Cell Culture and Production

Cheryl Scott

September 1, 2010

15 Min Read

Time to market, cost of goods, and reduction of financial risk are major challenges in protein manufacturing. Process intensification can help biotech companies achieve their goals. Already underway in several other industries, implementing this concept shrinks production facility sizes by 10–1,000 times using novel processes and products (e.g., single-use and isolator technologies in biotechnology) to reduce capital and operating costs. The results can be safer, more energy-efficient, and environmentally sustainable manufacturing processes. For example, Gerben Zijlstra (senior scientist in R&D at DSM Biologics) will report at the BPI Conference this year on his company’s XD process culture strategy, with viable cell densities >150 million cells/mL achieved by Chinese hamster ovary (CHO) and other cell lines in a combination fed-batch/perfusion process, yielding up to 27 g/L of high-quality product.

To compete in an increasingly cost-conscious world, the biopharmaceutical industry is reinventing itself. Product development and manufacturing technologies are critical strategic elements in this process. A process begins with the design and culture of recombinant cells expressing therapeutic proteins. Maintaining product quality and optimizing production through all phases of drug development is a challenge. Quality by design (QbD) offers a solution through a design space with predetermined ranges for each parameter tested to ensure acceptable process performance — and ultimately, product quality.


Cell Line Engineering Improves Product Yield and Quality

Timelines, throughput, and product quality are vitally important to biopharmaceutical companies. With increasingly diverse portfolios of therapeutic proteins, more efficient methods for creating and screening high-expressing stable cell clones are coming into use. Screening clones that express different proteins helps companies identify the best combinations for development. As Medimmune scientist Gareth Lewis will report at the BPI Conference, for example, shaking microwell and microbioreactor scale-down models can facilitate clone selection for increased predictability in bioprocessing.

With money in short supply, companies want to determine the manufacturability of biological product candidates early on. After evaluation of the drug-like properties of a given protein comes development of a production cell line. Significant progress has been made in recent years toward optimizing biopharmaceutical production in cell culture systems. Most effort has focused on improving host cell lines and expression engineering, including optimized selection processes that reduce development times as well as improving yields to ultimately improve productivity. There is growing emphasis on understanding the relationship between protein characteristics and clinical significance (e.g., biological activity, immunogenicity, and pharmacokinetics). So companies are interested in understanding and manipulating cellular mechanisms that can control product quality.

Cell line engineers modulate cellular pathways through genetic manipulation, using genomic/proteomic/metabalomic research to understand cellular mechanisms of product synthesis and secretion. Process engineers manipulate the physicochemical culture environments to control the characteristics of the recombinant product expressed. Case studies at the BPI Conference will demonstrate how companies are integrating information to improve the quality of protein therapeutics as well as expression yields.

For example, David Kocisko (principal scientist at Alnylam Biotherapeutics) will show how RNA interference technology can help reduce the presence of other proteins that affect quality, activity, and specific productivity of a therapeutic protein. He reports that 1-nM siRNA added directly to a 40-L CHO culture reduced levels of a selected protein >80%. This approach should be amenable to existing cell lines, in which the expression of multiple proteins can be reduced simultaneously.

Implementing Novel Media Development and Feed Strategies

The late-1990s “mad cow disease” scare catalyzed the transformation of cell culture processes from using old-school serum-based media to newer, better characterized alternatives. Now biopharmaceutical companies are producing higher protein titers using what once was considered inadequate (if at all possible): protein-free and/or chemically defined media without animal-sourced ingredients. Supplementation of cell culture media with plant-sourced hydrolysates has replaced serum to increase cell viability and productivity of recombinant biotherapeutics.

Although they represent a better option than animal-derived serum, hydrolysates are still a biologically based raw material that can introduce variability and certain risk to an already complex bioprocess. As Masaru Shiratori (a late-stage cell culture engineer at Genentech, Inc.) will show at the BPI Conference, even eliminating such additives may be possible in biopharmaceutical production. And Elizabeth Dodson (bioanalytical chemistry manager for bionutrients R&D at BD Biosciences) will report on her team’s deconstruction of hydrolysates using chromatography, peptide sequencing, and other biochemical techniques for high-resolution analysis by mass spectrometry to identify their unique components. Design of experiments approaches to those identified molecules led to formulation of a chemically defined supplement that can provide the same cell productivity properties of a hydrolysate for mammalian cell culture without its inherent variability.

Maintaining quality and product comparability while maximizing productivity creates challenges in bioprocess development, whether for innovators or makers of biosimilar products. Many companies are now investigating the effects of culture media on cell performance and product quality. Media composition affects cell growth, productivity, and posttranslational modifications. Zhaohui Geng (principal scientist in cell process development at Pfizer Biotherapeutics) will report on work in this area at the BPI Conference.

