Therapeutic Protein Expression System
Expression system refers to the system that synthesizes recombinant proteins through specific cells and transfected DNA vectors, which can translate the supplied DNA genetic information into the amino acid sequence specific to the target protein. After translation, these molecules are affected by post-translational modification (PTM). There are significant differences in the frequency and complexity of PTM between prokaryotes and eukaryotes, and the influence of PTM on the latter is more widespread. Although fungus, insect, and plant cells can undergo glycosylation, the structure of their glycoproteins is different from that of human proteins, which may result in undesirable immunogenicity.
The development of cell lines all begins with the construction of expression vectors. The vector is a DNA element capable of self-replication in which a fragment of foreign DNA is introduced. In addition to the transferred foreign genes, the vector usually includes some expression elements, such as promoters, enhancers, multiple cloning sites (MCS), intron sequences, transcription terminators, selective markers, and some DNA elements that regulate chromatin structure. In addition, epigenetic elements may be included to optimize protein expression processes and mitigate silencing effects.
Cell type, expression requirement, safety, economy, process scale-up, turnaround time, and regulatory demands should be considered when selecting vector and gene delivery methods. Methods for vector gene delivery include reagent-based, instrument-based, and virus-mediated technologies. Reagent-based methods cover cationic lipid transfection, calcium phosphate precipitation, diethylaminoethyl (DEAE)-glucan/polyethyleneimine (PEI)/polymer/dendrimer-mediated technology, etc. Instrument-based methods encompass electroporation and microinjection and virus-mediated means include gene delivery mediated by adeno-associated viruses and lentiviruses. Most of the non-viral gene delivery techniques have obtained regulatory approval and are the preferred methods in protein drug production.
- Reagent-based transfection methods
The common principle of reagent-based transfection methods is that a positively charged chemical forms a complex with a negatively charged nucleic acid, which is then attracted to a negatively charged cell membrane. The complex then enters the cell through endocytosis or phagocytosis.
Calcium phosphate precipitation and DEAE-glucan are the oldest DNA delivery technologies. Although the cost is lower, they often have the problems of low transfection efficiency and high cytotoxicity. In addition, the calcium phosphate method requires the use of a serum medium, which limits its application in the production of biological drugs.
PEI and other cationic polymers are non-cytotoxic reagents, with low cost, dyeing efficiency of up to 100%, and can be amplified to hundreds of liters, so they are widely used.
Another popular DNA delivery method is cationic lipid-based technology, which has the advantages of high transfection efficiency, user-friendly, minimal steps, and easy amplification. As its cost is relatively high, it‘s rarely used in large-scale transfection. However, the cationic lipid is commonly used in transfection in Petri dishes in the early stages of development because of its high efficiency and its suitability for high-throughput systems.
- Electroporation
Electroporation (electrotransfection) is a method that causes cells to absorb foreign DNA molecules by temporarily increasing the permeability of cell membranes through the action of a high-intensity electric field. The optimal electric field intensity, pulse length, buffer conductivity, waveform, pulse number, and other parameters can be used to ensure high transfection efficiency and high survival rate without changing the biological structure/function/host cell, and it is easy to use and has a wide range of cell line applicability. This method can be used to introduce foreign proteins, mRNA, siRNA, and other biomolecules, and is suitable for stable transfection and transient gene expression.
- Virus-mediated method
The virus-mediated method relies primarily on the mechanism by which the virus infects cells, unlike the chemical and physical transfection methods described above. Insertion capacity varies by virus type or even serotype of the same virus and is often used for transient gene expression. Recombinant virus vectors with replication defects are the first tool that can efficiently transfer non-cytotoxic genes into human cells. Due to the natural cell invasion ability and high gene delivery efficiency, viruses are the most commonly used methods of in vivo gene therapy. Among them, adeno-associated virus (AAV) vectors are the most well-developed. AAV is a persistent virus that induces low pathogenicity and virulence and can promote long-term expression of transgenes through chromosome integration. Some single-stranded (ss) or double-stranded (ds) RNA and DNA viruses are used in other viral delivery systems, such as adenovirus, alphavirus, flavivirus, herpes simplex virus, measles virus, retrovirus, lentivirus, etc.
When selecting the vector, it is necessary to focus on the packaging ability, toxicity, immunogenicity, cracking ability, long-term or short-term transgenic expression, infected non-dividing cells, specific replication ability in tumor cells, and other factors. In the past, researchers generally believed that the efficiency of non-viral gene delivery was low, and the expression time was short, so it‘s not widely used. Later, with the development of physical transfection methods and vector development techniques, non-viral vectors have become a new generation of gene transfer tools with great potential due to their low immunogenicity. As for viral vectors, although they are dominant in active gene therapy-related clinical trials, they also have some drawbacks, including more complex cloning strategies, biosafety concerns, and possible health risks associated with viral vectors and recombinant proteins.
