pump system without actually being there. Coupling this insight with existing plant line of single-use components knowledge gives operations greater flexibility on how they manage the line. is growing in importance.” We are now seeing customers asking for pumps with more —Gregg Johnson, connectivity and automation options such as cloud-based monitoring and control. Cloud-based record keeping is also Cole-Parmer a growing request from our downstream customers, as manufacturing floors align with market trends to move business When it comes to processing large volumes (e.g., large- operations to the cloud while also meeting the growing scale monoclonal antibodies [mAbs] or blood-plasma frac- demand from regulatory bodies for more rigor and account- tionation), single-use equipment has a defined limit in abilityBioPharm in operational Intl fill assys data. qtr May20.qxp_Layout ◆ 1 4/13/20 12:54 PM Page 1 terms of its scalability and economics. Here, stainless-steel equipment is for some applications the preferred choice. In addition, single-use processes are widely adopted in the Single-Use Filling Assemblies manufacture of mAbs from mammalian cell cultures; however, when it comes to microbial fermentation, stainless-steel for Aseptic Fill & Finish Applications equipment is mainly used.

CELL AND GENE THERAPIES BioPharm: How do fluid pumping requirements for the emerging areas of personalized medicine and cell and gene therapies differ from conventional biopharmaceutical processing? Johnson (Cole-Parmer): Personalized medicine/cell and gene therapies require lower volumes of fluid, which requires the fluid handling system to have much lower flow rates, and the pump can be physically smaller. Pumps that offer independent multi-channel control and flows in the range of microliter • Molded junctions improve per hour are needed to meet these new requirements. Frerix (Quattroflow): flow & reduce the risk of leaks & entrapment One big difference is the batch • Custom made to your specs size, especially because in autologous gene and cell therapy, • Sterilized & ready to use See us at the cells from individual patients have to be treated, modified INTERPHEX within different processes, and then administered back to the patient. This technology involves one individual process per patient, along with relatively low flow rates, the need for disposable equipment, and the handling of low volumes. In www.advantapure.com/bp4 888-579-0751 these types of applications, particulate release from the pump

MANUFACTURED BY NewAge Industries AdvantaPure® & NewAge® cannot be tolerated, as these particulates can cause health risks EMPLOYEE-OWNED SOUTHAMPTON, PA , USA reg. TMs NewAge® Industries, Inc. once the modified cells are returned to the patient.

www.biopharminternational.com May 2020 BioPharm International 31 Manufacturing

Straight Talk on Closed Aseptic Systems The industry’s aversion to risk has led to its treating closed aseptic processing systems as miniature cleanrooms, resulting in redundant and expensive practices.

JAMES AGALLOCO

ommercial production of sterile pharmaceutical prod- provided the formal demonstration of performance capability ucts has changed dramatically since the early 1900s for aseptic processing. The media-fill test, first introduced by the C (1), when most injectable products were filled manually, World Health Organization (WHO), eventually became a regu- without the accoutrements that are now considered essential to latory expectation for aseptic activities (1,2). protecting sterile products from contamination. Over time, the As manufacturers gained experience using this test, they industry recognized that physically separating operators from the sought adaptations that would accommodate the preparation of product led to improved patient safety. As a result, some installa- sterile APIs (3). This came in the form of industry documents, tions began to use gloveboxes that were designed to protect sterile prepared jointly by the Parenteral Drug Association (PDA) materials from operator-borne contaminants. and Pharmaceutical Manufacturers Association (now referred As sterile liquid-handling systems grew substantially in size, to as the Pharmaceutical Research Manufacturers Association closed systems of varying complexity were also used in the aseptic [PhRMA]), the second of which formally defined the closed manufacture of liposome formulations, suspensions, creams, and aseptic processing system (4,5). The 2006 revision to this docu- ointments. Because these systems can be reliably sterilized and ment outlined the design and performance attributes that estab- maintained in a closed state, regulators did not formally require lish a system as ‘closed’. internal environmental monitoring of closed systems or of their According to this document, a closed aseptic processing system background environments (2). is one that is designed to prevent the ingress of microorganisms Nevertheless, a gap has developed between existing indus- by means of physical separation of materials and product from try practices and the science behind closed systems, which JAMES AGALLOCO is principal of Agalloco & Associates. With are still restricted by use of design and control concepts that nearly 50 years of industry experience with companies that include were developed for open systems (Sidebar, p. 34). This gap Merck, Pfizer, Squibb and Bristol-Myers Squibb, he has served as an officer or director of PDA and is a member of Pharmaceutical poses important questions for manufacturers and regulators. Technology’s editorial board, [email protected]. Until the 1970s, sterility testing and environmental monitoring prescott09/Stock.Adobe.com

32 BioPharm International May 2020 www.biopharminternational.com Manufacturing

the surrounding environment. In order to • Connectable to other closed systems tor-staffed cleanrooms. Even though meet this definition, the system must be: while maintaining the integrity of regulators did not explicitly require • Constructed, installed, and all closed systems (e.g., via a rapid environmental monitoring, manufac- qualified in a way that shows that transfer port, steamed connection, or turers incorporated existing environ- integrity is maintained throughout other comparable means). mental monitoring requirements into the full range of operating • Safeguarded from any loss of integrity routine practices. conditions and over a time period by scheduled preventive maintenance. The design concepts that were that includes periods of the longest • Operated using sterilizing filters embodied in the 2006 document for expected usage (i.e., manufacturing whose integrity has been tested, closed aseptic processing systems rep- campaign). This qualification must and which can be traced back to resent two different approaches in follow a formal protocol using each product lot for sterilization of closed system design (see Table I). generally accepted engineering process streams (5). This divergence has resulted in sub- principles and practices, and must stantial differences in validation, mon- be documented. ENVIRONMENTAL itoring, and operation. • Sterilized in place, or sterilized while MONITORING Blind adherence to methods and prac- closed, prior to use of a validated As first conceived, this definition tices developed for use with open sys- procedure. embraced isolator systems, which tems makes no sense with closed systems, • Capable of being used for its intended had evolved from the earlier glove- which, regardless of their configuration, purpose without compromising the box technology. These systems were separate process and operator, and are integrity of the system. mainly used in the later stages of bulk designed to be fully functional indepen- • Capable of being adapted for fluid antibiotic production (e.g., during dry dent of the background environment. transfers in and/or out while powder handling), operations that had One question for the pharmaceuti- maintaining asepsis. previously been performed in opera- cal industry to consider is whether the

Ca++Pure-HA™ Media Unique Multimodal Media for Bioprocessing Puriﬁ cation

A Best-In-Class Hydroxyapatite Media from Tosoh Bioscience LLC

 High dynamic binding capacity  Increased durability  High ﬂ ow rate/pressure tolerance

Excellent for Puriﬁ cation of mAbs, DNA/RNA, Proteins, and Viruses Want to learn more? www.tosohbioscience.com

TOSOH BIOSCIENCE LLC • Customer service: 866-527-3587 • Technical service: 800-366-4875, option #3 Tosoh Bioscience is a registered trademark of Tosoh Corporation. Ca++Pure-HA is a registered trademark of Tosoh Bioscience LLC in the USA, EU, India, and Japan.

www.tosohbioscience.com

www.biopharminternational.com May 2020 BioPharm International 33 Manufacturing

Table I. Divergence of design elements in closed systems. HEPA is high efficiency particulate air. ISO is International Organization for Standardization. Large, single-use disposable bioreactors Core principle Tank-based (wet) Room-based (dry) Enabling greater efficiency and process Varied processes within Varied processes in air filled Primary use liquid phase background environment separation (7,8), these systems are often Isolators, pass-throughs, used in conjunction with support vessels Major design elements Tanks, valves, piping chambers for inoculums, media, buffer, and other Primary interior contents Liquids Air fluids. Many of the designs incorporate Secondary interior contents Headspace air Liquids and solids means for including operations such as Filters used Membrane filters HEPA filters mixing, in-situ analysis, addition and/or Sterilization method Steam Hydrogen peroxide removal of fluids, sampling, and filtration. As use of these bioreactors increases and Internal environment Unclassified ISO 5 their reliability is demonstrated, there is External environment Non-critical ISO 8 growing interest in locating them in less controlled environments (9–11). 2006 document is still applicable, given ing final product by membrane filtra- the many improvements that have been tion is not possible (e.g., for powders, Single-use made to closed system designs since then suspensions, ointments, creams, emul- disposable filling systems (Sidebar, p. 35). sions, and liposomes). Their prepara- Sterilizing filters, surge vessels, per- tion requires multiple vessels and a unit istaltic pumps, and needles (12-15) APPLICATIONS operations that include particle sizing, have been shown to simplify the Closed systems’ utility has resulted in an ultra-filtration, subdivision, and bulk sterilization and aseptic assembly of increasingly diverse array of applications packaging (6). The equipment train is the fill set. Use of disposables also in biopharmaceutical manufacturing. sterilized in-situ, and the overall system eliminates the risk of cross-contam- eliminates aseptic assembly and human ination. Because these systems are Sterile APIs and complex intervention. The formulation-focused ordinarily used for aseptic filling into pharmaceutical formulations systems are contemporaneous with and open containers with separate closure Closed systems have proven useful in similar in concept to the fixed-vessel systems, they are typically installed manufacturing sterile products in bulk configurations that were previously used in International Organization for containers, in situations where steriliz- for sterile bulk production. Standardization (ISO) 5 environments.

Risk-aversion remains an obstacle to innovation

In the early 1980s, the first pharmaceutical isolators The reason that these practices have persisted used in sterility testing were fitted with turbulent air- reflects manufacturers’ wish to avoid the regulatory flow systems and relied upon manual manipulation headaches triggered by process change applications. internally for test execution. High-efficiency particu- In the heavily regulated world of current good late air (HEPA) filter integrity and particle counts were manufacturing practices, change is anathema, and confirmed during system qualification, and internal process improvements using new technologies microbial testing was performed. This mimicked es- require multiple layers of review, documentation, and tablished practices for the traditional operator-run approval before the changes can even be submitted to sterility test suites that the isolators were intended regulators for approval. to replace, for the simple reason that making fewer The result has been a situation in which procedures changes to procedures and manufacturing processes demonstrating the absence of human contamination would make it easier to install and implement the new are still required for systems that prevent any contact technologies in existing production facilities and, of with operators in the first place. This is a bit like course, secure regulatory approval for their use. requiring training wheels on the bicycles used in the However, well before then, widespread practices had Tour de France. established that fixed vessels and piping systems in closed Reflecting the current environment, some sterility systems needn’t be monitored internally by any means test isolator vendors have even begun to offer other than process simulation. Despite this, manufacturers unidirectional airflow designs, ignoring evidence continued to impose these requirements on their isolator based on 40 years of global experience with isolators operations, and eventually they found their way into and millions of successful sterility tests using turbulent regulatory guidance. flow systems.

34 BioPharm International May 2020 www.biopharminternational.com Manufacturing

Isolation technology Closed-vial technology and vent sampling, and connection Today’s most advanced closed isolators In this approach, closed sterile contain- components that facilitate their use. are vastly upgraded versions of those first ers are penetrated, filled, and resealed These features can be fully integrated used for sterility testing in the 1980s (16). using an open needle. Using this with the closed system or incorporated Erroneous perspectives and aversion to approach, closed systems have evolved to as add-ons provided by others. These change resulted in the wholesale adop- utilize a filling needle which is closed at sub-systems are smaller and less com- tion of cleanroom-like environmental all times except when dispensing inside plex, and provide unique means for monitoring programs for isolators with- closed containers. In an open needle sampling and connection that reduces out consideration of their utility (10). The configuration, an ISO 5 environment or eliminates the potential for product integration of isolation technology with is required to protect the fill needles contamination. robotics has led to the gloveless isola- during setup and filling. The use of a tors in which all aseptic manipulations closed needle obviates the need to have SEPARATING OPERATORS are performed remotely and robotically. filling done in a classified environment. FROM THE PRODUCT These systems can include autoclaves, System performance can vary substan- support isolators, lyophilizers, and other Integrated systems tially, however, and not all of these operational elements that do more than Many of the previously described technologies may be suitable for use simply fill and seal sterile containers. closed systems include filtration, fluid in less well-controlled environments.

