BioProcess Theater: Cell-Line and Process Development

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7604416Process development forms the core of BioProcess International’s coverage and interests. From cell-line engineering through seed-train and production cultures, the results of which are harvested and purified to yield a bulk drug substance, every biologic therapy and vaccine begins its life at small scale in a research and development laboratory.Cell lines may be mammalian, insect, microbial, or even plant-sourced (1). They all require different types of culture media and supplements. And purification options are many and diverse. Process intermediates are collected and stored along the way, as are final bulk substances ready for formulation into drug products.

Whatever variations a specific product requires of its biomanufacturing process, all biologics must go through a series of well-documented stages with increasing regulatory stringency. Under the 21st-century regulatory paradigm, bioprocesses must be engineered to produce high-quality products by design. New technologies such as single-use systems and components, continuous processing options, and design of experiments are helping process engineers rise to that challenge through process optimization.

Protein Expression

Animal Cells: Mammalian cell lines — especially from Chinese hamster ovary (CHO) cells — are the dominant expression system in biopharmaceutical manufacturing (2). They can express correctly formed human proteins with complicated posttranslational modifications that are essential to their biological function. Insect cells have utility especially in vaccine production by means of the baculovirus expression vector system (BEVS) for transient protein expression. New viral vaccines are being made using the BEVS, although influenza vaccine makers are transitioning from egg-based methods of virus production to culture of mammalian cells. Some amazing scientific and technological advance- ments have led to high-expressing animal cell lines and culture systems (3). For example, cell-line engineering has taken monoclonal antibody (MAb) expression titers from hundreds of milligrams to several grams per liter of culture through a number of mechanisms. Selection markers and clone-picking instruments help developers identify the best cells to passage into master and working cell banks. Transient transfection in mammalian cells speeds early development by quickly making enough product to test (4). And small-scale studies help optimize the best growth media and supplements for healthy, productive cultures.

Microorganisms: Microbial fermentation for protein production has also been improved in the 21st century. Escherichia coli is the most prominent expression system after CHO cells (5). But some up-and-coming options include Pseudomonas fluorescens. Yeast expression platforms are also attractive because of their well-defined genetic properties, ease of genetic manipulation, and simple fermentation processes that promote rapid growth rates. As eukaryotes, they can perform some posttranslational modifications that other microbes cannot.

Culture Media and Supplements: No matter what kind of cells are being cultured, it’s best for regulatory compliance to use serum-free, protein-free, chemically defined, or other types of media that do not come from animal sources. This provides several advantages in bioprocessing: defined composition, reduced contamination risk, and lower costs. But these culture media often require supplementation with special ingredients that have particular functions as cell nutrients. Media were supplied in liquid form early in biotech’s history, but over time as companies scaled up to larger facilities, powdered media grew in popularity. Special media preparation efforts were worth the space savings in storage. However, in recent years more companies are going back to liquid media supplied on demand — particularly as upstream improvements make it possible to produce more protein in smaller cultures (6, 7).

Production Hardware

A high-quality product begins with efficient upstream process equipment. Whether glass, stainless steel, or polymer-based, bioreactors are the core of the production suite. In recent years, we’ve seen the full acceptance of disposables here, especially for seed-train and smaller-scale production. And with increasing expression titers and culture densities, extremely large-scale equipment may be a thing of the past. Most plants today combine sophisticated single-use components with stainless steel equipment. Biotech facilities of the future appear poised to have smaller footprints with flexibility and “just-in-time” production in mind.

The classic modes of bioreactor system operation (batch and fed-batch) are still going strong. But more and more companies are developing methods of continuous and perfusion processing. Single-use technologies is assisting here as well. And many suppliers are continuing to refine their single-use approaches even now.

Process Monitoring and Control: Development and optimization of cell culture processes requires powerful technologies for process monitoring. And quality by design (QbD) requires reliable process control. Methods based on near-infrared (NIR) spectroscopy and radiofrequency impedance sensors and probes are well established for measuring many parameters (some off-line, some on-line). Here, single-use technologies have actually complicated matters, but instrument suppliers are innovating with QbD in mind. Sensor technology has evolved from probes to single-use optical sensors. And to make use of all their data, automation is making inroads for process control.

