Cell Culture

After over three decades of progress, cell culture bioprocesses in the biopharmaceutical industry continue to evolve. From early laboratory culture methods to vaccine production in the mid-20th century, the first biopharmaceutical companies had a century of basic understanding to build on when they developed the first recombinant expression systems. Since those first efforts in the 1980s, a succession of cell culture biologists and engineers have improved processes using a series of products that have been increasingly specified for their use. Product titers rose exponentially, and the characterization of media and cell lines steadily improved. While even more knowledge is amassed in the literature as well as through personal experiences and company intellectual property, this evolution will be ongoing.

The BioProcess International Conference and Exhibition provides an annual update of progress in cell culture. This year’s program reflects that evolution in process and media optimization for antibodies, conjugates, and other protein products. How are advances in cell-line engineering and automation (from evolving assay methodologies to the recently published CHO cell genome) helping companies develop and scale up their culture processes? How are decisions made regarding disposables and culture modes? And what are vaccine manufacturers learning in turn from the biopharmaceutical experts who years ago adapted their culture approaches to the first “bioprocesses” in a burgeoning industry?

AUDIENCE: UPSTREAM PROCESS DEVELOPMENT, QA/QC, analytical

KEYWORDS: CHO cells, genomics, media, supplements, bioreactors, perfusion, disposables, vaccines, Characterization, cell lines

TAKE-AWAY: APPLY NOVEL APPROACHES TO IMPROVE PREDICTABILITY, ACCELERATE, DEVELOPMENT, AND CONTROL QUALITY.

Scientific Advancements

Perhaps the most exciting scientific publication in cell culture of recent years has been the Chinese hamster ovary (CHO) cell genome. Professor Michael Betenbaugh of Johns Hopkins University will report in his keynote address on Thursday, 3 November 2011, on the next steps. “A new era is about to begin in the biotechnology community,” he says. The majority of biopharmaceuticals are produced by CHO cells. Sequencing and characterizing CHO K1 and other related cell lines and tissues is being pursued in earnest by a variety of international groups. “The impact these events will have on biotechnology and bioprocessing is not yet clear,” says Betenbaugh. “But it is likely that the sequencing of the CHO genome will transform the way in which cells and processes are analyzed and improved in future decades.” In his presentation, he will discuss ongoing efforts to characterize the genome, proteome, and other “-omics” sources in the CHO community. He will suggest how these changes may transform the way biotechnology problems are solved, including representative examples from his laboratory and others.

“Reaping the greatest benefits of this information explosion will require the community to coordinate efforts in new and different ways,” he says. “As a result, we will discuss ongoing efforts to organize, distribute, and share the rapidly expanding CHO genomics knowledge base in a framework that will provide users with the tools that will provide the greatest benefit to the entire biotechnology community.”

Following Betenbaugh on Thursday morning, two other presenters will get into the subject with some detail. First, Iman Famili (senior director of R&D at GT Life Sciences, Inc.) will describe genome-scale metabolic models of CHO and NS0 cell lines that her company developed using an integrated computational and experimental platform. Her team used a reconstructed CHO model to develop proprietary media and novel selectable markers. Experimental implementation of these modeling strategies showed improvement in cell culture metabolism. This approach, Famili says, could also improve product quality and glycosylation.

Postdoctoral researcher Colin Clarke of the Irish National Institute for Cellular Biotechnology at Dublin City University will then present a predictive model of cell-specific productivity (Qp) in CHO bioprocess culture based on gene-expression data. His group used a machine-learning algorithm, partial least squares (PLS) incorporating jackknife gene selection, to model Qp, predicting it to within 4.44 pg/cell/day. In addition, they linked several genes constituting this model with biological processes relevant to protein metabolism.

Cell-Line Development: The 21st century so far has seen great advancements in the methods, materials, and results of cell-line engineering (1,2,3,4). Thursday afternoon’s cell culture track follows the morning logically with a discussion of novel approaches to cell-line development. Arnaud Perilleux (upstream biotech process scientist at Merck Serono SA in Switzerland) will also describe a use of predictive methods. Cell-line selection programs and early stage process development are currently undergoing major changes, says Perilleux. He will describe a robust, fed-batch, platform process using 96–deep-well plates to accurately predict bioreactor performance.