Is the 2006 Technical Document still relevant for contemporary closed systems?

Given the disparity between the principles of closed sys- manned environment and the predominant contamination tems and some current practices, it is useful to ask: Is the source, personnel, are excluded. 2006 definition of closed systems still applicable today, Is viable environmental monitoring inside a closed given improvements that have been made in the technol- system beneficial? No. Properly designed and ogy? Consider the following questions. maintained closed systems do not require internal viable What is a closed system isolator? It is a decontaminated monitoring. Its inclusion only adds risk due to added (internal sterilization of all surfaces is optional) air-filled system complexity and invasive manipulations, and thus enclosure, which is designed to provide and maintain should be avoided. a physical barrier between the interior and exterior at Is non-viable environmental monitoring inside a closed all times. A pressure differential is maintained during system useful? Depending on internal processes and decontamination and use. This would include isolators equipment, it can be useful to confirm that these are not that are equipped with gloves, but exclude those contributing particles to the materials. It can also serve where materials and component ingress and egress are as a more immediate indication that system integrity managed by air pressure differentials. has been lost. It would only be appropriate, however, in Is sterilization necessary for non-product contact systems that contain air (or other gases). surfaces of a closed system isolator? It’s not required, If the closed system is pressurized relative to the but preferred when viable monitoring is no longer external environment, how important are leaks? Leaks performed. When the entire interior is sterilized, then from clean (i.e., inside the closed system) to less clean the closed isolator is identical in process capability to a (i.e., external to the closed system) environments do not closed stainless-steel vessel. present a risk. Are high efficiency particulate air (HEPA) filters There is no evidence that microorganisms and particles sufficient to assure the integrity of a closed system can enter against an outward flow. This is a core premise isolator? HEPA filters can provide sterile air (or other in all manned aseptic processing operations. Periodic leak internal gas [e.g., nitrogen]).Integral HEPA filters remove testing is a closed system requirement to assess integrity microorganisms quantitatively. over time and identify performance decay. Does unidirectional air provide any benefit in a closed Does the external environment in which a closed system isolator? No. Unidirectional air is a carryover from system is located have significant impact? No. If the manned cleanrooms, where its purpose is to direct air flow system is compromised, it has failed and action is required from the supply past the cleanest environment (where regardless of the background environment. sterile materials are exposed) past dirtier environments Counterintuitively, fewer controls on the exterior (where personnel are present) on its way to the air return. environment can facilitate the detection of contamination In a closed system isolator, there is no reason to require because a breach may result in a greater impact on the use of unidirectional air flow because all internal surfaces interior, thereby shortening response time and facilitating can be sterilized, something that is not possible in a corrective action.

www.biopharminternational.com May 2020 BioPharm International 35 Manufacturing

Table II. Contemporary monitoring for closed systems. ISO is International Organization for Standardization. Closed system Primary contact surface Internal External Classification Monitoring Classification Monitoring Pre-2006 Vessels for sterile API Wet N/A None N/A None Closed isolators Dry ISO 5 Non-viable ISO 8 – CNC Varies Present Day API/complex liquid Wet N/A None N/A None formulation systems Bioreactors/biopharm Wet N/A None CNC Varies processes Single-use disposables Wet N/A None ISO 5 – CNC Varies Gloveless isolators Dry ISO 5 Non-viable ISO 8 – CNC Varies Viable & non- Closed gloved isolators Dry ISO 5 ISO 8 – CNC Varies viable Closed vial/open needle Viable & non- Wet / Dry ISO 5 ISO 5 ISO 5 filling viable Closed system filling Wet N/A None ISO 8 - CNC None

2. J. Agalloco, American Pharmaceutical Collectively, these technologies represent contamination from gaskets, seals, and Review, 7(4), p. 26-29, 139, (2004). the future of aseptic processing. Coupled other components. 3. World Health Organization, Annex with the benefit of achieving true separa- Differences in the way the two types 4, General Requirements for the Sterility of Biological Substances, tion between operator and product, it is of systems are sterilized should not Part A, Section 2,1973. difficult to imagine future pharmaceuti- matter if they have been properly val- 4. FDA, Guideline on Sterile Drug Products cal manufacturing operations that won’t idated. Differences in filtration, mem- Produced by Aseptic Processing, 1987. 5. FDA, Guide to Inspection of Sterile Drug involve greater use of closed systems. brane, or high efficiency particulate air Substance Manufacturers, 1994. However, increasing usage and diversity are also irrelevant (10). 6. PDA, TR #28, PDA Journal of of closed-system technology is often in Pharmaceutical Science and Technology, 52(4), Supplement (1998). direct conflict with process-monitoring INTERNAL MONITORING CONFERS NO BENEFITS 7. PDA, TR #28 revised, PDA Journal of expectations derived from more open pro- Pharmaceutical Science and Technology, cesses, Table II lists the suggested mon- Internal monitoring confers no benefits Vol. 60, Supplement S-2, (2006). 8. J. Akers, and J. Agalloco, PDA Journal itoring practices that are appropriate for on closed isolators, especially those that of Pharmaceutical Science and these newer technologies. can be operated without gloves. In fact, Technology, 54(2), pp.110-111, 2000. introducing sampling into these systems 9. J. Agalloco, Pharmaceutical 15(4), p. 34, (2016). WET VS. DRY SYSTEMS can increase the potential risk of con- Manufacturing, Table II, 10. ISPE, Baseline Guide, Biopharmaceutical Reviewing it’s evident that ‘wet’ tamination. In short, since closed aseptic Manufacturing Facilities, Second and ‘dry’ systems are considered differ- processing systems were first introduced, Edition, November 2013. 11. S. Chalk, BioPharm International, ently with respect to internal monitoring. they have found increasing use in the 26 (11) pp. 49-50, (2013). Systems that are used for liquids have pharmaceutical industry. There is growing 12. J. Agalloco and P. DeSantis, “Sterile Bulk never been subject to internal monitoring evidence as to their utility and reliability. Pharmaceutical Chemical Production”, chapter in Bulk Pharmaceutical Chemical despite the direct contact between the If the industry is to remove obstacles to Production, 2nd edition, edited by S. equipment and the contained liquid. increased use of closed systems, regulators Nusim, InformaUSA, New York, 2010. In marked contrast, isolator systems and manufacturers must address overly 13. S. Chalk, S., et al, BioPharm International, 24(8), pp. 44-59, (2011). (where the interior surface is not in restrictive and redundant practices that 14. E. Bohn, Pharmaceutical Technology, contact with the sterile materials) rely impede use of these technologies. 39(9), pp 54-55, (2015). heavily on viable and non-viable mon- 15. P. Estape, P., et al, Pharmaceutical itoring, adhering to practices derived REFERENCES Engineering, September-October 2017. 1. J. Agalloco, “Aseptic Processing: 16. J. Agalloco, and L. Mestrandrea, from cleanroom monitoring. This is State of the Art and A Vision of the Pharmaceutical Technology ebook, surprising, because ‘wet’ systems are Future”, presentation at AAPS Arden Vaccine Development and Manufacturing ◆ more susceptible to leaks, as well as House conference, February, 2007. 2017, pp 31-38, November 2017.

36 BioPharm International May 2020 www.biopharminternational.com Analytics

Building Better Bioassays Next-generation therapeutics and regulatory requirements create demand for complex, fit-for-purpose tests.

CYNTHIA A. CHALLENER ioassays for use in biologic drug development and is happening in the patient, in the best-case scenario the bioassay final product release must be robust and meet an will mimic what is relevant for the patient (i.e., what makes the B array of regulatory requirements. As the complexity drug efficient),” explains Ulrike Herbrand, scientific director, of next-generation biopharmaceuticals increases, so does global in-vitro bioassays at Charles River Laboratories. the complexity of the bioassays necessary for effective eval- Developing bioassays intended to measure the potency uation of their performance across a spectrum of activities. of biologics that truly reflect the relevant MOAs involves Collaboration is essential between drug developers, testing selecting appropriate indicator cell lines and relevant read- laboratories, and regulatory authorities to ensure that new outs that measure expected cell responses, adds Weihong bioassays consistently serve their intended purpose. Wang, technology development manager, cell and molecular biology services at Eurofins Lancaster Laboratories. “A MOA, STABILITY, VALIDATABILITY, AND MORE master and/or working cell bank should be created for indi- Regulators expect bioassays for any new biologic to reflect cator cell lines and be well-characterized in order to assure the mechanism of action (MOA) of the drug in question, meaningful interpretation of assay results and avoid artifacts have stability-indicating properties, and be validatable, as unrelated to drug effects,” she says. outlined in International Council for Harmonization (ICH) In addition, a quantitative method is generally expected Q2(R1) (1) and United States Pharmacopeia (USP) General for potency measurement and results are usually expressed as Chapter <1033> (2). “Assays need to be adequately robust, percent relative potency when compared to a reference stan- accurate, and precise to ensure consistency in batch manu- dard that is fully characterized, according to Wang. Sponsors facturing and to provide meaningful data to support stability should avoid using a surrogate assay unless they can provide claims,” asserts Sharon Young, scientific manager, analytical sufficient justifications that address scientific rationale (MOA) and formulation sciences with Thermo Fisher Scientific. MOA is important because it uncovers important clues— CYNTHIA A. CHALLENER, PhD, is a contributing editor to sometimes the most important clue—as to what is happening in BioPharm International. Caleb Foster/Stock.Adobe.com Caleb the patient in vivo. “While the MOA rarely mimics exactly what