Perfusion cell culture processes are gaining in popularity throughout the biopharmaceutical industry. Perfusion cultures can reach very high cell densities in a short time. As Robel Tezare (senior research associate in pharma technical development at Genentech) will report at the BPI Conference, inoculum train culture operated in perfusion mode provides the option of inoculating batch and fed-batch production cultures at higher seeding densities. That in turn can increase titers and/or speed production.


Wednesday, 22 September 2010
Integrating In-Line Process Monitoring and Control Technologies in Upstream Processing

Thursday, 23 September 2010
Plenary Session: Integration of Upstream and Downstream Processing

Accelerating and Optimizing Cell Line and Process Development

Host Cell Engineering to Improve the Yield and Quality of Biotherapeutics

Implementation of Novel Media Development and Feed Strategies

Friday, 24 September 2010
What Comes Next after Titer Increase?>
An Industrial View of Biopharmaceutical Comparability and Characterization

Approaches to Improve Product Quality and Achieve Process Optimization

Advantages of Using Mixed Mode Technologies
Overcoming Challenges of Producing Specific Proteins

At 8 AM on Thursday 23 September 2010, the BPI Conference will offer a plenary session titled “Integration of Upstream and Downstream Processing,” chaired by Konstantin Konstantinov (vice president of technology development for Genzyme Corporation). Presenters will discuss challenges and efficiencies gained by integrating process development (Gene Schaefer, senior director of large-molecule API development at Johnson & Johnson), optimizing interfaces and hand-offs (Jens H. Vogel, global CMC development team leader and head of isolation and purification in global biological development at Bayer HealthCare), and linking upstream and downstream (Jonathan Coffman, principal engineer III for Pfizer Biotherapeutics).

“Historically, perfusion processes have been primarily used to culture mammalian cells producing unstable molecules,” Chetan T. Goudar (head of cell culture development for Bayer HealthCare’s global biological development) told me. “Bioreactor residence time is a critical variable for such products, and this constraint can be elegantly met in a perfusion system through perfusion rate optimization. However, the advantages of perfusion cultivation extend far beyond residence time optimization and include the ability to reach very high cell densities, high culture viabilities and stable bioreactor operating conditions. Some of these attributes will be addressed at the BPI Conference by speakers from multiple companies.”


Anthony S. Lubiniecki (senior fellow in CMC strategy for large-molecule portfolio management at the Janssen Pharmaceutical Companies of Johnson & Johnson) will offer a presentation on Friday 24 September 2010 at 10:15 AM: “An Industrial View of Biopharmaceutical Comparability and Characterization.”

His presentation abstract says, “Process and product changes (including manufacturing site, container–closure, and analytical changes) are an inevitable part of biopharmaceutical development and have been made for every product currently on the market. Comparability studies are typically performed to assess whether the change(s) is likely to affect product safety and efficacy. Most of these have positive outcomes, but sometimes the results require additional nonclinical or clinical studies. Strategies for management of comparability study risks will be reviewed.”

BPI: Your topic is product comparability and characterization. What places it into the Cell Culture and Upstream track?

ASL: Most process changes that lead to comparability studies and physicochemical changes to the product molecules being detected in comparability studies occur in the upstream phase of manufacturing.

BPI: Do process transfers across borders present greater challenges when it comes to comparability?

ASL: No. Transfers across the street can be just as problematic as those across borders. Those transfers involving organizations with little/no previous experience in biologicals pose special problems, whether borders are involved or not.

BPI: Recent news suggests that large multinational companies already involved in biomanufacturing are likely to be the leaders in making biosimilars. Is this partly because of their proven expertise — in, for example, product characterization?

ASL: Yes

BPI: What brings you back as a presenter and attendee? What do you think of the location this year? Is there anything you’re particularly looking forward to at the 2010 meeting?

ASL: It is a good meeting, and I have not been able to attend for a few years due to other duties, so I am looking forward to it.

Goudar is chairing a session on mixed-mode technologies. The benefits of continuous processing can be offset by its complexity. In Goudar’s session, Timothy Johnson (senior bioengineering process development manager at Genentech) will discuss the current state of the art as well as special considerations necessary to achieve robust manufacturing based on perfusion technology. And Kesav Reddy (associate upstream process development scientist at CMC Icos Biologics) will describe different approaches for rapid development and evaluation of fed-batch and perfusion processes in parallel. “Crosstalk” between the two approaches helps his team improve productivity and product quality.