Adrian Haines (a principal process development scientist at Lonza Biologics plc) will also describe how his company has incorporated shaking 96-well plates into fed-batch culture evaluations. Haines will review some technologies available for such scaled-down fed-batch culture studies. As he reports, Lonza recently expanded the use of deep-well cell culture in “islands” of automation, technology it has integrated into One-Step cell line constructions.

Cell population heterogeneity is one characteristic being scrutinized in a number of laboratories. Two university researchers — David James of England’s University of Sheffield and James Piret of Canada’s University of British Columbia — will highlight this work at the BPI Conference. First, James shows how genetic heterogeneity in CHO cell populations can affect cell-line development. This topic is of particular interest to companies developing biopharmaceutical production platforms, no matter the cell line.

Piret then describes the use of microfluid arrays for clonal analysis, especially in the cell therapy arena. “Advances in stem cell research have led to cells being tested as therapeutic agents in an array of clinical trials,” Piret says. But “cell population heterogeneity poses a major obstacle to understanding complex biological processes. We have developed microfluidic devices containing thousands of nanoliter-scale bioreactors for the culture of single cells or colonies.”

While process development timelines necessarily shorten across the biotechnology industry, quality by design (QbD) initiatives are taking a more predominant role in bioprocess manufacturing. So companies need to generate cell culture data more quickly and efficiently than ever before. Someet Narang (a cell culture scientist at MedImmune LLC) will explain how the company evaluated two high-throughput bioreactor systems to better understand the balance between data quality and quantity. With all the advantages of a shake-flask–bas
ed system — e.g., low manual labor and cost — such milliliter-scale bioreactors provide a pH and oxygen-controlled environment critical for scalable prediction of product quality and volumetric productivity. Narang will summarize the methodology and factors considered in MedImmune’s comprehensive evaluation of systems from Pall and TAP Biosystems.

“We have found that statistical analysis of data, specifically the power calculations, is an effective method for assessing variability and resolution inherent to the system,” says Narang. “Results from such analyses (as well as certain other factors) play a critical role in determining which applications are best suited for a given system.” After those evaluations, the company assigned each system to an appropriate step in its cell culture process development cycle for effective, synergistic use of both. Media and Technologies

Today’s biopharmaceutical organizations must reduce timelines and manage shrinking resources (5). On the morning of Friday, 4 November 2011, Tiffany Rau (Pall’s global technology and technical manager) will present data showing the development of small-scale bioreactors for both mammalian and microbial cell line selection and process optimization activities. She plans to demonstrate the advantages of controlled “high-throughput” bioreactors for rapid, early stage process development that can shorten development timelines — and ultimately lower development costs.

Legacy Systems: As cell biology and production technologies progress, many “legacy” bioprocess facilities end up on the path to obsolescence. Process changes can be so difficult they become impractical, especially for large-scale commercial manufacturing and particularly when major equipment changes are considered. In Thursday afternoon’s plenary session, Thomas Daszkowski (vice president and head of process technology at Bayer Technology Services) will address some changes seen in bioprocessing and evaluate how modern facility concepts may be able to better address ongoing and future changes. Flexibility may well be the key to staying power (6).

Facility flexibility matters, according to Yael Hirsch (a manufacturing science and technology engineer for Genentech, Inc). His case study on Friday morning, 4 November 2011, describes how his company supported new technology implementations required for a phase 3 process. “Successful execution of a clinical campaign requires that a sufficient amount of drug of acceptable quality is produced,” Hirsch says, “in time to supply the clinical trial.” He will show what aspects of facility flexibility enabled process transfer to introduce a novel seed train strategy, chemically defined media formulations, and a modular viral clearance step within project timelines.

Media and Culture Feeding: Perhaps the most common changes considered in cell culture processes involve culture media and supplementation (7,8,9,10,11,12,13,14,15,16). Lauren Feeney (a late-stage cell culture research associate in pharma technical development at Genentech, Inc.) will show on Friday afternoon, 3 November 2011, how her company used amino-acid supplementation to eliminate a low-level sequence variant (<2%) caused by tyrosine misincorporation in cell culture.