www.biopharminternational.com May 2020 BioPharm International 37 Analytics

and comparability to traditional assays method in a GMP-compliant [quality additional bioassays that really reflect that might be practically challenging/ control] QC lab,” she explains. the biological mechanism(s) of action less robust. and then show that results using the A bioassay should also be able to REPORTER-GENE ASSAYS reporter-gene assay are comparable, or determine the stability of a drug and SEE GROWING USE better, to those bioassays that mea- for how long it will be stable. Stress Reporter-gene assays are a category of sure the cellular response(s) directly. tests are conducted over a prolonged surrogate assays, notes Wang, that have “Bridging results need to include a large time at moderate conditions, including been used more often in the past sev- number of lot release samples as well the recommended storage conditions, eral years, especially in place of assays as stability and force-degraded samples. and the results help determine how a that require primary cells where assay Bridging studies should also include drug should be stored (at what tem- isolated impurities in these compar- perature and for how long), according As demand for isons to directly demonstrate that all to Herbrand. She also notes that accel- potential changes in the molecule can erated stress testing at various stress bioassays increases, be detected in the reporter-gene assay conditions (e.g., temperature, oxidation, comparably, or better, than in the direct deamidation, shear-stress, repeated laboratories look to assay(s),” she observes. freeze-thaw cycles) is required to deter- Wang highlights that the increasing mine which types of alterations the simplify processes use of reporter-gene assays is a good chosen bioassay is capable of detecting example that mechanism-of-action is by showing altered activity. with ready-to-use the first and foremost important charac- “Another key expectation for a teristic, and when this feature is proved, bioassay is fulfillment of validation cell banks and solid method performance including requirements by meeting the preset robustness is another key element of a validation acceptance criteria, which automated tools successful bioassay. is required for lot release and stability testing of the biologic at the and processes. SIMPLIFICATION TREND end of clinical Phase II at the latest,” The top trends in bioassay devel- Herbrand observes. performance can be quite variable and opment observed at Thermo Fisher Young adds that bioassays should lack robustness desired as a QC testing Scientific include the implementation employ statistical monitoring to detect method. They are particularly becoming of ready-to-use cell banks and auto- any shifts in performance over time common in early development and are mation. “Thaw-and-use cell banks can and to detect any changes in the ref- increasingly being accepted by regula- be thawed and plated directly in the erence standard potency in real time. tory authorities, adds Young. bioassay, eliminating the need to spend The use of more advanced statistical As an example, Wang points to anti- a large amount of resources on main- tools such as computational simulation body-dependent cell-mediated cyto- taining actively growing cultures. It also can raise questions from regulators toxicity (ADCC) reporter-gene assays, simplifies monitoring of assay perfor- because they may seem unnecessarily which have been used to measure Fc mance because impact from the age complex, but such approaches can pro- receptor mediated response for mono- (passage number) of the culture need vide a much larger data set to deter- clonal antibodies (mAbs) and in place not be tracked and can reduce method mine assay acceptance criteria (3), she of traditional peripheral blood mono- variability attributable to variations in cites. The use of animal models is nuclear cell (PBMC)/primary natu- culture conditions between testing occa- discouraged, Young comments, but is ral killer (NK) cell-based cytotoxicity sions,” Young explains. sometimes required and is acceptable if assays. “Instead of looking at direct cell Laboratory/assay automation, from reasonably justified. killing (by primary NK cells), the assay simple pipetting robots to end-to- Ensuring the bioassay is suitable for measures activation of signal transduc- end automation, is helping to increase transfer to other labs is not exactly a key tion associated with the cellular event,” throughput and reduce the risk of lab- requirement from a regulatory perspec- she explains. The resulting reporter oratory error, according to Herbrand. It tive, but nevertheless, Herbrand says gene assay is easy to perform with much also allows for miniaturization, which it is an important point to consider. “A shorter assay duration and in general she says enables performance of six to bioassay that is ridiculously difficult for delivers excellent assay performance. seven tests simultaneously, affording other labs to use can delay programs The key to successful use of reporter- higher throughput, the need for fewer since it takes too long to establish the gene assays, adds Young, is to have reagents and lower cost.

38 BioPharm International May 2020 www.biopharminternational.com Analytics

For instance, the use of automated tion of the expressed protein should be clear, particularly when a complex liquid handlers is becoming increas- be confirmed, for example, by enzy- assay matrix is proposed, and the assay ingly common in bioassays because it matic assays, or receptor binding, etc., must be shown to be capable of reject- significantly reduces assay variability depending on the MOA. Therefore, ing a sub-par batch, she adds. attributable to pipetting, Young says. often times several assays are needed to One of the main challenges, asserts “Automation also mitigates variabil- fully address all aspects of the product Herbrand, is that there currently are no ity attributable to positional bias by characteristics,” explains Wang. clear guidelines on how to evaluate bio- enabling randomization of plate lay- Potency assays for cell therapy prod- activity for cell-therapy and gene-ther- outs within and between plates without ucts also have their unique challenges. apy products. “For now,” she says, “these concern for analyst error, which sig- “First of all,” states Wang, “it may be therapies are being considered on a nificantly mitigates plate position and more difficult to correlate in-vitro case-by-case basis by regulators. Many sequence (order of addition of reagents) potency measurements to clinical effi- of the gene therapy companies are cur- sources of variability,” she remarks. cacy compared to typical recombinant rently running tests that more or less protein therapeutics.” Second, depend- reflect what would be required for the CELL-THERAPY AND ing on the product, she notes that it is development phase, but they haven’t GENE-THERAPY sometimes difficult to truly establish nailed down what they need for QC CHALLENGES a “reference standard” to which sub- release of a marketed drug.” With the fast growth of the gene- and sequent lots may be compared. Third, What isn’t in question is the com- cell-therapy pipeline, more potency performance of functional assays, in plexity of cell and gene therapies. assays for this category of product general, tends to be less robust. “What is understood is that the saf- are being submitted to the regulatory Because of these issues, setting est course to take is to make sure we agencies, according to Wang. Potency specifications can also be challenging, reflect that the drug is doing its job assays for cell- and gene-therapy prod- according to Wang. “Last but not least,” on all relative levels. Twenty years ago, ucts are generally more complicated she observes, “some cell-therapy prod- we pondered the same questions with than recombinant therapeutic proteins, ucts require fast delivery to patients; mAbs and what was required for lot as they need to demonstrate several therefore the balance between thor- release. Today, at least we have impre- characteristics of the product. ough characterization and short assay cise guidelines,” Herbrand comments. These products, according to turn-around time may need to be care- Herbrand, require bioactivity testing, fully considered.” BISPECIFIC ISSUES often in a multistep approach. For the Because multiple assays may need Many molecules have more than one first step, molecular methods such as to be deployed, from phenotypic (e.g., mode of action, particularly bispecific a quantitative polymerase chain reac- flow cytometry), to functional con- antibodies, and regulators increasingly tion (PCR) or droplet digital PCR are firmation (e.g., cytokine release and/ want a single assay that can reliably detect often the methods of choice to confirm or cytotoxicity for chimeric antigen changes in potency through those mul- the desired re-expression or suppres- receptor T-cell therapies), these assays tiple mechanisms, according to Young. sion effect in vitro at the RNA level. need to be developed as part of a “Combined approaches measuring the In addition, she says the re-expression ‘matrix approach’ in order to fully char- activities of both epitopes within the or suppression of a gene of interest acterize the product and assure efficacy same assay rather than reporting two can be shown on the protein level in order to gain regulatory acceptance,” independent results for the activities of using enzyme-linked immunosorbent Wang says. the individual arms of the bispecific have assay (ELISA) testing, or in the case of A recent approach noted by Young an excellent chance of being accepted by receptors, via flow cytometry. In some that has gained approval is flow cytom- regulators,” Herbrand agrees. The reason: cases, a reporter-based method might etry detection of biomarkers that it is important that bioassays for potency be applicable as an MOA-reflecting, correlate with function, such as cell of bispecific molecules reflect any addi- cell-based bioactivity assay to confirm survival or differentiation. “Analysis tive or synergistic effects of the bispecific the effect on the functional level. of the selected biomarkers must be mechanisms of action. “An assay for a gene therapy usually able to detect unacceptable behavior “This need,” Young notes, “can involves transfecting/infecting a cell of the cells, and not simply rely on create challenges in developing the line followed by measurement of the cell identity markers that are unlikely method, particularly in cell-line selec- expression of the intended target gene to do so under conditions that affect tion, as well as in interpreting the at the nucleic acid or protein level. In cell function,” she notes. The rationale results. Results for a bispecific with a addition, whenever possible, the func- for selection of the biomarkers must synergistic effect will show changes

www.biopharminternational.com May 2020 BioPharm International 39 Analytics

in EC50 as well as in asymptotes, “These 3D systems are often a bet- the regulatory filing and months of for which tests of parallelism are not ter reflection of the in-vivo situation delays,” Herbrand explains. appropriate. Differences in asymp- than what is observed for traditional totes is traditionally unacceptable per two-dimensional cell-culture-based INCREASING BIOASSAY USP guidance, as sample curves must assays and provide better information COMPLEXITY REQUIRES be similar to reference curves for cal- regarding what is occurring in the CONSIDERATION culation of relative potency.” patient,” agrees Herbrand. She does AND CAUTION Currently, although the ideal assays note, though, that it can be tedious to Caution is warranted in general when from a regulatory and a sponsor’s validate such tests, and they are often it comes to newer bioassays, according standpoint would be dual reporter still time-consuming. Young also says to Young. “As cell culturing techniques systems, it is still more common to that it can be challenging to control advance and increasingly complex mul- independently test the bioactivity variability, find suitable methods of tifunctional molecules are developed in using two different assays to measure quantitation for potency results, and the biopharmaceutical industry, bioas- each arm of the bispecific antibody, implement such complex techniques say development will become increas- Herbrand remarks. “While easier to into a QC environment. ingly complex as well,” she says. develop, they do not allow one to One way the industry has fully see the synergistic effects and POTENTIAL FOR PROTEIN responded, according to Wang, is to combined activity of the antibodies,” TAGGING AND HUMAN develop bioassays to support ther- she notes. PRIMARY CELL ASSAYS apeutic biologics as early as possi- Young observes that at the CASSS Protein tagging is based on peptide ble, particularly when more complex Bioassays 2016 conference, Bhavin sequences that are attached to pro- products are involved. “While science Parekh presented an approach for cal- teins to facilitate easy detection and continues to be the driving force culating relative potency/efficacy for purification of expressed proteins, for bioassay development, sponsors a bispecific product with synergistic according to Herbrand. They can also are encouraged to engage regula- activity that he developed based on be used to identify potential binding tory agencies early in order to obtain the field of pharmacology (4). She partners for the protein of interest. guidance on their assay development thinks this simple approach to reach a The tags, which Herbrand notes are approach and avoid unnecessary sur- discrete value to compare concomitant typically applied using CRISPR/Cas9 prises and delays in their product shifts in EC50 and asymptotes may gene editing technology, are of partic- development programs,” she says. be able to establish a precedent for ular interest because they are highly Young adds, “As an industry, we regulatory acceptance. sensitive down to the endogenous are constantly trying to find a bet- level of the protein, often with over ter model to assess potency of our 3D CELL CULTURE seven logs of dynamic range. molecules, but we must be cautious IS GARNERING Because human primary cell assays about the complexity of bioassays. SOME INTEREST are closer to the situation in patients, Bioassays are already complex, which Common in drug discovery, three-di- Herbrand suggests they may be more results in increased variability and sta- mensional (3D) cell culture models translatable than cell lines, which are tistical uncertainty and can be dif- have recently been attracting interest immortalized/modified in their key ficult to control between labs. We among biologics manufacturers for use characteristics and thus could exhibit must be careful to avoid unnecessary in bioassay development as well. “The modified cell behaviors. “However,” challenges in method execution from use of 3D cell cultures and 3D bio- she cautions, “human primary cell- over-complicating bioassays.” printing has the potential to enable based assays are not without risks.” assays in more complex cellular envi- Human primary cells are subject to REFERENCES 1. ICH, Q2(R1) Validation of Analytical ronments, more like tissue structures,” lot-to-lot variability and availability Procedures: Text and Methodology, Young explains. “Such models,” she of suitable sourcing material. In addi- Step 4 version (1995). adds, “are ideal for mimicking the tion, even if it was possible to identify 2. USP, USP General Chapter <1033>, “Validation of Biological complex cancer microenvironment as the perfect lot, it wouldn’t last forever. Assays” (Rockville, MD, 2010). well as for culturing cell lines that rely “There are no guarantees you won’t 3. M. Labant, Gen. Eng. extensively on the extracellular matrix have to adjust the assay. And even if News, 36(10), (2016). 4. B. Parekh, “Bioassay Development environment for growth, differenti- you manage to get the assay running for Complex Molecules,” ation, and function, such as skeletal again, you have to revalidate it, at least presentation at CASSS Bioassays muscle cells.” partially, which requires a change in (Silver Spring, MD, 2016). ◆

40 BioPharm International May 2020 www.biopharminternational.com An In-Depth Look at Intact Biotherapeutic Characterization and Quantitation in Biological Matrices