Automation, Process Monitoring, and Control Technologies

The first element in a product’s lifecycle under QbD is development of a cell culture process that can deliver therapeutic proteins with defined and acceptable critical quality attributes (CQAs). Maintaining performance and improving productivity and efficiency become key challenges that require the most current and relevant data. Process monitoring and control must become routine. Processes based on animal cell culture in particular currently require labor-intensive operator effort in monitoring and maintenance of culture conditions as well as data compilation. The dawning era promises to bring automation to their rescue.

Biosensors are already finding application in monitoring cell growth and cell metabolism. Li Malmberg (director of technical operations in biologics manufacturing at Abbott Laboratories) will report at the BPI Conference on his team’s successful implementation of biosensors in production reactors. When compared with off line measurements, their results were shown to be more consistent. The on-line sensors enabled tighter process control and reduced variability, improving process robustness.

Process and product changes are an inevitable part of each biotech product’s lifecycle, and according to Anthony S. Lubiniecki (senior fellow in CMC strategy for large-molecule portfolio management at Johnson & Johnson’s Janssen Pharmaceutical Companies), they have been made for every biopharmaceutical on the market. Comparability studies are typically performed to assess whether a change(s) is likely to affect product safety and efficacy. Most have positive outcomes, but sometimes the results require additional nonclinical or clinical studies. Lubiniecki will present strategies for management of comparability study risks at the BPI Conference.

Current bioreactor monitoring strategies rely on a limited set of process variables as proxies for cell physiology. Often such information is insufficient and disconnected from real process outcomes. Karthik P. Jayapal (a cell culture process development scientist at Bayer HealthCare) will discuss the use of “-omics” tools to study intracellular mRNA, protein, and metabolite concentrations that are more likely to reflect a cell’s true physiology. Such tools could be applied to more precisely define good cell culture processes.

Overcoming Challenges of Specific Proteins

Use of platform production formats to reduce resource requirements for early stage clinical biologic development is now common practice. Platforms have proven themselves effective for antibodies and certain other protein families. But the one-size-fits-all approach lacks flexibility, so it is not the right tool for every job. Proteins are complex biomolecules, and their complexity allows for great variation — in structure, function, stability, and preferred conditions.

Case studies presented at the BPI Conference will address the challenges of some recalcitrant products. For example, alternative scaffolds are small stable proteins often made through bacterial expression. Subina
y Ganguly (associate director and CMC team leader for Johnson & Johnson’s Redscript Ventures) will report on development of a scaffold his team is producing at high levels in a soluble form without periplasmic expression or secretion. Certain process modifications are required to ensure a homogeneous product.

A manufacturing scientist at BioMarin Pharmaceutical, Guru R. Thuduppathy, will describe his company’s production enzyme replacement therapies using perfusion cell culture. With experience gained from over six years of commercial Naglazyme manufacturing, he will discuss the differences between perfusion and fed-batch cell culture and summarize differences between the manufacture of enzymes and antibodies.

And Yao-ming Huang (senior engineer III in cell culture development at Biogen Idec) will describe his company’s application of a platform process to the production of a small protein therapeutic. Yield improvements are possible through optimizing process parameters and feed strategies, but critical product quality attributes may be affected.

As biotherapeutic production shifts into the fast lane, cell line and process engineers have become invaluable to manufacturing production and drug development. In response to a perceived capacity crisis, the first decade of the 21st century brought rapid advancements in expression titers and bioreactor technology. Science solved a business problem. Our new challenges are financial — and the same people are turning their formidable talents toward those as well.

We asked presenters about their experiences with the BPI Conference series and what they were looking forward to this year.

Subinay Ganguly (associate director and CMC team leader for Johnson & Johnson’s RedScript Ventures) is presenting, “Protein Engineering of an Alternative Scaffold for Improved Development and Production” on Friday 24 September 2010 at 3:30 PM.

“I have not presented at the BPI conference before, but I attended the last time in 2004. However, my group members have been attending and presenting all along. I’ve presented at the First European IBC Cell Line Development and Engineering meeting in 2005. This year, I’m looking forward to learning more about bioprocess development of complex proteins that are not monoclonal antibodies in nature.”

About the Author

Author Details
Cheryl Scott is senior technical editor of BioProcess International.


1.) Abts, H, and S. Arain. 2008. Process Monitoring in Suspension-Adapted CHO Cell Cultures: Noninvasive Online Detection of pH and Oxygen. BioProcess Int. 6:64-66.

2.) Anicetti, V. 2009. Biopharmaceutical Processes: A Glance into the 21st Century. BioProcess Int. 7:S4-S11.

