Monoclonal Antibodies: From Screening to Production

The specific methodology for hybridoma creation was devised in the 1970’s and has been refined and extended over the decades. The steps from clone creation to final product follow a standard path for protein products derived from modified mammalian cell lines.

The main steps in monoclonal antibody manufacturing include:

• Creation and selection of the hybridoma clone for a master seed
• Preparing seed train in either T-flasks, shake flasks or roller bottles
• Moving to bench-scale bioreactors (option bags) to increase seed yield
• Production up to 20,000 L
• Downstream processing and storage

Selection of the hybridoma clone for a master seed

At the beginning of the hybridoma culture process, a clone must be generated and selected. The best clones are stable and good producers with high antibody production.

Monoclonal antibodies are produced by immunizing an animal (usually a mouse) with a specific antigen. Subsequently B-lymphocyte plasma cells from the spleen of the immunized animal are removed. Since these B-cells are a primary cell line and have a limited lifespan in-vitro, they are fused with an immortal myeloma cell line, providing the ability to be sub-cultured indefinitely. The myeloma cell line can be derived from mice, other mammals, or human cells. Variations of standard monoclonal antibodies include bi-clonal, polyclonal, and chimeric versions e.g., up to 90 % human, 10 % mouse to improve acceptability for therapeutic applications.

Hybridoma creation selection and expansion to create a master culture can be done at the bench-scale in static culture. The expansion phase of the most stable, productive clones is typically performed in serum-free media in T-flasks. Master cultures can be prepared from this material and stored in liquid nitrogen for revival to start the seed train of a bioprocess. The alternative would be an intensification step in a small bioreactor to produce cultures with a high cell density for direct inoculation into a pilot or production bioreactor. These master cultures will be periodically tested to ensure the specificity and productivity if the clone remains constant.

Seed train from vial to shake flasks

Seed train development is the gradual increase in cell density in a series of steps which cascades the cells produced from one step to the next largest. Each transfer can introduce its own potential problems in terms of contamination, failure to grow and loss of productivity. Minimizing the number of steps is clearly desirable. The latest developments in process intensification can remove many intermediate steps.

An incubator shaker with CO₂ control designed specifically for cell culture is often used for early-stage seed development. Culture volumes can be as small as 20 mL at the start, moving in two or three steps to a litre or more. Multiple flasks are used at each stage to allow for potential problems and/or bulking of contents to provide the inoculum for the next stage. Of course, multi-deck systems can have several stages progressing in parallel.

A typical protocol for hybridoma cells [1] can look like the following:

Protein free media is available commercially and fill volumes for shake flasks should be around 20-25% (higher for some optimal growth flasks).  To move from a 1 ml vial to 1 L of culture in a shake flask may require at least two intermediate steps in terms of flask capacity.

Scale-up to bench-top and pilot bioreactors

The bioreactor phase of scale-up typically involves bioreactors in a range from 10-50 litres. Often bench-scale bioreactors are running in a perfusion mode to produce high-density cultures making many of increasing volume steps unnecessary.

Typical specifications for a cell culture bioreactor include:

• A round bottom or dished vessel.
• Short aspect ratio e.g., 2:1.
• A marine impellor for low shear.
• Low gas flow rates and sparging with small bubbles.
• A gas mix system for 3 or 4 gasses, including CO₂ for pH control.
• Slow-speed motor to provide gentle mixing.
• If applying continuous mode, a spin filter or similar is needed for continuous separation of supernatant from cells.

Hybridoma culture in bioreactors can be cell line and conditioning dependent. These guidelines are, at best, an approximation to provide a starting point for bench-scale cultures [2]:

Fed-batch operation can be achieved with daily feeding or, medium perfusion can be set at the rate of 0.5 – 1 working volumes per day. Batch, fed batch and perfusion cultures (using a spin filter or ATF filtration) can last for 7-14 days or longer after a batch phase of 3-4 days, depending on the process mode and clone selected.

Production scale

This can be in the thousands of litres and represents a significant investment in terms of time, resources, and costs. The high value of the products justifies the outlay but any efficiency improvements to the process would have a large impact on reducing the costs.

Typical scale-up times can be from 3-5 months from beginning to end, depending on the yield of the clones and the amount of antibody required. This includes creation of a suitable clone from a purified antigen, cultivation in shake flasks and bioreactors, followed by downstream processing.

The bioprocess for all stages will typically comply with GMP regulations and the process could require a full validation process for therapeutics intended for human use. This is more a matter of certification of materials and following the correct protocols rather than a special construction.

Bioprocess software becomes important if process validation is required. User management and process analytics capabilities are needed alongside the usual tools such as recipes, planning and comparison options.

Downstream processing

The supernatant from the culture is processed in a series of steps to provide purified monoclonal antibody i.e.

• Separation of cells from supernatant by low-speed centrifugation.
• Further clarification with depth filtration for high particulate supernatants.
• Direct column chromatography using protein A (or similar) for purification and some concentration. Protein A is a bacterial surface protein with binding sites for antibodies.
• Elution at low pH from the column (also provides virus inaction).
• Ion exchange chromatography polishing – two steps (anion and cation) to bind specific proteins based on charge, while removing impurities and residual media components.
• Virus filtration using membrane filters with a specific pore size, having variants for batch or continuous processing.
• Ultra-filtration or diafiltration to concentrate the finished product ready for bagging, testing, and dispensing as required.

Combined with the upstream steps, this forms a coherent process platform for production of monoclonals, as only the starting antigen need vary. Scale can be varied, and advantage taken of opportunities to make the process more parallel and use several smaller columns.

Summary

The production of monoclonal antibodies using hybridoma cells just pre-dates the commercial use of recombinant DNA technology for manufacture of human insulin. It is therefore a prime example of a modern bioprocess involving skills in multiple scientific disciplines. However, once the specifics of the creation of a productive clone are complete, the subsequent steps in the bioprocess make a good example of the concept of a process platform. Once the antigen of interest has been identified, purified, and used to create a hybridoma, everything downstream of this can remain close to constant. A change to another antigen needs only minimal changes to the rest of the process.

The key areas covered are:

• A brief overview of creation of a stable, productive hybridoma clone
• Seed train development in shake flasks
• Scale up through laboratory and pilot bioreactors
• Production scale in bioreactors with reference to regulatory requirements and process improvement
• Downstream processing from harvest to purified, polished product ready for filling

For a more detailed overview on the critical parameters for successful cell culture process transfer have a look at our blog post: Everything You Ever Wanted to Know About Cell Culture Process Transfer.

References:

[1] Supplementary Materials for Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell–driven tumor immunity .  Lucas Ferrari de Andrade et.al. Science 359, 1537 , (2018)
[2] A Single Dynamic Metabolic Model Can Describe mAb Producing CHO Cell Batch and Fed-Batch Cultureson Different Culture Media. Robitaille J, Chen J, Jolicoeur M, PLoS ONE 10(9), (2015)