Register for this free webcast at: ON-DEMAND WEBCAST: www.chromatographyonline.com/lcgc_p/biological_matrices Aired: Thursday, April 23, 2020 Event Overview This webcast discusses a highly sensitive and automated workflow for intact biotherapeutic characterization and quantitation from biological matrices. We will also discuss native protein characterization and its challenges. Automated sample preparation, sample analysis and data interpretation Presenter will be presented as well as pitfalls encountered. Performance and robustness of the workflow will be discussed. Finally, future expansion of methods and techniques will be discussed. In particular, we will discuss: • An optimized workflow from sample preparation, separation, data acquisition to data analysis • A high throughput and reproducible method with limited sample prep that enables various mixtures of mAbs can be Christian Klein LC/Q-TOF and IM-QTOF Product Manager quantitated simultaneously Agilent Technologies • A robust workflow with excellent dynamic range of over 3 orders of magnitude and low picogram in sensitivity

Key Learning Objectives • How to prepare samples using automated liquid-handling Moderator • How to optimize HPLC methods with columns, for better MS sensitivity and reproducibility? • How to achieve the best sensitivity using LC/Q-TOF for intact mAb analysis • How to setup various data processing methods for accurate intact mAb quantitative analysis

Who Should Attend Laura Bush BioPharma Lab Managers, Discovery and Development Scientists Editorial Director LCGC

Sponsored by Presented by

For questions or concerns, email [email protected] Peer-Review Research

Shaping IR Spectroscopy into a Powerful Tool for Biopharma Characterizations

DIPANWITA BATABYAL, LIBO WANG, JEFFREY ZONDERMAN, AND MATS WIKSTRÖM

ABSTRACT Microfluidic modulation spectroscopy (MMS) is a novel automated infrared spectroscopic technique with high sensitivity and repeatability. Here, the authors present a series of experimental studies showcasing the performance of MMS in the secondary structure characterization of biopharmaceutical products and compare the MMS results with the conventional Fourier transform infrared data.

he successful development of terization of biopharmaceuticals proteins (e.g., biopharmaceuticals involves immunoglobulin G1 [IgG1] and IgG2) (15). the study of their higher order Microfluidic modulation spectroscopy structure, a critical quality attrib- (MMS) represents a novel automated tech- ute, to ensure a therapeutically active mole- nique that directly addresses the current lim- cule in appropriate formulation conditions itations with both conventional FTIR and

molekuul.be - Stock.adobe.com - molekuul.be T (1–3). Robust structural characterization of far-UV CD by shaping IR spectroscopy into the biopharmaceutical products is impor- a far more effective analytical tool in biop- tant throughout the development process. harmaceutical product characterization (16). For instance, comparability studies are per- This article presents a series of experimental formed to ensure that a manufacturing pro- studies showcasing the performance of MMS cess change during clinical and commercial applied to challenges in the characterization development does not have an adverse effect of the secondary structure of biopharmaceu- on quality, safety, and efficacy (4–6). tical products including comparisons with Infrared (IR) spectroscopy is a powerful conventional FTIR data. Dipanwita Batabyal*, method for characterizing the secondary [email protected], MATERIALS AND METHODS is scientist at Attribute structure of proteins (7–14). However, the Sciences—Higher Order lack of automation of conventional Fourier Conventional FTIR and MMS were used Structure Group, Amgen transform IR (FTIR), along with relatively to determine the secondary structure of two and Mats Wikström*, high sample concentration requirements, biopharmaceutical samples: a monoclo- [email protected], is are major limitations with this technology. nal antibody (mAb) and a bispecific T cell principal scientist at Higher Far ultraviolet circular dichroism spectros- engager (BiTE, a registered trademark of Order Structure, BR&C, copy (far-UV CD) is an important alterna- Amgen) (17) molecule (Amgen, Thousand Attribute Sciences, Amgen. tive for the characterization of secondary Oaks, US). The BiTE molecule represents Libo Wang is principal structure, but it also has major drawbacks. a fusion protein, created by linking the vari- application scientist and Measurement is necessarily carried out at able light and heavy chain corresponding to Jeffrey Zonderman is chief low concentrations, typically at 0.5 mg/mL two antibodies. Polysorbate (PS) 80 was pur- commercial officer at RedShift Bioanalytics. but down as low as 0.1 mg/mL, which can chased from Fluka (Cat#: 59924-100G-F undermine the relevance of the resulting data. Lot: BCBC1232). * To whom correspondence The presence of certain excipients in the for- Conventional FTIR measurements were should be addressed. mulation buffer can also significantly inter- carried out using a Bruker Vertex 70 spec- PEER-REVIEWED fere with the measurements. Furthermore, trometer equipped with an Aquaspec trans- Submitted: Dec. 9, 2019 far-UV CD and conventional FTIR have mission cell that requires manual injection Accepted: March 9, 2020. been shown to lack sensitivity in the charac- of the sample and reference buffer at room

42 BioPharm International May 2020 www.biopharminternational.com Peer-Review Research

Figure 1. Conventional Fourier transform infrared (FTIR) and microfluidic modulation spectroscopy (MMS) measurements for a monoclonal antibody are highly comparable at a concentration of 10mg/mL. Unlike conventional FTIR, MMS can also measure with high sensitivity at a concentration of 0.5 mg/mL. Acceptable quality data (similarity score >95%) is not possible at 0.5 mg/mL using conventional FTIR.

RedShiftBio MMS And Traditional FTIR Data Are Highly Comparable 10 mg/mL, using RedShiftBio 10 mg/mL, using Bruker Vertex 70

Second derivative spectra Arbitrary

RedShiftBio MMS can be used at low concentration with high sensitivity (similarity scores for >98% ) 0.5 mg/mL, using RedShiftBio

Similarity score of samples from MMS

*Similarity score is calculated by comparing the area of overlap (AO) and the mean of the three runs at 10 mg/mL is used as reference.

temperature. The reference spectra for ple. The data were analyzed using the similarity in the conventional FTIR buffer blank were subtracted from the software AQS3 delta analytics package was calculated using the QC compare protein spectra according to previously to produce the final spectra and analysis function from the OMNIC software. established criteria (18). Spectral sim- results. For each experiment, interpo- The spectral similarity from MMS data ilarity was quantitatively determined late differential absorbance spectra, abso- was calculated by comparing the area of using the Thermo OMNIC software lute absorbance spectra, and the second overlap of each sample replicate to the quality control (QC) compare function. derivative spectra were obtained, and the mean area of overlap of all three repli- MMS measurements were con- similarity score was calculated using the cates. In general, there was very good ducted at ambient temperature using area of overlap in the amide I band region agreement in similarity scores between an AQS3pro system (RedShiftBio, (1700 cm-1–1600 cm-1). the two methods. The MMS data were Burlington MA, US) with multi-sample further analyzed using the QC com- RESULTS automation. A microfluidic transmission pare function from OMNIC, and the cell of approximately 24 µm pathlength Case 1: testing instrument sensitivity results showed consistency between the was used. Streams of protein samples and This section discusses testing instru- similarity scores obtained by the two reference buffers were introduced into the ment sensitivity at different protein con- approaches. flow cell alternatively at a back pressure centrations and with different modalities. At lower concentrations of the mAb of 5 psi and a modulation rate of 1 Hz. To compare the relative sensitivity of (0.5 mg/mL), acceptable quality data (> Simultaneous modulation of the sample MMS and conventional FTIR, mAb 95% similarity score between the repli- and an appropriate buffer enabled a real- samples were analyzed using the two cates) were not obtained by conventional time subtraction of the buffer background methods at concentrations of 0.5 mg/ FTIR, whereas the MMS data showed and allowed the differential absorbance mL and 10 mg/mL in acetate buffer. high repeatability at low concentrations, measurement. Thirty-one discrete wav- At 10-mg/mL concentration, the data indicated by the high similarity scores enumbers across the amide I band from from MMS matched well with the (> 98%) between the three replicate runs. 1590 cm-1 to 1714 cm-1 were scanned, and conventional FTIR. Both techniques The similarity score in MMS is calcu- the differential absorbance spectra of showed high repeatability quantified by lated by comparing the area of overlap samples were collected. Triplicate meas- high spectral similarity scores (> 99% (AO) of the replicates using mean AO of ALL FIGURES COURTESY OF THE AUTHORS. THE OF COURTESY FIGURES ALL urements were carried out for each sam- for both, see Figure 1). The spectral the three replicates as reference.

www.biopharminternational.com May 2020 BioPharm International 43 Peer-Review Research

Figure 2. Microfluidic modulation spectroscopy data for the BiTE sample show excellent repeatability at low protein concentration (1mg/mL). Panel on the left shows overlaid absolute spectra, panel on the right shows overlaid second derivative spectra.

A BiTE molecule sample was ana- the runs. The contributions from the sition but with different amounts of lyzed at 1.0 mg/mL to further assess the different secondary structure elements polysorbate (PS) 80 (0.01%, 0.05%, and sensitivity of MMS at concentrations (referred to as the higher order struc- 0.1% w/v, respectively). lower than those accessible with conven- ture analysis, or HOS analysis) were fur- As shown in Figure 3, the abso- tional FTIR, with a different modal- ther determined using Gaussian peak lute absorbance spectra and the second ity. As shown in Figure 2 (left panel), assignment from known correlations derivative spectra of the three replicates the absolute absorbance spectra gener- with absorption at specific wavenumbers are very closely matched, suggesting ated through triplicate measurements within the amide I band (19). As shown that different amounts of PS 80 have are almost indistinguishable, indicating in Table II the BiTE molecule antibody no effect on the secondary structure of high repeatability of the MMS meas- consists predominantly of β-sheets (58.67 the mAb. Similarity scores of the three urements. The second derivative spectra ± 0.80%) along with some contributions replicates in all three buffers are > 99% of the three replicates (Figure 2, right from β-turns (31.38 ± 0.48%). (Table III), indicating high repeata- panel) overlay very well, indicating the bility. In Table IV, the HOS analysis high consistency between measurements Case 2: buffer excipient shows that the secondary structure of the further quantified by comparing the sim- This section assesses the impact of the mAb consists predominantly of β-sheets ilarity score. Overall the second deriva- buffer excipient on secondary structure. (approximately 61.5%), with β-turns at tive spectrum exhibits a strong β-sheet To investigate the impact of buffer approximately 29%. When compared peak at around 1639 cm-1 together with composition and different excipients, to the BiTE molecule HOS analysis a β-sheet peak at 1689 cm-1. The sim- an MMS test of a mAb sample (5 mg/ (Table II), there is a relative higher pro- ilarity score of three replicates for the mL) was carried out and analyzed in portion of β-sheet content and a lower BiTE molecule are all > 99% (Table I), three different buffers—Buffer A, B, proportion of β-turns in the secondary indicating high repeatability between and C—with the same base compo- structure analysis of the mAb samples.

Table I. Similarity scores of the BiTE sample measured by microfluidic modulation spectroscopy. SD is standard deviation. Sample conc. (mg/mL) Similarity (%) of replicates Mean±SD 1.0* 99.01 99.20 98.97 99.06±0.12

*Similarity score is calculated by comparing the area of overlap (AO), and the mean AO of the three replicates is used as the reference.

Table II. Higher order structure (HOS) contents (%) of the BiTE molecule sample determined by microfluidic modulation spectroscopy.

