Protein production and bioprocessing

Therapeutic proteins are commonly engineered in pharmaceutical laboratories to generate human protein therapeutics. The first protein therapeutic was insulin derived from recombinant DNA in 1982. Bacteria expression systems and mammalian cell lines such as Chinese Hamster Ovarian (CHO) are used to produce therapeutic proteins, antibodies, enzymes or hormones that can be injected into humans or animals to treat diseases.

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Applications

Protein Production & Bioprocessing

Recombinant Protein Production Using CHO Media System

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 for starting protein production

Protein production starts by first defining what protein you want to produce. Such proteins selected are used to treat specific diseases. The drug development steps for the discovery and production of novel protein drug candidates are:

Target identification
The first step of drug discovery is to identify the biological origin of a disease, and the potential targets for intervention.
Target validation
The second step of the drug discovery process is to prove if an identified target is directly involved in a specific disease and if modulation of this target is likely to have a therapeutic effect.
Hit identification and lead generation (H2L)
The next early drug discovery step utilizes a high throughput screening (HTS) process to evaluate and optimize promising lead compounds previously identified as potential targets. These lead compounds undergo more extensive optimization in subsequent steps of drug discovery in the lead optimization (LO) step.
Lead optimization
The objective of this drug discovery phase is to synthesize the lead compounds in order to improve properties. Lead optimization includes experimental testing for the confirmation of the identified compound(s). Optimization refers to optimizing binding affinity, selectivity and/or efficacy. Once this process is confirmed, these drug candidates can be registered or patented for intellectual property protection.
Preclinical testing
In this preclinical step it is vital to determine if the drug candidate has promising activity against the biologic target relevant to the disease of interest. Additionally, sufficient safety and drug-like properties prior to human testing must be identified. Defining safe dosing for human studies is a key aspect of preclinical testing process.
Preclinical results can be submitted to regulatory authorities (e.g. US - FDA) as an Investigational New Drug (IND) and must be finalized before moving to the clinical phase.
Clinical phase
Clinical studies consist of 4 phases:
1. Phase I – first human studies – mainly safety testing on healthy persons
2. Phase II – testing different doses on patients
3. Phase III – expanding test panel, efficacy, and meeting primary and secondary endpoints
4. Phase IV – posting marketing safety study, tackling 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.

The TheraPRO^® CHO Media System

The TheraPRO^® CHO Media System is a chemically-defined, animal component-free formulation proven to optimize productivity and product quality when used in conjunction with the GS-CHO cell line, the prevalent protein expression system worldwide for therapeutic proteins and antibodies.

TheraPRO® CHO Media System

Which expression system to choose for protein production

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

Characteristics	Mammalian cells	Insect cells / Baculovirus	Yeast	E. coli
Cell growth	Slow	Slow	Rapid (±90 min)	Rapid (±30 min)
Complexity of growth medium	Complex	Complex	Simple	Simple
Expression level	Low-moderate	Low-high	Low-high	High
Extracellular expression	In medium	In medium	In medium	In periplasm
Posttranslational modifications
Protein folding	Proper folding	Proper folding	Refolding may be required	Refolding usually required
Glycosylation	++	+	+	-
Phosphorylation	+	+	+	-
Acetylation	+	+	+	-
Acylation	+	+	+	-
Gamma-Carboxylation	+	-	-	-

Why is E. coli not always the ideal choice when producing recombinant proteins?

Microorganisms, like E. coli are easy to grow and express high levels of protein. 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). 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

Transient transfection is ideal for the rapid production for small scale antibody (Ab) production. Transient protein production can be realized in 5-14+ days, depending on the method.

Stable 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 conjugate 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)

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Protein production methods

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 decreases 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.

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. (see table)

Perfusion protein production table

Operation mode	Batch*	Fed batch*	Perfusion*
Media Addition	Initial	Periodic	Continuous
Benefits	Less cell debris, lower risk of contamination	High titer	High productivity
Disadvantages	- Fair productivity - Frequent equipment turnover	- Maintaining sterility in bioreactor - Medium cell debris	- Maintaining sterility in bioreactor and in the primary clarifier - High cell debris

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