“The occurrence of recombinant protein sequence variants is a challenging issue in CHO cell line development,” Feeney says. Her group discovered a tyrosine– phenylalanine variant at low levels in 28 sites within a certain recombinant product, and a process change to fix the problem was deemed appropriate.

The decision to make a raw material or manufacturing process change for a marketed biopharmaceutical product can involve consideration of a large number of variables, so according to Michael Titus (director of quality management and regulatory compliance at BD Biosciences) it is not one to be taken lightly. Regulatory requirements need to be addressed concerning every proposed change even as regulatory pressures to remove animal products from bioprocessing continue to rise. The risk of known and unknown viral and transmissible spongiform encephalopathy (TSE) contaminants can outweigh the benefits of using animal-sourced legacy products. Replacing undefined raw materials can also improve process consistency and reduce total biological drug manufacturing costs.

In a session on Thursday afternoon, 3 November 2011, Titus will focus on removal of undefined raw materials used to produce marketed drugs and replacing them with chemically defined substitutes. He will discuss the regulatory concerns raised when contemplating such a change, and approaches that can be considered. As Titus points out, media suppliers can be helpful to companies making such changes.

One reason for considering media changes is the fact that variability in animal-sourced media components can affect the quality of final products. Christopher Crowell (a principal scientist in process development at Amgen Inc.) will follow Titus with a case study of technology transfer challenges encountered for a monoclonal antibody (MAb) produced by CHO cell culture due to a medium’s variable trace element content. Initial experiments focused on understanding and controlling the impact of such variability on product quality differences when a process was transferred from Amgen to a contract manufacturer. The two companies found their cell culture metabolism to be sensitive to levels of a particular trace element that required additional controls to be put in place.Cell Culture Sessions

Cell Culture Sessions ()

In another Amgen case study, a high-yield chemically defined medium was developed for commercial manufacturing use. Henry Lin (senior scientist in cell science and technology at Amgen Inc.) will describe how his group implemented and optimized that medium at large scale to improve cell culture scale-up and product quality. According to Lin, preparation and treatment of such media can profoundly affect cell culture performance and scale-up from laboratory scale to manufacturing. Understanding the impact on media of operations such as filtration, high-temperature short-duration treatment, hold stability, and storage is critical, says Lin. So they need to be taken into consideration during process transfer and scale-up. He will report on strategies developed and implemented to mitigate the risk of those operations for fitting the process into a new facility.

Implementing Disposables: One key aspect of modern, flexible production facilities is single-use technology (6, 17,18,19). In a stepwise process, single-use bioreactors, medium storage bags, sampling assemblies, and other disposables are eliminating the need for glass and steel in some production areas. But Charles Sardonini (associate director of cell culture development at Genzyme Corp.) cautions that “a number of bottlenecks remain before the autoclave can be put to rest and a 100% disposable process can be achieved.” On Wednesday af
ternoon, 2 November 2011, he will review the migration of single-use technology into a pilot-scale cell culture manufacturing facility for early clinical trials. And Joachim Bär (associate director of upstream manufacturing science at Boehringer Ingelheim GmbH & Co Kg) will describe challenges encountered when implementing disposable bioreactors.

Disposables are often used for liquid media storage, handling media before cell culture inoculation, and for cell culture operations. Masaru Shiratori (a late-stage cell culture engineer at Genentech, Inc.) reports in the same session how his company investigated single-use technology for cell culture media applications. They sought to better understand and prevent negative effects on cell growth and product yields correlated with specific disposables and the conditions of their use.

Disposable bioreactors are also being implemented in new viral vaccine manufacturing processes. Jean-Francois Chaubard (director of industrial viral bulk production at GlaxoSmithKline Biologicals in Belgium) reports on the morning of Thursday, 3 November 2011, how his company performed an in-depth testing of commercially available single-use bioreactors in preparation for choosing one. After selecting a specific technology for developing and scaling up new viral vaccines — and having used this technology for viral vaccine production for over two years — the company can now present its status in terms of process efficiency, robustness, field of application, revised cost of goods, and biosafety. Vaccines and Other Proteins

Vaccines are undergoing an industrial renaissance, most notably due to new production options involving mammalian and insect cell culture (20,21). So this year, a special session on 3 November 2011 highlights new types of vaccines and updates their production methods. First, John Aunins (executive scientific director at Merck Research Laboratories) overviews the state of the art in vaccine development and manufacturing, current issues, and future trends. “Vaccines are the crown jewel in the accomplishments of biotechnology,” he opines, “impacting far more lives at far less cost than any other medical intervention.” Aunins will emphasize the evolution of bioprocess and manufacturing requirements relating to new and potential products.