Sample conc. HOS% (mean±SD) of replicates (mg/mL) Beta Turn Unordered Alpha 1.0 58.67 ± 0.80 31.38 ± 0.48 7.64 ± 0.78 2.32 ± 0.36

44 BioPharm International May 2020 www.biopharminternational.com Peer-Review Research

Figure 3. Microfluidic modulation spectroscopy data of monoclonal antibody samples with different amounts of polysorbate 80 (PS 80) in buffer. Buffer A: 0.01% (w/v) PS 80, Buffer B: 0.05 % (w/v) PS 80 and Buffer C: 0.1 % (w/v) polysorbate 80. The left panel shows the overlaid absolute spectra and the right panel shows the overlaid second derivative spectra.

DISCUSSION Case 3: higher reference. Overall, > 99% similarity protein concentrations was observed at all protein concentra- The criticality of HOS makes measure- This section discusses test consistency, tions (ranging from 5 mg/mL to 100 ment essential throughout biopharma- precision, and accuracy of MMS at mg/mL) indicating high repeatabil- ceutical development. Robust formulation higher protein concentrations. ity of the MMS measurements. The and process development relies on mea- In this experiment, the same mAb high consistency of measurement is suring the impact of concentration—of sample that was used in the case 2 retained even though measurements different buffers and of processing condi- study was analyzed by MMS at con- are made over multiple days. The tions as a drug candidate proceeds toward centrations of 50 mg/mL and 100 mg/ data further confirmed that nei- commercialization—with the different mL in the base buffer without any PS ther the protein concentration nor types of therapeutic molecules that are 80. Tests were performed on differ- the buffer excipient PS 80 impacts increasingly part of the new drug pipeline. ent days, and the resulting data were the secondary structure of the mAb. All analytical techniques have strengths compared to check for consistency and Further HOS analysis giving the and limitations when assessed against this precision of MMS measurements. secondary structure component also informational need. For example, unlike The similarity scores are shown in reflects the high consistency that was conventional FTIR or near-UV CD, Table V and were calculated using observed in the similarity score data MMS is not a general-purpose platform mean AO of a 50 mg/mL sample as (data not shown). at this time. MMS has been optimized

Table III. Similarity scores of the monoclonal antibody sample in different buffers measured by microfluidic modulation spectroscopy. SD is standard deviation.

Samples @ 5 mg/mL Similarity (%) of replicates Mean±SD In Buffer A* 99.62 99.79 99.76 99.72 ± 0.09 In Buffer B 99.76 99.64 99.52 99.64 ± 0.12 In Buffer C 99.66 99.55 99.69 99.63 ± 0.07

*Similarity score is calculated by comparing the area of overlap (AO), and the mean AO of sample replicates in Buffer A is used as the reference. Buffers A, B, and C contain 0.01%, 0.05%, and 0.1% (w/v) polysorbate 80, respectively, in the same base buffer.

Table IV. Higher order structure (HOS) contents (%) of the monoclonal antibody sample in different buffers determined by microfluidic modulation spectroscopy. SD is standard deviation. HOS% (Mean±SD) of replicates Samples in Beta-sheets Beta-turn Unordered Alpha-helix Buffer A 61.41±0.09 29.40 ± 0.11 6.90 ± 0.01 2.29 ± 0.03 Buffer B 61.67±0.15 29.22 ± 0.06 6.87 ± 0.10 2.24 ± 0.10 Buffer C 61.63±0.13 29.29 ± 0.07 6.80 ± 0.10 2.27 ± 0.15

www.biopharminternational.com May 2020 BioPharm International 45 Peer-Review Research

Table V. Similarity scores of a monoclonal antibody sample analyzed in different buffers and at different concentrations. PS 80 is polysorbate 80. SD is standard deviation.

Sample Conc. Buffers Similarity (%) of replicates Mean±SD Base Buffer 50 mg/mL* 99.45 99.75 99.60 99.60 ± 0.15 (no PS 80) Base Buffer 100 mg/mL 99.37 99.36 99.37 99.37 ± 0.01 (no PS 80) Buffer A 5 mg/mL 99.30 99.31 99.17 99.26 ± 0.08 (0.01% PS 80) Buffer B 5 mg/mL 99.18 99.28 99.06 99.17 ± 0.11 (0.05% PS 80) Buffer C 5 mg/mL 99.36 99.26 99.23 99.28 ± 0.07 (0.1% PS 80)

*Similarity score is calculated by comparing the area of overlap (AO) and the mean AO of 50 mg/mL sample replicates, which were used as the reference. Buffers differ in the amount of PS 80 in the same base buffer. for all types of protein- and peptide-based The data show that MMS allows routinely exhibits instrument drift. This secondary structural analysis, which is the determination of secondary struc- characteristic is due to the way in which of interest in biopharmaceutical devel- ture over a much wider concentration MMS generates differential data via a opment and manufacture, but not other range than conventional FTIR, thus process of continuous auto-referencing structural features (i.e., tertiary) or mole- removing the requirement of either that eliminates the issue of background cule types. dilute or concentrated samples for drift. High repeatability contributes In contrast, the application of measurement. In this study, MMS directly to the ability of a technique multiple techniques to characterize measurements were successfully made to detect difference and indicates that HOS, a necessity when combining across a concentration range from 0.5 MMS will exhibit greater sensitivity to conventional FTIR and far-UV CD mg/mL to approximately 100 mg/mL. changes in protein structure. to cover the range of conditions of In contrast, conventional FTIR meas- CONCLUSION interest, is inherently problematic. urements require a minimum concen- Such an approach can introduce tration of approximately 10 mg/mL to MMS is a powerful new technique for uncertainty, where there is overlap acquire data of acceptable quality. the assessment of the secondary struc- between techniques and discrepan- The capability to measure at low con- ture of proteins. The results presented cies in the results produced as well centrations means that MMS is not lim- here show how it enables accurate, highly as complicating analytical workflows. ited to studies of mAbs, but can also repeatable characterization across a wider Any requirement for sample prepara- be applied to other protein therapeutic concentration range than conventional tion can also undermine data integrity modalities, such as BiTE molecules, FTIR, and measures with high sensitivity because proteins are labile, changing which are typically measured at prod- with different buffers. These capabilities in response to their local environment. uct concentrations below the minimum offer potential to streamline the routine The adoption of techniques that can required for conventional FTIR. For analysis associated with biopharmaceuti- be applied directly, to a broad range low-concentration measurements, far-UV cal development for various protein ther- of sample types, is therefore techni- CD would typically be the technique apeutic modalities, including mAbs and cally advantageous. of choice, but it can be unreliable for BiTE molecules. The results from this study clearly formulations containing chromophores REFERENCES demonstrate the performance of other than those associated with the drug 1. J. D’Antonio, et al., J. Pharm. Sci. 101 (6) MMS. A direct comparison with con- entity, necessitating filtration or dilution 2025–2033 (2012). ventional FTIR illustrates a number of the sample prior to the measurement. 2. Y. Jiang, et al., J. Pharm. Sci. 100 (11) of ways in which MMS is a superior The data showing the repeatability of 4631–4641 (2011). 3. C.H. Li, et al., J. Pharm. Sci. 100 (11) presentation of IR spectroscopy for mAb measurements in solutions with 4642–4654 (2011). this application, while the ability to different buffer concentrations are help- 4. FDA, Guidance for Industry, Q5E measure with high sensitivity and pre- ful in demonstrating the ability of MMS Comparability of Biotechnological/ cision at high concentrations and in to address this limitation. Biological Products Subject to Changes in the Manufacturing Process, (CDER, the presence of different buffers high- Finally, the results show that MMS CBER, June 2005). lights the potential of MMS relative to data are highly repeatable with high pre- 5. B.M. Teska, et al., Anal. Biochem. 434, far-UV CD. cision, unlike conventional FTIR, which 153–165 (2013).

46 BioPharm International May 2020 www.biopharminternational.com Peer-Review Research

More on biopharmaceutical characterization

For more on biopharmaceutical characterization, read the following articles on BioPharmInternational.com: • How Advanced Mass Spectrometry Technologies and Workflows are Delivering Comprehensive Protein Characterization www.BioPharmInternational.com/how-advanced-mass-spectrometry-technologies-and-workflows-are-delivering-comprehensive-protein-chara • Biopharma Analysis Benefits from New Technology and Methods www.BioPharmInternational.com/biopharma-analysis-benefits-new-technology-and-methods • New FT–IR Spectrometer Enhances Protagen Protein Services’ Analytical Capabilities www.BioPharmInternational.com/new-ft-ir-spectrometer-enhances-protagen-protein-services-analytical-capabilities-0 • Accepting the Challenge of Protein Characterization www.BioPharmInternational.com/accepting-challenge-protein-characterization • Protein Analyzer Offers Ability to Measure Protein Titer Directly from Bioreactor www.BioPharmInternational.com/protein-analyzer-offers-ability-measure-protein-titer-directly-bioreactor

6. N.N. Ding, et al., Anal. Biochem. 464, 12. S.J. Prestrelski, A.L. Williams Jr., and 17. P.A. Baeuerle and C. Reinhardt, Cancer 60–62 (2014). M.N. Liebman, Proteins Structure Res 69 (12) 4941–4944 (2009). 7. W. Wang and C.J. Roberts, Aggregation Function and Bioinformatics 14 (4) 440– 18. A. Dong and W.S. Caughey, Methods of Therapeutic Proteins (Wiley, 2010). 450 (1992). Enzymol. 232, 139–175 (1994). 8. A. Elliott and E.J. Ambrose, Nature 165, 13. W. K. Surewicz, H.H. Mantsch, and D. 19. J. K. Koenig and D. L.Tabb, “Infrared 921–922 (1950). Chapman, Biochemistry 32 (2) 389–394 9. H. Susi and D.M. Byler, Methods (1993). Spectra of Globular Proteins in Enzymol. 130, 290–311 (1986). 14. J.L.R. Arrondo, et al., Prog. Biophys. Mol. Aqueous Solution,” in Analytical 10. W. K. Surewicz and H.H. Mantsch, Biol. 59 (1) 23–56 (1993). Applications of FTIR to Molecular and Biochim. Biophys. Acta 952, 115–130 15. J. Wen, et al., J. Pharm. Sci. 109 (1) 247– Biological Systems, J.R. Durig, Ed. (D. (1988). 253 (2020). Reidel Publishing Company, Dordrecht, 11. A. Dong, P. Huang, and W.S. Caughey, 16. E. Ma, L. Wang, and B. Kendrick, Netherlands, 1980), pp 241–255. ◆ Biochemistry 29 (13) 3303–3308 (1990). Spectroscopy 33 (7) 46–52 (2018).

