Monoclonal antibodies: From screening to production

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.

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 liters. 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]:
  
  

  Temperature	37°C
  pH	7
  DO	50%
  Stirrer speed	60 – 120 min-1 (a higher speed will reduce any cell leakage via a spin filter)
  Gas mix	2 gasses for air plus CO2 for pH control

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.

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 monoclonal antibodies, 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)