Then BioProcess International editorial advisor Scott Wheelwright (president of Strategic Manufacturing Worldwide, Inc.) addresses the differences between manufacturing vaccines and biotherapeutics. Whereas the basic techniques are essentially the same, he points out, certain aspects of vaccine production deserve special consideration. Plasmids, live viruses, and allantoic fluids all present challenges that don’t come up in manufacturing most biotherapeutics.

Host-cell proteins are a concern in production of all recombinant proteins. When vaccines are made using similar technology to that applied to biotherapeutics, regulatory authorities expect that immunoassays for detecting residual host-cell proteins supplement traditional measures of product purity and process consistency such as sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Marc Thorsteinsson (a research fellow in vaccine analytical development at Merck and Co., Inc.) will discuss strategies for immunoassay development in recombinant vaccine production processes. He will emphasize reagent generation strategies, assay development, and validation in the context of regulatory expectations for these highly complex assays.

Finally, two case studies will be presented that illustrate nonmammalian culture applied to vaccine manufacture. Manon Cox (president and CEO of Protein Sciences Corporation) will discuss the use of the insect cell baculovirus expression vector system (BEVS) in making a new recombinant influenza vaccine. The company filed a biologics license application for its FluBlok product in April 2008, and Cox will describe interactions with the FDA during the product review cycle as well as how Protein Sciences is preparing for its product launch.

Then Scott Winram (program manager of the health sciences business unit life-sciences operation at Science Applications International Corporation) reports on development and preclinical optimization of a circumsporozoite protein vaccine candidate against malaria. Operating as an unbiased “virtual-pharma” company to support the US National Institutes of Health (NIH) and National Institute of Allergy and Infectious Disease (NIAID), SAIC is pursuing manufacture using a Psuedomonas platform. After production of soluble protein demonstrated efficacy of the system, formulation studies were applied early in scale-up to prevent downstream problems. Winram will provide an update of his company’s progress and parallel preclinical evaluations of adjuvants, virus-like particles (VLPs), and nanoparticle technologies.

Beyond Antibodies: Thanks to several high-profile clinical and commercial successes, MAbs are the most familiar biotherapeutics. Because they share certain structural features and general functions, over the years their production and processing have evolved an accepted platform approach. But other recombinant proteins are less inclined to fit into a generic production platform. For example, the many types of vaccines — from subunits to VLPs to whole viral organisms — all require specific unit operations and attention to different details. Within those classes, however, limited manufacturing platforms may be possible. The same holds true for nonantibody proteins and new antibody variations.

On the morning of Friday, 4 November 2011, a BPI Conference session will examine different types of “next-generation” antibody products. Alane Wentz (process development senior scientist at Abbott Bioresearch Center) will report on a challenge study of antibody platform processes using hydrolysate-based and chemically defined media with a new class of bispecific antibodies: dual–variable-domain immunoglobulins (DVD-Igs). Abbott targeted three unique DVD-Igs with distinctive growth behaviors for process development and scale-up in its cell culture platforms. The team analyzed mRNA levels to reveal possible mechanisms responsible for the broad cell-specific productivity differences they observed, and Wentz will report the results.

With certain diseases, targeting a single protein is not sufficient to achieve full drug efficacy. That problem has prompted development of innovative antibody formats that are selectively cross-reactive. One company is working with a novel therapeutic bispecific MAb format: Kappa Lambda antibodies. Nicolas Fouque (process development and bulk manufacture section head at NovImmune) will address specific technical considerations his company has taken to face manufacturing constraints and challenges for this new product class.