Call for Peer-Review Papers

BioPharm International accepts four types of peer-review papers that are Peer-Review Research considered: standard data-driven, novel research; topical literature or Therapeutic Potential of Green, Synthesized patent review; technical case studies/technical application notes; and Gold Nanoparticles SATABDI RAUTRAY AND A. USHA RAJANANTHINI science-based opinion papers. ABSTRACT Gold nanoparticles (AuNPs) are important components for biomedical applications and are widely employed for diagnostics and therapeutics. Nanoparticles are mainly synthesized through chemical and physical methods, which are often costly and potentially harmful. Synthesis of nanoparticles using plants, however, is less toxic and more effective. Manuscripts for peer-review papers are accepted on an ongoing basis. Recently, researchers have been focusing on green synthesis of AuNPs. This study aims to use plant-leaf extract for the green synthesis of AuNPs and to evaluate their antibacterial and antioxidant activity. The results indicated that AuNPs can be synthesized using a simple method with extracts from Adiantum capillus veneris (ACV) and Pteris quadriureta Publication priority is given to papers in the order they are accepted (PQ) leaves. The characterization of the AuNPs was done by ultraviolet- visible spectroscopy, powder X-ray diffraction, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy. The nanoparticles of ACV and PQ were seen at the wave length of 573 nm and 520 nm, Kateryna_Kon - Stock.adobe.com - Kateryna_Kon for publication. Submitted papers are initially screened by the editors, respectively. The nanoparticles of both ACV and PQ leaves extract showed antioxidant, antibacterial, and antifungal activities. ACV nanoparticles showed increased antioxidant and antimicrobial activity compared to PQ. Taken together, the results reveal that the AuNPs synthesized from leaves of ACV and PQ possesses antioxidant and antimicrobial activity. then submitted for formal review by a member of the editorial advisory

anotechnology is the most Nanoparticles show novel and improved active area of research in the properties based on particular characteristics, field of biotechnology (1). such as size, distribution, and morphology board, who will review the article for technical interest and content in a Nanobiotechnology applies (7). Metal nanoparticles, especially gold nan- the nanoscale principle to help understand oparticles (AuNPs), show tremendous ther- Nand modify biosystems, including living apeutic potential against pathogens (8–10). and non-living, by using biological mate- The sizes of AuNPs vary from 1 nm to 8 rials to make new strategies (2). Several μm, and the shapes are spherical, octahedral, double-blind review process. Article acceptance is conditioned on the A. Usha Rajananthini, methods are applied to synthesize nanopar- sub-octahedral, decahedral multiple twinned, [email protected], and ticles including chemical, physical, electro- icosahedral multiple twinned, irregular shape, Satabdi Rautray, rautray. chemical, sonochemical, irradiation, and nanotriangles and nanoprisms, tetrahedral, [email protected], are biological methods. Among these, biolog- hexagonal platelets, and nanorods (11–12). with the Department of ical methods, by way of microorganisms Researchers in the fields of medicine, cosme- reviewer’s approval. Once accepted for publication, a paper typically is Biotechnology, Mother (microbial nanosynthesis) and plants (phy- tology, biology, clinical chemistry, and phar- Teresa Women’s University, Kodaikanal, Tamil Nadu, India. tonanosynthesis), are the most preferable. macology (13–17) have focused their attention The synthesis of nanoparticles using plant on AuNP synthesis because of the particles’ Correspondence should be extract provides advancement over other distinctive properties, including physiochemi- addressed to both authors. published within three to five months. Peer-review papers are published methods because it is simple, one step, cal and biological (18–20). Gold nanoparticles PEER-REVIEWED cost-effective, environmentally friendly, are also used for the diagnosis and treatment Submitted: July 30, 2019. relatively easy to reproduce, less toxic, and of several diseases, such as diabetes, cancer, Accepted: Oct. 15, 2019. more effective (3–6). Parkinson’s, Alzheimer’s, HIV/AIDS, tuber- in the print and di gital editions of BioPharm International, and on www. 30 BioPharm International January 2020 www.biopharminternational.com BioPharmInternational.com. Links to the online versions of peer-review papers also are featured in e-newsletters distributed to the publication’s audience. Manuscripts may be sent to Editorial Director Rita Peters at [email protected]. To learn more about the peer-review submission process, click the Submission Guidelines link on www.BioPharmInternational.com. We look forward to hearing from you.

www.biopharminternational.com May 2020 BioPharm International 47 Quality/Regulations

Good Automation Practices for Remote Operations Having remote operations in place is crucial to maintaining good automation practices.

LAUREN LAVELLE

ioPharm International spoke with Casey Snodgrass, the price of complexity. Remote operations may require market segment leader for Pharmaceutical Sciences more extensive programming and project management. The B at the Hamilton Company; Cynthia Pussinen, vice- stakeholders need to have a clear vision of the system’s goals, president and general manager, Life Sciences and Specialty along with an incredibly detailed map of the requirements, Chemicals for Honeywell Process Solutions; and Bruce Kane, including interactions with other equipment and the laboratory industry technology consultant for Rockwell Automation, information system. A remotely operated system is far different regarding the proper procedures for remote operations when than a stand-alone automation platform for a dedicated workflow. complying with good automation practices. However, the term ‘remote operations’ can be a bit misleading, because a facility still needs people—let’s call them super users— PRACTICALITY AND SAFETY physically interfacing with a liquid handling system to load BioPharm: How practical are remote operations in tips, consumables, and reagents onto a robotic system, and also manufacturing facilities in terms of good automation practices? providing periodic maintenance and troubleshooting, or error Snodgrass (Hamilton): In practical terms, remote handling response. It is possible to configure an automated operations minimize the number of people that directly interface system with convenient remote access for users that are with the system. This is especially useful when working with trained in creating a job, and at the end of the run, remotely hazardous materials or highly infectious pathogens, such as retrieve the data or send samples downstream. Facilities can SARS-CoV-2, to limit the number of people with potential also link Hamilton’s dissolved oxygen (DO), pH, cell viability, exposure to infectious agents spending valuable time with and conductivity sensors to a biocontrol system for remote hands-on training, and reduces the amount of personal monitoring and automatic event triggering. protective equipment (PPE) used at the facility. To balance Pussinen (Honeywell): Remote operations in this perspective, convenience and practicality often come at manufacturing facilities are extremely pragmatic, concerning Sikov/Stock.Adobe.com

48 BioPharm International May 2020 www.biopharminternational.com Quality/Regulations

automation practices. A good reference also use the power of Honeywell Forge on-site, we offer safety features that prevent document for the life-sciences industry, offerings to proactively detect issues and accidental access to a remotely operated authored by the International Society bring them to the attention of both the system. Error handling is also a critical of Pharmaceutical Engineers (ISPE), customer and a Honeywell expert. safeguard to enable frequent unattended The Good Automated Manufacturing With these three methods, we are use while preventing major disruptions Practice (GAMP) Guide for Validation able to keep customers safe while also in the form of unplanned downtime. of Automated Systems in Pharmaceutical maintaining business continuity. The Hamilton’s systems can automatically Manufacture (1), describes a set of industries we serve include: recover from many error scenarios and principles and procedures that help ensure • Life sciences send remote notifications to authorized that pharmaceutical products have the • Pulp and paper/flat sheet personnel. For serious issues, some on-site required quality. manufacturing intervention may still be necessary. The use of remote operations in life • Gas terminals sciences settings will likely evolve rather • Oil and gas. EQUIPMENT quickly as the industry seeks to ensure In fact, we have employed these AND INSTRUMENTS it is able to deliver life-saving therapies unique technologies within some of BioPharm: What equipment/ to patients around the world, without our own plants. instruments does your organization rely interruption, in case of natural disasters, Kane (Rockwell): It depends on on when operating remotely? inclement weather, pandemics, and/ how the systems are designed, but, yes, Snodgrass (Hamilton): Hamilton or in other situations where colleagues modern systems employing current best Robotics has a number of automated are not able to be physically present at a practices in security, engineering, and liquid handling workstations that can be manufacturing facility. safety controls are very safe and secure. used in a remote operation, integrated Kane (Rockwell): Good automation There are several perspectives that should with other devices into larger work practices are not exclusive to remote be considered when safeguarding remote cells, and linked to other operations via operations, or vice versa. Instead, good operations. The most basic safeguard a laboratory information system. With automation practices are centered is that connections are secure and not different platform sizes, configurations around building quality controls into the open to attacks or hacks. Following and capabilities, it’s easy for biopharma manufacturing process. Remote capabilities good network design and remote access manufacturers to fine-tune a solution can serve to enhance this quality. Remote principles help ensure this safety of specific to their needs. Hamilton Storage technologies that enable safer, more communication. Instrument Society of offers automated storage systems along frequent, and more timely monitoring America/International Electrotechnical with automated cappers and decappers and intervention into process disturbances Commission 62443 also has that can further enhance automated are of great value to ensuring product recommendations for good and secure workflows in remote operations. In a quality. The technologies that allow both network design. One should also consider remote environment, Hamilton’s sensors interactive, remote, and full remote are the nature of the remote operations monitor in real-time whereas many well evolved and suited for a wide variety and their impact on local personnel, others only provide a before and after of remote-use cases. We believe that, as for example, signaling that indicates snapshot of the activity. By monitoring the technology continues to evolve and equipment is under remote operation. even tiny fluctuations in real time, users augmented visualization technologies We recommend something similar know exactly what’s happening in the become more robust, there will be an to a process hazard and safety review be process, and make rapid determinations increase in their uses throughout a wide conducted on any remote operations before about next steps instead of having to fail cross section of facilities. implementation to ensure safe operation. an entire batch, which as you can image, is BioPharm: Are remote operations in For example, some equipment may require a huge risk for a multi-week process. manufacturing facilities safeguarded? a line of sight to visualize that equipment Pussinen (Honeywell): Honeywell Pussinen (Honeywell): Yes, during start up and be able to immediately provides solutions for our customers to absolutely. Customers around the world respond if there is a problem. In order to support remote operations—cybersecurity are also looking for Honeywell to provide follow the same intention with a remote- is often a critical element, and a variety remote support in light of restrictions on start operation, additional equipment of solutions and services are available. personnel traveling and entering a site. might need to be installed to provide One such offering, Managed Industrial Honeywell has deployed multiple remote the visual checks in the absence of local Cybersecurity Services with security service options to continue providing operators. analytics, device management, and expert support via remote connectivity Snodgrass (Hamilton): For those regionalized support services helps to our equipment. These remote options interacting with Hamilton’s workstations customers modernize industrial

www.biopharminternational.com May 2020 BioPharm International 49 Quality/Regulations

control system (ICS) capabilities while home orders.’ In some cases, quarantines begin to use it remotely. Next, make sure minimizing operational issues caused by of key individuals might impact the to implement error handling protocols cybersecurity incidents. availability of skilled resources at the site. and detailed personnel trainings as they Additionally available are real-time In response, Honeywell has issued new are both critical to successful remote process supervisory control and data software that enables process operations operations. Clearly define the workflow acquisition (SCADA) solutions delivered to be monitored or even executed from and support it through standard as a secure and scalable service. A member remote locations outside of the plant operating procedures (SOPs). Obviously, of Honeywell’s suite of cloud-enabled facility—in other facilities or from don’t skimp on documentation. By their solutions for operations technology home—depending on specific needs, this nature, biopharma companies already and information technology (OT/IT), might present a very valuable option. have a wealth of documentation to SCADA provides cybersecure access and Kane (Rockwell): Remote support their audit trails. Scheduling is collaboration anywhere, while ensuring operations are applicable for use in important; dedicate time for preventative regulatory compliance. almost any manufacturing environment. maintenance and for cleaning and Kane (Rockwell): Rockwell I can’t think of one where it would not reloading the system. Key steps like these Automation is an automation system be applicable. Provided good engineering can ensure smooth operations in the long provider, and in a sense our whole and safety practices are followed, there run. Consider ways to further minimize business is about providing remote is no reason why remote operations risks, like redundant equipment or control to process, whether it be sending could not be employed. In fact, I can devices and spare parts, or training a signal to a valve to open across the plant, think of a few reasons why remote in-house service personnel. or around the world. Our business has operations would be preferred: first, a Kane (Rockwell): Process hazard always been about removing the operator reduction of personnel in an area reduces analysis, process workflow analysis, and from having to be physically present at the sterile load on an area. Second, engineering studies. By thinking about the valve to open it. We are using some of remote operations support centralized how to identify candidate operations the newer and more exciting technologies management and resource sharing. And for remote control, such as reviewing available for remote operation. Those lastly, remote operations allow more operations for opportunities to remove involve using highly flexible screen timely responses to disturbances. local actions and engineering new sharing capabilities with Thin Manager, BioPharm: How do you suggest processes with remote operations. This a FaceTime-like conferencing with other organizations implement remote is a very broad concept to consider, augmented reality and 3D persistent operations into their biopharma but focus should be on the question, whiteboarding (Chalk). And for sharing manufacturing facilities (e.g., as applies ‘What problem am I trying to solve work methods and training remotely, we to workflow, day-to-day operations, with remote operations? Is it simply have some unique augmented reality/ cleaning/sterilization, etc)? remote operations, or is it inspection or virtual reality knowledge management Snodgrass (Hamilton): This goes troubleshooting? And will an operator systems (Knowledge Capture). along with the best practices, and we be local or not?’ These are general already touched on a few of these points. questions to ask when implementing RECOMMENDED USE I can’t stress enough just how important remote operations into facilities. AND IMPLEMENTATION planning is, including trying to envision BioPharm: Do you recommend use every possible case scenario. For example, REFERENCE 1. K.Vaishnavi, Good Automated of remote operations in biopharma it’s not feasible for a user to program a Manufacturing Practice (GAMP), 225 manufacturing? liquid handling workstation in a vacuum, (2012). ◆ Pussinen (Honeywell): Yes, taking so to speak, and expect that others can advantage of remote operations is a More on automation great way in which to facilitate business continuity when it is not possible to For more on automation, read the following articles on have colleagues physically present at a BioPharmInternational.com: manufacturing facility, and to allow • Automating Bioprocesses visibility into one’s operations at a contract www.biopharminternational.com/automating-bioprocesses manufacturing organization. Given the current day challenges posed with • Automation in the Lab: What to Consider COVID-19, companies increasingly have www.biopharminternational.com/automation-lab-what- fewer individuals working at an operating consider-0 process location, in adherence to ‘stay at

50 BioPharm International May 2020 www.biopharminternational.com Outsourcing

Biomanufacturing: Demand for Continuous Bioprocessing Increasing But are innovations sufficient to increase adoption? CMOs are demanding better continuous bioprocessing options.