Cell Therapy Systems: Human therapeutic cells are not like the immortalized cell lines used for protein production. Very few can be adapted to suspension culture, so most need a surface on which to attach themselves and grow. Because of that, cell therapy culture vessels may be scaled out (with more small batches in parallel) rather than up (larger batches in bigger vessels). That can greatly increase production costs because it requires more equipment with increased handling and automation. Microcarriers can allow for cell expansion in suspension culture, however, and many other options are in the works for this still nascent segment of the biotech industry (8–12).

Cell-Line and Process Development Companies

Here are some companies offering biologic development products and services that will be part of the BioProcess Zone at the 2015 BIO International Convention.
Contract Development and Manufacturing Organizations
AAI Pharma
Ajinomoto Althea
Aragen Bioscience
Aseptic Technologies
Catalent Pharma Solutions
Charles River Laboratories International
Cobra Biologics
Cytovance Biologics
Emergent BioSolutions
Fujifilm Diosynth Biotechnologies USA
Gallus BioPharmaceuticals
Goodwin Biotechnology
Integrity Bio
Kemwell Biopharma
Lampire Biological Laboratories
Masy Systems
Phage Consultants
Piramal Pharma
Pyramid Laboratories
Samsung Biologics
Therapure Biopharma
Equipment and Materials Suppliers
Ajinomoto Small Pack
American Plastics Technologies
Diba Industries
DV Biologics
EMD Millipore
Emerson Process Management
Meissner Filtration Products
Puritan Products
Red Bag Solutions
Spectrum Laboratories
Worthington Biochemical

Here are some companies offering biologic development products and services that will be part of the Discovery Services Zone at the 2015 BIO International Convention:
Bioneer Corporation
BioXcel Corporation
Contamination Prevention Services
Menarini Biotech
MFG Tray Company

Here are some companies offering biologic development products and services that will be part of the Contract Services Zone at the 2015 BIO International Convention: Advantar Labs
Avid Bioservices
Bachem Americas
Coldstream Laboratories
Cook Pharmica
Germfree Laboratories
GlaxoSmithKline Biopharmaceuticals
Jubilant HollisterStier
KBI Biopharma
LSNE Contract Manufacturing
Lyophilization Technology
Paragon Bioservices
PolyPeptide Group
WuXi AppTec

Harvest and Clarification

Clarification of the reactor “soup” full of cellular debris and process impurities is the first step in a downstream process. It can involve significant risk of product degradation, bioburden concerns, or process errors — and sometimes requires mechanical cellular disruption for microbial products that aren’t secreted.

Harvesting methods and equipment are chosen based on the type of cells, product being harvested, and process fluid properties. Traditional techniques include membrane microfiltration, tangential-flow filtration (TFF, or crossflow filtration), centrifugation, and depth filtration, as well as specialized solutions coupling either microfiltration or centrifugation with TFF or depth filtration.

The trend toward smaller-scale batches is changing primary recovery requirements (13). Some new solutions include glass-fiber and polyethersulfone membranes. Some companies are looking at old technologies in new ways (e.g., diatomaceous earth filtration), and others are considering novel ideas such as chromatin-directed clarification (14–16).

As titers have increased, so too has the importance of efficient product recovery. Clarification of high-density cell cultures presents unique challenges. Ever-increasing amounts of cell debris need to be removed quickly to prevent process bottlenecks. This is driving increased interest in perfusion systems and continuous processing.


Liquid chromatography’s power to separate molecules in solution based on their size, electrical charge, or other physicochemical properties makes it the cornerstone of every biopharmaceutical downstream process. This is one technology that has been problematic for conversion from reusable to single-use formats. Glass or stainless steel columns are regenerated a number of times to offset their cost, as are the separation media packed inside them — which is good because they can be very expensive depending on their construction and chemistry. But a growing number of vendors are offering disposable, prepacked columns, and automation is finally becoming a reality for packing and regeneration of columns as well as running chromatographic operations (17, 18).

Protein A and Beyond: When it comes to expensive media, the most notorious (and celebrated) are protein A affinity resins. Because of the strong affinity of antibodies for binding protein A, it is so useful that despite its cost, high-yield protein A affinity capture is often the first in line for a MAb purification platform.

Even so, the associated expense combined with engineering and practical difficulties is making some process developers in this era of high-titer production wonder whether some alternative might be a better choice than protein A (19–22). Others are simply investigating ways to use it more efficiently (22, 23). Meanwhile, chromatographers and their suppliers haven’t stopped improving these technologies. New media are always arriving on the scene, with multimodal and multicolumn approaches showing particular promise (24, 25).


