What is protein production?

Protein production systems, also referred to as an expression system, are commonly utilized by the pharmaceutical industry to produce novel medicines. Protein production is the biotechnological process of generating a specific protein. It is achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This expression process includes the transcription of the recombinant DNA to messenger RNA (mRNA). When the mRNA is translated into polypeptide chains, the chain folds into functional proteins and can then be targeted to specific subcellular or extracellular locations.

Protein production, to produce a protein or antibody of interest, is a multi-step process.

Steps to take before starting protein production

• Phase I – first human studies – mainly safety testing on healthy persons
• Phase II – testing different doses on patients
• Phase II – expand test panel, efficacy, and meet primary and secondary endpoints
• Phase IV – post marketing safety study, tackle safety concerns, different populations, and sometimes-rare side effects

Market ready

When the biologic is approved for market release; protein production on a large scale is required.

Which expression system to choose for protein production

Many organisms can produce proteins. See the table below for major organisms and important cellular characteristics.

Microorganisms, like Escherichia coli (E. coli) are easy to grow and express high levels of protein. So why is E.coli not always the ideal choice when producing recombinant proteins? The limitation lies within the protein folding process and the ability to produce complex proteins such as glycosylated proteins. Therefore, mammalian cell platforms are often utilized for complex protein production (posttranslational modifications – PTM). Although these mammalian cells may not proliferate as rapidly and may not yield as much protein as E.coli, these platforms are often selected for complex protein production to deliver protein therapeutics (i.e. biologics) in the biotech sector.

Transient or Stable transfection for protein production

• Transient transfection is ideal for the rapid production for small scale antibody (Ab) production. Transient gene expression results are often realized in 6-10 days from the initiation of DNA transfection.
• Stable transfection often begins transiently but through a process of careful selection and amplification, stable clones are generated. Within stable transfected cells, the foreign gene becomes part of the host genome and is therefore replicated. Descendants of these transfected cells express the foreign gene and become a stable cell line. Because this transfection process is complex and time consuming, it is more often used for large scale Ab production. 

Products produced using protein production

There are a variety of products that can be produced via protein production: 

• Modified human proteins (protein-protein fusion products, drug-toxin conjugates, PEGylated protein drugs)
• Monoclonal antibodies (humanized or chimeric monoclonal antibodies, monoclonal antibody fragments, single chain antibodies, bispecific antibodies, antibodies to conjgate to a toxic payload (ADCs).
• Growth factors and cytokines (colony stimulating factors, interferons, interleukins) 
• Hormones (insulin, erythropoietin, growth hormones)
• Blood products (blood clotting factors, thrombolytics, fibrinolytics, albumin)

Protein production methods

A protein can be produced in different ways:

Batch protein production

This is a large-scale closed culture system where cells are expanded in a fixed volume of medium with no additional additives. Since fresh media is not added during the incubation period, the concentration of nutrition decrease throughout expansion and various toxic metabolites accumulate. A batch culture will follow the characteristics growth curve with lag phase, log phase, stationary phase and decline phase. 

*These processes are only appropriate for proteins that are excreted into the media, ie this method is not applied to intracellular expression proteins.

Fed-batch protein production

A semi closed system for protein production where one or more nutrients (feeds) are added in intervals into a bioreactor. The product(s) remain in the bioreactor throughout the production process.

Perfusion protein production

Perfusion carried out by continuously feeding fresh medium into the bioreactor and simultaneously removing the cell-free spent medium as the cells expand in the bioreactor. The cell density remains constant by maintaining a constant dilution and flow rate.