ERIC S. LANGER

n nearly all other manufacturing technologies, cost con- batch processing, with manufacturing batch fluids essentially siderations dictate that continuous production will be moving incrementally en-masse from one process step and I the rule. But in bioprocessing, the normal evolution from set of equipment to the next. batch to continuous operations has not moved as quickly as Downstream processing continues to create bottlenecks in many had expected. production, and improvements in batch processing are not Continuous processing upstream has been around for really emerging. Therefore, the industry continues to seek decades as perfusion (e.g., fiber-based perfusion bioreac- solutions from innovators for better continuous processes tors for fused-cell hybridoma culture in the 1980s). But that offer further process intensification and lower costs. In that’s essentially the only continuous-adapted upstream unit fact, 70.6% of bioprocessing professionals are either testing process, with such things as culture media and additives continuous bioprocessing downstream technologies or con- preparation still done in batch processing. In some respects, sidering them. This is up from 68% based on data from our perfusion has overall been a commercial failure. Sales of the 2016 Annual Report (1). leading alternating tangential flow (ATF) perfusion systems According to BioPlan’s 17th Annual Report and Survey on from leading suppliers, after more than 15 years, are under Biopharmaceutical Manufacturing Capacity and Production (2), $20 million. And continuous processing downstream is still there has been a slow increase in assessment of the various con- largely lacking and, where implemented, involves just a few tinuous bioprocessing options over the past five years, and the of the many unit processes involved in downstream process- ERIC S. LANGER is president and managing partner at ing. Multi-column, countercurrent, and other variations of BioPlan Associates, Inc., a biotechnology and life sciences continuous chromatography units are just starting to enter marketing research and publishing firm established in Rockville, MD in 1989; [email protected], the market. The classic and still predominant approach to +1 301.921.5979. bioprocessing, both upstream and downstream, remains valdis torms/Stock.Adobe.com

www.biopharminternational.com May 2020 BioPharm International 51 Outsourcing

Figure 1: Facilities evaluating continuous bioprocessing (downstream) technologies in CMOs than biomanufacturers (40% vs the next 12 months (2016–2020). 28%) are indicating they want vendors to focus greater efforts on developing continuous upstream technologies (1).

BUDGETS FOR ADOPTION OF CONTINUOUS BIOPROCESSING BioPlan’s annual report for 2020 also evaluated adoption of bioprocessing technologies based on new technology purchases. When evaluating new expenditures, industry decision-makers were asked about new technologies they were budgeting for. Of the nearly 20 technologies identified, the top technologies this year included single-use bioreactors (noted by 45.9% of respondents), followed by cell cul- Figure 2: New expenditures, 2020. ture media including optimization, and then continuous bioprocessing (upstream), and continuous bioprocessing (downstream), according to preliminary data. BioPlan data in general indicate that the direction of the industry is more toward single-use novel devices, those that allow rapid transitioning from project to project, and options for continuous bioprocessing. Some of these technologies also support the increasing demand for biologics that may be called for in smaller quantities. Figure 2 shows the economic commitment decision-makers are focusing data support ongoing interest in the com- OUTSOURCING AND on continuous bioprocessing, as evi- ing year (see Figure 1). Approximately CONTINUOUS denced by companies’ top three new 55% of facilities surveyed are actively or BIOPROCESSING expenditures including both upstream informally evaluating continuous process- Contract manufacturing organiza- and downstream continuous biopro- ing technologies in the coming year. tions (CMOs) are often on the lead- cessing equipment, which was noted Although there are a number of tech- ing edge of new technology adoption. by a robust 25.9% and 16.5% response nologies providing process intensifica- For continuous bioprocessing and pro- from decision-makers. tion and continuous purification steps, cess intensification, BioPlan’s Annual it appears that more robust continu- Report shows that significantly more TRENDS MAKING ous chromatography technologies, such CMOs will be testing these technolo- CONTINUOUS as simulated moving bed (SMB) and gies over the next 12 months (53% of BIOPROCESSING ATTRACTIVE periodic countercurrent chromatogra- CMOs will be evaluating downstream Several technological advances and phy, are generally not yet ready yet for options, vs 38% of biomanufacturing related trends are making continuous commercial-scale adoption (other than facilities). On the upstream side, again bioprocessing attractive. Some estab- adoptions performed using single-use it is the CMO outsourcing organi- lished bioprocessing facilities are being upstream equipment generally limited zations that are seeking better prod- retrofitted and upgraded for more con- to 2000-L scale). ucts and more improvements. More tinuous operations. ALL FIGURES COURTESY OF THE AUTHOR.

52 BioPharm International May 2020 www.biopharminternational.com Outsourcing

Figure 3: Single most important biomanufacturing trend (2014–2020) (Selected Findings).

There are many benefits to operat- • Increased flexibility: Continuous tems that use bolt-on-type technol- ing bioprocesses continuously rather manufacture enables more ogy, which retrofit components unit than in batch mode, with many of adaptability and efficient facility operations for existing systems. Other these similar and complementing utilization, similar to the conventional downstream continuous those of single-use and modular sys- advantages of single-use devices. adaptable technologies, such as cen- tems: Bioprocessing also becomes much trifugation, will also see increasing • Reduced costs: Operating more portable, and facilities more adoption in coming years. Potentially continuously allows use of cloneable. revolutionary capillary fiber perfusion significantly smaller-scale Many upcoming continuous bio- bioreactors and other new technolo- equipment, with a smaller volume processing technologies are very gies, including those for downstream bioreactor. novel. For example, a single 50-L bioprocessing, will be likely coming • Increased productivity: Because reactor is expected to be able to man- online and be more widely adopted much of the bioprocessing ufacture the same quantity of product, for commercial manufacture over the equipment is operated often at better quality, comparable to next 10 years. continuously, there is little need a 5000-L bioreactor over the same for large transfer/storage vessels time period. Case studies and other CONTINUOUS PROCESSING and no halts between processes. reports of such performance will fur- TRENDS IN BIOPROCESSING Bioprocessing thus tends to move ther promote rapid adoption. There When respondents were asked about much more smoothly. will be increasingly rapid adoption their ‘single most’ important bioman- • Improved quality: Biological of single-use systems for new com- ufacturing trend, or operational area molecules are expressed mercial manufacturing over the next on which the industry must focus its continuously, and compared to five years; and continuous bioprocess- efforts, upstream and downstream batch culture, continuous culture ing, particularly upstream processing, continuous bioprocessing declined tends to be more controllable, less is expected to follow a similar tra- dramatically over the past six years, intense and stressful, including jectory. Use of continuous biopro- from 9.1% to 1.25% for upstream, and less shear and media nutrient cessing is likely to further increase 10% to 4.7% for downstream continu- levels kept constant. with the arrival of more hybrid sys- ous bioprocessing (Figure 3). www.biopharminternational.com May 2020 BioPharm International 53 Outsourcing

Figure 4: Perspectives on continuous bioprocessing and process intensification bioprocessing have been resolved in (Selected Data) (2). recent years through the application of innovative technologies, including new developments in single-use equipment. On the other hand, perfusion processing is now significantly less complex, less prone to contamination, and more readily scalable than previously. Negative assessments from within the industry of continuous perfusion fed- batch processing overall may reflect a lack of direct exposure or experience with continuous technology. In BioPlan’s annual report, for example, key areas where most respondents reported they perceive perfusion as presenting more concerns While this might imply that interest of perfusion for early stage vs. large/ (vs. fed-batch) included: in continuous bioprocessing is waning, commercial-scale manufacturing. • Process operational complexity combined with the increased expendi- BioPlan studies have shown that few (perfusion noted by 72% as more tures in the area, it suggests that con- processes are scaled-up, particularly operationally complex vs. batch) tinuous bioprocessing is becoming a for commercial good manufacturing • Contamination risks more mainstream bioprocessing area, practice (GMP) manufacture, using • Upstream development and and therefore, less trend-relevant, thus, perfusion in continuous upstream biocharacterization time the lower trend ‘score.’ processing CP USP. Perfusion adds • Process development control considerable mechanical complexity challenges IMPLEMENTATION OF and regulatory uncertainties (i.e., it • Process development general CONTINUOUS is avoided for GMP manufacturing, challenges BIOPROCESSING expert staff are needed, etc.), as well • Validation challenges Although this is beginning to change, has having limited equipment options • Need for greater process control implementation of continuous bio- and universal industry inertia restrain- • Cell line stability problems processing is and has been slow (see ing adoption. • Ability to scale-up process. Figure 4). At best, a few unit pro- Large-scale continuous down- Interestingly, while approximately cess/steps both up- and/or downstream processing, particularly chro- 76% believe downstream continuous stream have been implemented as matography operations, remain rare. bioprocessing will be a long time in continuous by a minority of facilities. Even where continuous downstream coming, 66% believes that perfusion Some commercial biopharmaceuti- processing has been implemented, it systems will be adopted by most bio- cal products that essentially require involves at best only one or few out processing facilities. This shows the perfusion’s generally milder/less of the usual multiple chromatography expectation that continuous biopro- intense processing conditions, includ- and other downstream processing unit cessing is here for the long haul, but ing Factor VIII (the largest recom- processes/steps having been imple- widespread adoption may not be in binant molecule biopharmaceutical) ment as continuous. the near future. and coagulation factors, have been Survey data suggests that biopro- manufactured for decades using per- cessing professionals may believe con- REFERENCES 1. BioPlan Associates, 16th Annual Report fusion (other products use continuous tinuous processing is more ready for and Survey on Biopharmaceutical centrifugation). broad adoption for more unit pro- Manufacturing Capacity and BioPlan studies have shown cesses than it currently is. Notably, Production (Rockville, MD, April 2019). 2. BioPlan Associates, 17th Annual Report approximately 5% of bioreactors that continuous processing equipment and Survey on Biopharmaceutical are over desktop-size use perfusion, manufacturers and users report that Manufacturing Capacity and mostly for feeder, not production, many of the problems long associ- Production, Preliminary Data (Rockville, MD, March 2020). ◆ bioreactors. There is more adoption ated with perfusion and continuous

54 BioPharm International May 2020 www.biopharminternational.com Implementing Attribute Monitoring Analyses for Biopharmaceutical Development and QC

ON-DEMAND WEBCAST: Aired Wednesday, April 29, 2020

Register for this free webcast at: http://www.chromatographyonline.com/lcgc_p/monitoring_analyses All attendees will receive a free executive summary of the webcast!