Mammalian cell expression system
Mammalian cell production technology is costly, difficult to scale up, and has the risk of microbial contamination. However, due to their ability to undergo complex post-translational modifications, they are still widely used, and the vast majority (70%) of recombinant proteins on the market are produced using mammalian cell culture processes.
The most commonly used selectable markers in mammalian cells are glutamine synthetase (GS), dihydrofolate reductase (DHFR) genes, and antibiotic resistance genes. After transfection and selective culture, the surviving cells are subjected to single-cell isolation and used to generate clonal populations. Cloning screening is then carried out to select the most productive and stable clones to create a cell bank for large-scale protein production.
Non-mammalian cell expression system
Although most FDA-approved recombinant glycoproteins are produced by mammalian cells, non-mammalian expression systems such as bacteria, yeast, insects, and plant cells are still required in many cases. The advantages of the latter are high yield, easy scaling up, low cost, and less susceptibility to contamination by mammalian pathogens. However, these heterologous expression systems have a common disadvantage which is the lack of a mechanism required to synthesize glycosylation and other human-like PTMS. Therefore, non-mammalian systems are the most economical option if the therapeutic proteins required are small and their clinical function does not require human-like glycosylation.
Bacteria are the most commonly used host cells expressing simple heterologous proteins (without PTM) and are the most popular expression system for non-mammalian cells due to their low cost of culture-medium, rapid division, easy scaling up, and high yield. Currently, about 30% of therapeutic proteins are produced by bacterial hosts. However, due to the formation of inclusion bodies during production, proteins tend to accumulate and be inactivated. In addition, as mentioned earlier, there is insufficient PTM in the system to produce complex therapeutic proteins. Escherichia coli is the first expression host used in biological drug production and is currently used as a mass expression system for simple proteins.
Similar to bacteria, yeast cells also have the advantages of rapid cell division and high protein expression. However, the proportion of mannose-oligosaccharide in the protein produced by the yeast cell is higher, so its clearance rate in vivo may be faster. Some recent glycosylation engineering techniques have enabled yeast to produce humanized sialoglycoprotein, but human-like glycosylated protein is still difficult to express. One such yeast system, Saccharomyces cerevisiae (S. cerevisiae), expresses therapeutic recombinant proteins including insulin peptides, hepatitis vaccines, human serum albumin, and virus-like particles. The other is P. pastoris, which can produce recombinant protein products on the gram scale. At present, P. pastoris is mainly used for the production of insulin, human serum albumin, α-IL6 receptor single-domain antibody fragments, and other small proteins.
Baculovirus expression vector system (BEVS) can also be used to produce recombinant proteins with complex sugar types. It is an expression system established by using insect baculovirus as a foreign gene carrier and insect cells such as Sf9, Sf21, and BTI 5B1-4 as receptors. Since the translation and post-translational modification patterns of insect cells are similar to those of mammalian cells, the biological activities of the recombinant proteins produced are similar to those of natural proteins in terms of antigenicity, immunogenicity, and function. Moreover, its cost is low, easy to achieve mass production. However, the proportion of suspected immunogenic sugar structures like mannose-oligosaccharide in the protein produced by the protein is high, and it could not be modified by sialylation. In addition to recombinant proteins, the system is also widely used to produce vaccines, virus-like particles, viral vectors, and other products.
- Plant expression system
Like insect cells, plants cannot undergo sialylation. Immunogenic glycans such as α (1,3)-fructose and β (1,2) -xylose are synthesized by the system. Therefore, plant expression systems are mainly used to produce recombinant proteins that do not contain sialic acid (e.g., acid beta-glucosidase).
Summary
In addition to the various expression systems mentioned above, attempts have been made in recent years to produce simple protein molecules using cell-free protein synthesis systems (SFPS) to reduce the complexity of the protein expression process and shorten the development time. Each expression system has its relative advantages and disadvantages in terms of efficacy and safety. For instance, human-like PTM in mammalian cells is more functional but has a higher risk of viral contamination. Non-mammalian cells grow faster and have high yields, but express proteins that may contain immunogenic epitopes of the potential risk to patients. Therefore, it is often necessary to select the most appropriate expression system based on the molecular properties, quality requirements, efficacy, and safety of the product.