Filters are not only used as downstream unit operations in themselves, but also in support of nearly every other step in bioprocessing, from incoming air and feeds for cell culture operations to cleaning the circulating media in perfusion processes, harvest clarification, and removing buffers from chromatographic eluate. Filters are used in formulation work and in fill–finish, as well as in environmental monitoring and process waters. And they have expanded their utility even more with the advent of membrane adsorbers. And sterile filtration is particularly indispensable in viral safety and Mycoplasma removal (26, 27).

Filter cartridges were among the first disposable items used in bioprocessing. Their manufacturers led the charge for single use technologies. Disposable membrane sorbers combine typical filtration action with chromatographic binding ability (27–29). Available chemistries cover the range of liquid chromatography formats familiar to bioprocessing from ion exchange to reverse-phase to hydrophobic interaction — and even some affinities. Lately, their adoption is expanding as vendors have addressed early challenges (28).

Related Educational Sessions

A number of educational program tracks at the 2015 BIO International Convention
might appeal to those involved in clinical and commercial manufacturing: Next-
Generation Biotherapeutics; Oncology, Orphan and Rare Diseases, Personalized
Medicine and Diagnostics, Primary Care/Chronic Diseases, Infectious Diseases,
Intellectual Property, Regulatory Science, and Translational Research. Some sessions
stand out in particular:
Next Generation Biotherapeutics
“Nucleic Acid and Cell Therapy: Insights, Successes and Challenges” (10:15–11:45 am 16 June 2015, Room 107AB)
“Harnessing the Power of Antibodies” (2:00–3:15 pm 16 June 2015, Room 107AB)
Orphan and Rare Diseases
“Achieving Successful Marketing Authorization for Orphan Products: Experiences from
Both Sides of the Atlantic” (2:00–3:15 pm 17 June 2015, Room 108A)
Personalized Medicine and Diagnostics
“The Future Is Now in Precision Medicine: Developing Targeted Oncology
Therapeutics” (10:15–11:30 am 17 June 2015, Room 105AB)
Infectious Diseases
“Global Emergency Case Study: Ebola” (10:15–11:30 am 17 June 2015, Room 113A)
“After Ebola, What’s Next — and What Should Be Done Now?” (9:00–10:15 am 18 June 2015, Room 113A)
“China and Novel Vaccine Development for Emerging Markets” (10:30–11:15 am 18
June 2015, Room 113A)
Intellectual Property
“Antibody Therapeutics: May I Have Them All?” (10:15–11:30 am 16 June 2015, Room 109AB)
“The Impact of USPTO Inter Partes Review Proceedings on (Bio)Pharma” (2:00–3:15 pm 16 June 2015, Room 109AB)
“Celebrating Innovation: Retelling the IP Story of Crixivan, an Award-Winning Life
Saver” (3:30–4:45 pm 16 June 2015, Room 109AB)
“The Evolving Landscape of Patentable Subject Matter” (9:00–10:00 am 17 June 2015, Room 109AB)
“Inventions Patentable: Evaluating Proposed Amendments to Section 101” (10:15–
11:30 am 17 June 2015, Room 109AB)
“Patentable Subject Matter Around the World: Life Science Inventions in Canada,
Europe, China, Australia and New Zealand” (2:00–3:15 pm 17 June 2015, Room 109AB)
“IP Sponsor” (3:30–4:45 pm 17 June 2015, Room 109AB)
Regulatory Science
“Global Regulatory Trends for Biosimilars and Biotherapeutics” (10:15–11:30 am 17
June 2015, Room 106AB)


1 Marconi PL, et al. Mathematical Model for Production of Recombinant Antibody 14D9 By Nicotiana tabacum Cell Suspension Batch Culture. BioProcess Int. 12(1) 2014: 42–49.

2 Martin EM. How an Obscure Asian Rodent Took Over Biotechnology. BioProcess Int. 10(8) 2012: 76.

3 Rader RA, Langer ES. 30 Years of Upstream Productivity Improvements. BioProcess Int. 13(2) 2015: 10–14.

4 Agrawal V, et al. A High-Yielding, CHO-K1–Based Transient Transfection System: Rapid Production for Therapeutic Protein Development. BioProcess Int. 11(1) 2013: 28–35.

