Great strides have been made in fed-batch culture and feed strategies with new tools and strategies introduced regularly. In addition to improving cell growth and viability, our optimization focus has grown to include strategies for influencing protein quality, such as glycosylation profiles. This article and accompanying interview will explore current feed design strategies and will look ahead to the future of fed-batch culture.

Introduction

Fed-batch culture as a mode of production developed rather gradually and organically. Years ago, engineers noticed that cultures depleted media of the main carbon sources, glucose and glutamine. To extend culture life, they added more of each, initially in a timed schedule but eventually in closed-loop (feed-back) based control. However, concern soon grew regarding concomitant production of the by-products lactate and ammonia having a negative effect on pH and osmolality, and consequently upon cell activity, production and product quality. This led to the development of new approaches for controlling this rational supplementation of glucose and glutamine in a fed-batch approach, and the use of alternative carbon sources.

Since those early days, process developers have been able to measure many more primary and secondary metabolites. They can also now correlate these requirements to more features, such as culture vitality, peak viable cell density and culture longevity. Also, it has been observed that many of the required additions are most beneficial when added at different times, periods and concentrations. All this, and the fact that component grouping allows higher stock concentrations, has caused most operators to employ several feeds added at different times and rates of addition.   So now, we employ recombinant null clones with beneficial metabolism, advanced media and feed designs providing additional or alternative carbon and nitrogen sources, along with an improved process control in the feeding process design.

Feed strategies can be developed in-house, through development partnerships or many media suppliers are now offering off the shelf media and feed combinations that permit a more simplified optimization. In these situations, a base media is offered with a selection of different feed options. Engineers can then conduct a smaller Design of Experiments (DoE) to test the base media with the different feeds offered. This reduces the amount of screening that needs to be done to find a media and feed that works well for a particular cell line.

Current feed optimization strategies and future development opportunities

I was recently able to interview William Whitford, an expert in the field of media development and feed design about current feed optimization strategies and future directions. I have included the transcript of our interview below.

What are the current methods employed in developing feeding strategies?

Process developers now have a much better understanding of such coarse and fine product expression regulators as transcription factors, enzyme binding proteins, metabolite transporters, metabolic flux and secondary pathways. Concurrently the costs of genomic sequencing, ‘omics data generation, and computing resources are decreasing rapidly. The resulting increased knowledge has been powerfully combined with high-throughput screening assays and DoE designs in small-scale multi-well studies.

One example of a powerful new method was introduced last fall: dynamic flux balance analysis (DFBA), was employed to examine the relationship of media supplementation with amino acids, increased product yield, extended growth phase and increased cell density. The study showed that culture feeding could support greater culture performance while not changing, including negatively, the overall metabolism of the cells or product glycosylation. DFBA also showed, rather counterintuitively to fed-batch designers, that the metabolic state varies more at the beginning of culture, and less by the middle of culture [Reimonn].

Another advancement is that final development work can now be accomplished using automated cell-free sampling and multiplexed at-line and operator-independent analytics in benchtop bioreactors. This includes popular membrane and non-membrane-based multi-analyte analyzers as well as newer NIR, Raman, and 2D-fluorescence. Being able to model these complex operations at the bench permits a better understanding of large scale manufacturing with more efficient methods.

In addition to a better understanding of the cellular biology, operators now know more about the specific characteristics of their bioreactors. Through such techniques as CFD and MS, engineers understand peak sheer forces and the mass transfer coefficient and capacity of their bioreactors in application. Such advances in process analytical technologies and bioreactor CPP have synergised with developing computational power and data management infrastructures.

Finally, feed-forward model-based controls supporting automated control loops are changing how efficiently we can use all that information. In fact my company, GE Healthcare, is moving forward rapidly toward this state in biomanufacturing applications of their Digital Twin . The result of all this is that operational efficiency is improved, product quality becomes more consistent, data management, transfer and reporting becomes automatic, and previously unmanageable procedures and operations can be entertained.

How do product quality attributes figure into feed strategy design and implementation?

We just spoke of improving culture vitality and longevity, but biopharmaceutical products have numerous quality attributes that can potentially impact safety and/or efficacy of the product. Such attributes as protein aggregates, biological potency, pharmacokinetics and immunological activity can vary due to consequences of culture conditions.

As more structure-function relationships are established, product structural components’ becoming critical to control include post-translational (PT) modifications. These features include glycosylation, charge isoforms, phosphorylation, oxidation, amidation, free sulfhydryl and disulphide bridge structure, isomerisation, fragmentation, and glycation, acetylation and methylation. We’ve known for years that the relative levels of some forms can be modulated by actively manipulating the process conditions, including the metabolites and co-factors, in the production media. Now, both entity sponsors and biotechnology material/service suppliers are employing a more comprehensive, “enhanced approach” to process development. Here, a better understanding of both product quality attributes and cell biology support advanced process design and control strategies.

Levels of particular glycans on the product have been linked to the modulation of critical intermediate’s biosynthesis caused by variations in the extracellular level of such simple components as glutamine. Under different culture conditions, some glycan levels can also vary from the effect of such waste products as ammonia upon glycosyltransferases and transporter molecules. Levels of even extracellular glucose and glutamine have been found affect critical processing enzymes’ intracellular availability [Fan]. More recently researchers from GE demonstrated that the distribution of acidic charge variants could be influenced by adjusting the process time, media pH and the level of such culture media components such as identified sugars and iron chelates [Björkman].