Event Overview There is an ongoing progression to extend high-performance LC-MS Presenters technologies beyond traditional characterization roles into product attribute monitoring assays deployable across development, manufacturing and Scott J. Berger, Ph.D. quality control organizations. This webcast will cover the use of high- Senior Manager, performance LC-MS for biopharmaceutical analysis, including transitioning Biopharmaceutical Markets product characterization knowledge into monitoring assays for product and Waters Corporation process related quality attributes of biopharmaceuticals. Angelo Palmese, Ph.D. A case study presented from Merck Serono will: Head of Characterization and • Detail their assessment of analytical technologies and workflows for Innovative Analytics Unit multiple attribute monitoring (MAM) assay development Merck Serono - • Assess the benefits of these new assay formats Guidonia Site, Italy • Reveal plans for deploying these assays within development, manufacturing, and QC functions within their organization Moderator

Key Learning Objectives Rita Peters Editorial Director During this webcast, you will learn about the following: BioPharm International • Realize the capabilities of high-performance LC-MS, for biopharmaceutical analysis

• Recognize that LC-MS technologies have become more readily For questions or concerns, email deployable and can now be operated by non-MS experts [email protected]. • Learn how attribute-based analysis can supplement product knowledge gained through traditional assays, and potentially streamline testing for process monitoring and product Sponsored by Who Should Attend • Academics, R&D, Lab Directors • Biopharmaceutical organizations, CXO, and research institutes focusing on large molecule drug development and manufacture • Analytical scientists supporting biologics development, manufacturing Presented by and QC • Lab managers, Directors, VPs in charge of characterization and monitoring strategies • Biopharmaceutical regulatory authorities PRODUCT SPOTLIGHT

Pre-Packed Columns Supercritical Fluid Tosoh Bioscience introduced the SkillPak 1 mL and 5 mL Chromatography System pre-packed columns for The Nexera UC Prep, a fast method development preparative supercritical and resin screening of fluid chromatography monoclonal antibodies, (SFC) system from antibody constructs, Shimadzu, works to oligonucleotides, proteins, provide maximum use and viruses. of lab resources through The columns come pre-packed with Toyopearl, TSKgel, flexible system configurations in a compact design that ++ or Ca Pure-HA process chromatography media and are requires low installation space. ready for use immediately. Additionally, the high performance, The system can be configured based on user scalable columns are available with short delivery times specifications, which include chiral or achiral purifications, in affinity, anion and cation exchange, hydrophobic single injections, stacked injections, and fraction collections interaction, and size exclusion chromatographic modes. Peak from microliters to liters. Additionally, the system comes performance is guaranteed, according to the company, and equipped with LotusStream gas-liquid separator technology the columns can be operated directly with low- or medium- for high recovery and low carryover, and Prep Solution pressure liquid chromatography systems. software for streamlining operations. The SFC solution also reduces the need for solvents, Tosoh Bioscience shortens purification run time and dry down time, reduces the www.tosohbioscience.com total size of the recovery system, and allows for easy rinsing.

Shimadzu www.shimadzu.eu

ReNu Single-Use Technology Mass Flow Controllers The SLA5800 Series Biotech and the SLAMf Series Biotech Cartridge Assemblies are two new mass flow controller (MFC) models from ReNu SU (single-use) Technology cartridge assemblies from Brooks Instrument designed specifically for improved gas Watson-Marlow Fluid Technology Group are designed for the flow control in biotechnology development and production applications. The new models of personalized medicines. allow biotech equipment Available with CPC AseptiQuik, manufacturers to reduce the Pall Kleenpak Presto, or GE number of MFCs they need for Readymate aseptic connectors, their equipment, which allows the devices can be integrated them to simplify their designs directly into a customized along with reducing system fluid path, allowing for fast costs, purchasing complexity, bioprocess integration while and spare parts inventory eliminating alignment errors. requirements. The cartridge assemblies are equipped to work with the The MFCs have an enhanced control valve with a leak company’s quantum peristaltic pump, which allows for a rate of 0.005 sccm for flow rates between 5 sccm and 150 change in pump technology by providing higher accuracy slpm, and a rate of 15.6 sccm for higher flow rates. The low with flow linearity without back pressure. Additionally, leak rate potentially eliminates the need for a separate shut- the cartridge assemblies offer lot traceability on every off valve in the gas delivery system, reducing component component, ISO Class 7 cleanroom manufactured and purchasing costs and saving engineering, testing, and packaged assemblies, and product sterility of 10-6 SAL per validation time. The MFCs accommodate a wider range of ANSI/AAMI/ISO 11137 guidelines. gas flows and control up to four different gas flows including air, carbon dioxide, nitrogen, and oxygen. Watson-Marlow Fluid Technology Group www.wmftg.com Brooks Instrument www.brooksinstrument.com

56 BioPharm International May 2020 www.biopharminternational.com Ask the Expert

Ask the Expert — Contin. from page 58 Soon to be unveiled.... a course with an approved training company. If however, a new control system designed for upstream processing... you want to get a basic understanding of the freeze-drying process, you will easily find free tutorials online. Should you need hands-on experience, then training courses PendoTECH® Perfusion Controller offered by universities or industry associations with in- convert any bioreactor to a house training centers will be the right choice. perfusion bioreactor

SWITCHING CAREERS Features Include: PendoTECH Single What if you have been in the industry for a while, but • Vessel level control Use Sensors would like to change positions and/or area of exper- • Perfusion pump control • Filter feed pump control tise? Very often this is less of a question of opportunity, • Pressure monitoring of external perfusion but more of a question of an individual’s preferences. device, such as a hollow fiber filter (including There are equally excellent subject matter experts filtrate pressure monitoring to indicate filter clogging) with PendoTECH Single Use Pressure who never strayed from their vocation (say regulatory Pressure SensorsTM affairs, quality control, or manufacturing) and who • Optional monitoring of temperature in perfusion loop with PendoTECH Single are perfectly happy in their jobs, and there are those Use Temperature Sensor who worked in different departments to become more • Graphical User Interface with trending of process data and logging of data to a file universal experts. Pharmaceutical companies probably • Built-in data server to exchange data with Temperature look more for experts in a particular subject, whereas OPC client software such as PI from OSIsoft ® • Filtrate scale to measure perfusion volume per day service providers, such as consultancies or contract • Additional inputs to log data from optional sensors research organizations, may have a need for experts with more varied backgrounds. We may not always find the job we want, but we can always learn from what we do, and it will always be a ISO 9001:2015 Certified • www.pendotech.com • Princeton, NJ USA beneficial personal and job experience. ◆

Ad Index

Company Page Company Page

ADVANTAPURE 31 PHARMAFLUIDICS 25

AGILENT TECHNOLOGIES 41 ROCHE CUSTOM BIOTECH 13

CATALENT 60 SARTORIUS STEDIM BIOTECH 59

CYGNUS TECHNOLOGIES LLC 11 SINGLE USE SUPPORT GMBH 20–21

EAGLE STAINLESS 19 TOSOH BIOSCIENCE 33

MILLIPORE SIGMA 15 USP 7

NYSE GROUP INC 2 WATERS 55

PDA EUROPE 23 WUXI BIOLOGICS 5

PENDOTECH 57 WYATT TECHNOLOGY 29

May 2020 www.biopharminternational.com BioPharm International 57 Ask the Expert

Starting a Career in the

Bio/Pharmaceutical Industry Siegfried Schmitt, PhD, is Having a better understanding about compliance will be of vice-president, technical, Parexel Consulting. benefit when looking for a job or for furthering one’s career.

While working on a variety of projects Q: in three different continents, I had the We may not always find opportunity to meet and work with young, enthu- siastic newcomers to the industry. They were from the job we want, but we a variety of different professional backgrounds, including pharmacists, engineers, and chemists. During our conversations, most of them asked the can always learn from following types of questions: • The college or university I graduated from did what we do, and it will not provide courses on compliance or industrial operations—how can I fill this gap? • Though I applied for many positions, I have always be beneficial. been unable to find a permanent position through portals such as LinkedIn, Bing, or similar yet—how can I improve my chances? sites that have a good reputation with industry and • On the Internet, I found several courses on job agencies. Also, it’s important to write a succinct good manufacturing practices (GMP), for and well-thought out curriculum vitae, and there compliance experts, or similar. These are rela- is a lot of great advice available for free online on tively expensive. Are they worth investing for how to do this. The Internet is the place to research someone like me (a beginner)? jobs, but often also for potential employers to find • Should I work my way up within a particu- the right candidate. lar department or would it be better if I gain Having a better understanding about compli- experience in different departments? ance will surely be of benefit, whether looking for a job or for furthering one’s career pros- The following are not exhaustive or the pects. Whether you are lucky enough to have A: only answers to these questions, but your employer pay for external courses, be sub- they will give some insight. sidized (e.g., by a state job center), or have to It is true that few graduates have seen indus- pay yourself, in all cases you should scrutinize trial operations by the time they graduate, but the courses offered: that doesn’t mean that they don’t come equipped • How relevant are they to your current or pro- with many of the basic skills needed in the indus- spective work? try, such as team working, presentation skills, • Do they provide references? Is there feedback analytical thinking, and the ability to self-study. available? Companies will provide training, as a minimum • Are the certificates merely proof of attendance on the applicable and relevant internal processes or are they widely recognized by the industry? and procedures, which will cover both the opera- • Is it just classroom learning or is there also a tions and the compliance side of the business. A practical element? lot will be learning on the job, from peers and • Do they provide comprehensive documen- often also from mentors. tation? Finding your first permanent job can be a frus- The answers to these questions will help you trating experience, but persistence usually pays off. determine if the course is right for you. For exam- Gaining experience through temporary voluntary ple, if you want to become a certified auditor (be engagements, placements, or positions is what it for GMP or ISO 9001), you will have to pay for helps improve the chances for long-term or per-

manent employment. And don’t forget to network Contin. on page 57 Stock.adobe.com - freshidea

58 BioPharm International www.biopharminternational.com May 2020 Biostat STR® Generation 3 and Biobrain

Engineered for Ultimate Upstream Performance

The Biostat STR® single-use bioreactor system is designed for rapid process development and seamless scaling to commercial manufacturing. It is powered by the new Biobrain automation platform and BioPAT® analytical tools to deliver outstanding speed, quality, and productivity.

For more information, please visit www.sartorius.com/biostat-str Your one integrated biologics partner. For Biologics and Gene Therapy, we have the passion to help you accelerate, simplify and de-risk from development and biomanufacturing, to ﬁll/ﬁnish, analytical, clinical supply and commercial launch

25+ 600+ commercially antibodies & 80+ approved products recombinant through fill/finish proteins developed

5000+ 13 clinical trials marketed products supplied using gpex® technology

60+ 120+ gene therapy clinical trials using programs gpex® cell lines

Biologics | Biosimilars | Gene Therapy | Oncolytic Virus | Next Gen Vaccines

Comprehensive solutions. Advanced technologies. Better biologics.™ us + 1 888 SOLUTION (765-8846) eur 00800 8855 6178 biologics.catalent.com

Integrated Biologics_BioPharma.indd 1 4/21/20 1:41 PM