5 Pandiripally V, Lee J, Rader RA. A Novel Solid-Media E. coli Platform: Comparison with Standard Fermentation Processes. BioProcess Int. 12(8) 2014: 80–82.

6 Hudson J. Liquid or Powdered Media? BioProcess Int. 11(4) 2013: 18–23.

7 Langer ES, Rader RA. Powders and Bulk Liquids Economics of Large-Scale Culture Media and Buffer Preparation are Changing. BioProcess Int. 12(3) 2014: 10–16.

8 Simón M. Bioreactor Design for Adherent Cell Culture: The Bolt-On Bioreactor Project, Part 1: Volumetric Productivity. BioProcess Int. 13(1) 2015: 28–33.

9 Janas ML. Perfusion’s Role in Maintenance of High-Density T-Cell Cultures. BioProcess Int. 13(1) 2015: 18–26.

10 Szczypka M. Single-Use Bioreactors and Microcarriers: Scalable Technology for Cell- Based Therapies. BioProcess Int. 12(3) 2014: 54–64.

11 Hambor JE. Bioreactor Design and Bioprocess Controls for Industrialized Cell Processing. BioProcess Int. 10(6) 2012: 22–33.

12 Hemmerich J, Kensy F. Automation of Microbioreactors: Operating 48 Parallel Fed- Batch Fermentations at Microscale. BioProcess Int. 11(8) 2013: 68–76.

13 LeMerdy S. Evolving Clarification Strategies to Meet New Challenges. BioProcess Int. 12(9) 2014: insert.

14 van der Meer T. Diatomaceous Earth Filtration Innovative Single-Use Concepts for Clarification of High-Density Mammalian Cell Cultures. BioProcess Int. 12(8) 2014: S25–S28.

15 Schreffler J, et al. Characterization of Postcapture Impurity Removal Across an Adsorptive Depth Filter. BioProcess Int. 13(3) 2015: 36–43.

16 Gagnon P. Emerging Challenges to Protein A: Chromatin-Directed Clarification Enables New Purification Options. BioProcess Int. 11(9) 2013: 44–52.

17 Quinlan A. Advances in Chromatography Automation. BioProcess Int. 13(1) 2015: 16–17.

18 Chollangi S, Jaffe NE, et al. Accelerating Purification Process Development of an Early Phase MAb with High-Throughput Automation: Part 1 of a Technical Report. BioProcess Int. 12(3) 2014: 48–52.

19 Lain B. Protein A: The Life of a Disruptive Technology. BioProcess Int. 11(8) 2013: 29–28.

20 Gagnon P, et al. IgM Purification with Hydroxyapatite: A Primer. BioProcess Int. 12(2) 2014: 40–51.

21 Champagne J, et al. A Salt-Tolerant Anion-Exchange Chromatography Sorbent for Flexible Process Development. BioProcess Int. 11(6) 2013: 50–57.

22 Franzreb M, Müller E, Vajda J. Cost Estimation for Protein A Chromatography: An In Silico Approach to MAb Purification Strategy. BioProcess Int. 12(9) 2014: 44–52.

23 Holstein M, Cotoni K, Bian N. Protein A Intermediate Wash Strategies. BioProcess Int. 13(2) 2015: 56–62.

24 Müller-Späth T, et al. Purifying Common Light-Chain Bispecific Antibodies: A Twin-Column, Countercurrent Chromatography Platform Process. BioProcess Int. 11(5) 2013: 36–44.

25 Ulmer N, et al. Affinity Capture of F(ab’)2 Fragments: Using Twin-Column Countercurrent Chromatography. BioProcess Int. 13(2) 2015: 22–29.

26 Genest P, et al. Artifacts of Virus Filter Validation: Best Practices to Maintain the Scaled-Down Model. BioProcess Int. 11(5) 2013: 54–60.

27 Metzger M, et al. Evaluating Adsorptive Filtration As a Unit Operation for Virus Removal. BioProcess Int. 13(2) 2015: 36–44.28 Langer ES. Industry Adoption of Membrane Adsorbers: Fastest-Growing Little Segment in Bioprocessing. BioProcess Int. 11(9) 2013: 16–19.

29 Han XJ, et al. Performance of a Salt-Tolerant Membrane Adsorber in Flow-Through Mode. BioProcess Int. 11(2) 2013: 28–39. c

Cheryl Scott is cofounder and senior technical editor, and Maribel Rios is managing editor of BPI.

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