Because such PT features as glycan structures are the product of many intracellular enzymes, it is difficult to understand the effects genetic manipulations or nutrient additions on glycan structures. Some have built quantitative models of the process to be able to predict the relationship of culture media components to product glycosylation. Model-based results can even predict glycosylation features of the cell lines not previously published [Frederick J. Krambeck].

These types of activities have led understandings allowing such specific technologies as glycoform altering feed strategies to be specifically developed in response to individual needs. There are even some generic cocktails commercially that can have a beneficial effect in some cases. So, we can now address the issues of product quality as well as culture vitality, duration and peak cell concentration by specifying the number of feed solutions, their component types and concentrations, as well as the timing and mode of their addition. Entity sponsors and contracted manufacturers now have a choice of developing a customized process in-house from an array of previously commercialized products, or contracting for a solution through a premiere bioprocess materials and service vendor.

For some time, we have been hearing about the improvements in cell culture titers and the bottlenecks it has caused in downstream. How important is increasing productivity vs. other objectives, such as protein quality, etc. and how does this impact feed design?

It’s true that for many standard production platforms and pharmaceutical entity demands, the volumetric titer in fed-batch mode issue is solved. Not that we couldn’t design a process around a platform that could robustly produce 50 grams of quality product per liter. But for most platforms and modes of production, the economic drive to spend time improving current titers, beyond the existing potential from new recombinant and process technologies, often isn’t there. Goals driving cell culture media and feed development now include:

• New requirements for biopharmaceutical raw materials
• Different quality and regulatory requirements supporting such new product types as adoptive cell transfer
• New bioproduction expression platforms such as human and avian cell lines, as well as, cell lines recently converted to suspension
• New modes of production, such as perfusion for either intensified batch or continuous manufacturing
• Growing understanding of supporting or controlling product post-translational modifications

As an industry, we have made big improvements in upstream bioprocesses with cell culture productivity increases, implementation of more single-use technologies and better control over protein quality, what do you think will be the next big improvements?

One area for improvement is in continued expansion of the application of single- and limited-use systems. This includes designing systems that are integrated, qualified, and flexible. Expanded single-use also enables other improvements, such as modular, turnkey processes and more flexible facilities.

Future manufacturing efficiency improvements include employment of more advanced manufacturing execution systems and worldwide discrete manufacturing execution and distribution systems. Included in this is more efficient product transfer and packaging platforms, and shipping control. In addition, strategies like continuous biomanufacturing, intensified batch-mode processes, and in-line conditioning of buffers might aid in critical manufacturing challenges and bottlenecks. We might even see in-line cell culture media prep in the future.

One space where we have seen considerable advancements is in process monitoring and control and there is still room for improvement here as well. There are many new types of improved bioreactor probes and process analytics becoming commercially available. Better and more comprehensive process controls are also being developed by employing digital biomanufacturing techniques (Whitford, Digital Biomanufacturing Will Enable Tissue Bioprinting, The Cell Culture Dish).

Additionally there is room for improvement in defining product attributes. More defined and controlled product quality attributes like post translational processing consequences and host cell debris are on the horizon, as are limited material contaminant levels in such categories as unrelated substances, undesired metals, particulates and isomers.

Groups at GE have applied microarrays to discover genes that appear to be important for high cell-specific production rates. This type of information could be useful in cell line engineering or in the design of high producing Chinese hamster ovary (CHO) media (Transcriptome analysis reveals strategies for CHO cell culture media design and feed-spiking strategy to improve batch culture, The Cell Culture Dish).

Lastly, there are opportunities to improve vaccine manufacturing including moving classical vaccines to cell-culture based and plant based production. There are also several novel vaccine types being explored.

Summary

We are seeing great advances in fed-batch culture and feeding strategies due to advances in a number of arenas. These include:

• increased understandings in cell biology and ‘omics data
• the appearance of more flexible and robust equipment and facilities
• heightened bioprocess monitoring, modeling and control capabilities
• greater knowledge in the molecular characteristics of a quality product
• continued progress in the understanding and abilities of media and feed suppliers

And, finally, it’s exciting to observe that, in some of these areas, there does not appear to be an end to the gains we’ve been seeing.

Additional Reading on Feed Design Strategies:

“Transcriptome analysis reveals strategies for CHO cell culture media design and feed-spiking strategy to improve batch culture,” The Cell Culture Dish

“Development of DoE based fed-batch strategies for high-producing CHO cell cultures,” The Cell Culture Dish

About the Interviewee:

William Whitford, Strategic Solutions Leader, Bioprocess, GE Healthcare

Bill Whitford is a Strategic Solutions Leader at GE Healthcare in Logan, UT with over 20 years experience in biotechnology product and process development. He joined the company as an R&D Leader developing products supporting protein biological and vaccine production in mammalian and invertebrate cell lines. Products he has commercialized include defined hybridoma and perfusion cell culture media, fed-batch supplements and aqueous lipid dispersions. An invited lecturer at international conferences, Bill has published over 250 articles, book chapters and patents in the bioproduction arena. He now enjoys such activities as serving on the Peer Review Board for the European Medical Journal.

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