3.) Carrier, C, L Donahue-Hjelle, and MJ. Stramaglia. 2009. Banking Parental Cells According to CGMP Guidelines: A Practical Approach to Developing Stable Cell Lines. BioProcess Int. 7:20-25.

4.) Clarke, HRG, and BJ. Compton. 2008. Comparing Mammalian Expression Systems: The First Rate-Limiting Step in Making Products for Clinical Testing. BioProcess Int. 6:S24-32.

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

6.) De Jesus, MJ, and FM. Wurm. 2009. Medium and Process Optimization for High Yield, High Density Suspension Cultures: From Low Throughput Spinner Flasks to High Throughput Millilitre Reactors. BioProcess Int. 7:S12-S17.

7.) Fike, R. 2009. Nutrient Supplementation Strategies for Biopharmaceutical Production, Part 1: Identifying a Formulation. BioProcess Int. 7:44-51.

8.) Fike, R. 2009. Nutrient Supplementation Strategies for Biopharmaceutical Production, Part 2: Feeding for Optimal Recombinant Protein Production. BioProcess Int. 7:46-52.

9.) Gerber, MA. 2008. Integrated Strategies for Clone and Media Formulation Selection. BioProcess Int. 6:58-63.

10.) Gryseels, T. 2008. Considering Cell Culture Automation in Upstream Bioprocess Development. BioProcess Int. 6:12-16.

11.) Idusogie, EE. 2008. Development of an Antibody Screening Assay for Selection of Production Cell Lines. BioProcess Int. 6:20-33.

12.) Jain, S, BM Schilling, and A.A. Shukla. 2008. Determining the Effect of Raw Materials on Manufacturing-Scale Cell-Culture Performance: An Application of Mechanistic Mathematical Models. BioProcess Int. 6:36-39.

13.) Liu, X. 2010. Isolation of Novel High-Osmolarity Resistant CHO DG44 Cells After Suspension of DNA Mismatch Repair. BioProcess Int. 8:68-76.

14.) Mardirosian, D. 2009. Scaling Up a CHO-Produced Hormone–Protein Fusion Product: From Robotic Optimization to Large-Scale, Single-Use Stirred-Tank Bioreactors. BioProces Int. 7:S30-S35.

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

16.) Peppers, S. 2009. DoE Helps Optimize a Cell Culture Bioproduction System. BioProcess Int. 7:S24-S27.

17.) Rader, RA. 2008. Expression Systems for Process and Product Improvement: A Perspective on Opportunities for Innovator and Follow-On Product Developers. BioProcess Int. 6:S4-S9.

18.) Rosin, L. 2008. Moving On in Cell Culture: Flow Cytometry and Alternative Expression Systems. BioProcess Int. 6:76.

19.) Salmén, A. 2009. Efficient Development of Stable High-Titer Cell Lines for Biopharmaceutical Manufacturing: Automation Increases the Efficiency and Reliability of the Development Process. BioProcess Int. 7:34-39.

20.) Schmidt, J. 2008. Monitoring ATP Status in the Metabolism of Production Cell Lines. BioProcess Int. 6:46-54.

21.) Schwertner, D, and M. Kirchner. 2010. Are Generic HCP Assays Outdated?. BioProcess Int. 8:56-61.

22.) Scott, C, and L. McLeod. 2010. The Time Has Come for Automation in Bioprocessing. BioProcess Int. 8:16-25.

23.) Seamans, TC. 2008. Cell Cultivation Process Transfer and Scale-Up in Support of Production of Early Clinical Supplies of an Anti IGF-1R Antibody, Part 1. BioProcess Int. 6:26-36.

24.) Seamans, TC. 2008. Cell Cultivation Process Transfer and Scale-Up in Support of Production of Early Clinical Supplies of an Anti IGF-1R Antibody, Part 2. BioProcess Int. 6:34-42.

25.) Simula, T, S Grosvenor, and C. Scott. 2009. Rethinking Media Performance: Optimizing with Defined, Animal-Free Supplements. BioProcess Int. 7:48-59.

26.) Tsuji, S. 2010. An Efficient Thermoinducible Bacterial Suicide System: Elimination of Viable Parental Bacteria from Minicells. BioProcess Int. 8:28-40.

27.) West, L. 2008. Automated Closed-Loop Solution for Bioreactors and Fermentors: Redefining Automation in Process Development, Pilot, and Manufacturing Applications. BioProcess Int. 6:62-64.

28.) Whitford, WG, and JJS. Cadwell. 2009. Interest in Hollow-Fiber Perfusion Bioreactors Is Growing. BioProcess Int. 7:54-63.

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