Antibody–drug conjugates are of increasing interest for many biopharmaceutical companies (22). In a keynote address on Thursday afternoon, 3 November 2011, Morris Rosenberg (executive vice president of process sciences at Seattle Genetics) will examine their process development and manufacturing. The basic concept is to use an antibody that delivers a cytotoxic drug directly to a selected physiological target, such as a tumor-associated antigen. “Conjugates represent a broadly applicable approach to enhance the antitumor activity of antibodies,” says Rosenberg, “and improve the tumor-to-normal tissue selectivity of chemotherapy.” As a growing number of such products move through clinical development, they offer unique challenges and opportunities in process development and manufacturing. Rosenberg will focus on those issues and their potential solutions. Developments Are Ongoing

Five years ago, cell-line and production process engineers were proudly touting their response to a manufacturing
capacity shortage in the industry that was identified around the turn of the century (1,2,3). They’d managed to increase expression titers from levels measured in micrograms and milligrams per liter to multiple grams per liter of culture. Little did we know then that the best may be yet to come. Optimization is now the order of the day — and all their knowledge and experiences are helping others answer new questions of their own.

About the Author

Author Details
Cheryl Scott is senior technical editor of BioProcess International. Quotes not otherwise attributed are from presentation abstracts.

REFERENCES

1.) Kayser, K. 2006. Cell Line Engineering Methods for Improving Productivity. BioProcess Int. 4:S6-S13.

2.) Ludwig, DL. 2006. Mammalian Expression Cassette Engineering for High-Level Protein Production. BioProcess Int. 4:S14-S23.

3.) Kim, HY. 2006. Improved Expression Vector Activity Using Insulators and Scaffold/Matrix-Attachment Regions. BioProcess Int. 4:S24-S31.

4.) Lee, CC. 2006. High-Throughput Screening of Cell Lines Expressing Monoclonal Antibodies: Development of an Immununoprecipitation-Based Method. BioProcess Int. 4:S32-S35.

5.) McLeod, LD, C Scott, and SA. Montgomery. 2008. Special Report: 2008 in Review — Look Both Ways Before Proceeding. BioProcess Int. 6:34-45.

6.) Rios, M. 2010. Special Report: Flexible Manufacturing — Evolving Technologies Combine to Enable a New Generation of Processes and Products. BioProcess Int. 8:34-45.

7.) Johnson, T. 2006. Promises and Pitfalls of Cell Line Adaptation. BioProcess Int. 4:S52-S56.

8.) Kloth, C. 2008. An Inoculum Expansion Process for Fragile Recombinant CHO Cell Lines. BioProcess Int. 6:44-50.

9.) Seamans, TC. 2008. Cell Cultivation Transfer and Scale-Up. BioProcess Int. 6:34-42.

10.) Decaria, P, A Smith, and W. Whitford. 2009. Many Considerations in Selecting Bioproduction Culture Media. BioProcess Int. 7:44-51.

11.) Alahari, A. 2009. Implementing Cost Strategies for HuMab Manufacturing processes. BioProcess Int. 7:S48-S54.

12.) Paul, WC. 2009. Maintaining Product Titer While Replacing Undefined Components in a CHO Culture System. BioProcess Int. 7:30-38.

13.) Simula, T, S Grosvenor, and C. Scott. 2009. Rethinking Media Performance. BioProcess Int. 7:48-59.

14.) Fike, R. 2009. Nutrient Supplementation Strategies for Biopharmaceutical Production. BioProcess Int. 7:44-51.

15.) Fike, R. 2009. Nutrient Supplementation Strategies for Biopharmaceutical Production, Part 2. BioProcess Int. 7:46-52.

16.) Fike, R. 2010. Nutrient Supplementation Strategies for Biopharmaceutical Production, Part 3. BioProcess Int. 8:24-31.

17.) Kapp, T. 2010. Road Map to Implementation of Single-Use Systems. BioProcess Int. 8:S10-S19.

18.) Hanley, K. 2008. Integration of Disposable Technology. BioProcess Int. 6:S42-S46.

19.) Scott, C.A 2010. Convergence of New Products and Technologies Changes the Game. BioProcess Int. 8:S10-S15.

20.) Scott, C. 2008. Biotech Leads a Revolution in Vaccine Manufacturing. BioProcess Int. 6:S12-S18.

21.) Whitford, WG. 2010. Using Disposables in Cell-Culture–Based Vaccine Production. BioProcess Int. 8:S20-S27.

22.) Scott, C. 2010. Special Report: Protein Conjugates — Facing the Challenges of Their Development and Manufacture. BioProcess Int. 8:28-37.