Overview of the Mammalian Expression of Recombinant Antibodies

Recombinant protein expression is the process of introducing exogenous genes into host cells through gene recombination technology, utilizing the host cell's biosynthetic mechanism to express the target gene within the host cell. The commonly used recombinant antibody expression systems include Escherichia coli expression system, mammalian cell expression system, insect cell expression system, and yeast expression system. Among them, the E. coli expression system is the primary choice for prokaryotic expression. When expressing eukaryotic proteins, the corresponding expression system will be selected based on the characteristics of eukaryotic proteins and different expression needs of eukaryotic proteins. Mammalian cell expression systems can provide a cell environment similar to natural cells, suitable for the expression of structurally complex eukaryotic proteins. Mammalian protein expression systems can synthesize proteins with structures and functions similar to human proteins. Recombinant proteins produced by recombinant protein mammalian expression systems can undergo correct post-translational modifications, such as glycosylation. The glycosylation of recombinant proteins is closely related to their biological functions and can affect the safety and efficacy of the proteins. In addition, the lactation expression system can efficiently express various types of recombinant antibodies, such as recombinant IgG, Fab, VHH nanobodies and VHH nanobody fusion proteins, scFv and scFv fusion proteins, humanized antibodies, and chimeric antibodies. In recent years, the preparation of recombinant antibodies through mammalian expression systems has been widely applied in fields such as drug development and medical research.

Fig. 1 Flow chart of recombinant protein expression

Design of recombinant antibodies

Antibodies are immunoglobulins produced by B lymphocytes and play an important role in humoral immunity. Nowadays, through genetic engineering technology, antibody libraries containing a large number of different antibody sequences can be constructed. Afterwards, high-throughput screening techniques such as flow cytometry can be used to select antibodies with specific binding ability from the antibody library. A complete protein expression system includes target genes, vector plasmids, host cells, etc. The principle of recombinant protein expression is mainly to clone the target gene into an expression vector and transform it into the host cell, after which the target protein is produced through transcription and translation processes. When producing IgG antibodies, commonly used expression vectors include plasmids and viral vectors, which can be inserted with promoters, transcription stop signals, etc. to ensure efficient transcription and translation of genes. The gene of IgG antibody is inserted into the appropriate position of the vector, and then the constructed vector is transfected into host cells. Stable expression cell lines of IgG antibody can be screened using labeling for large-scale recombinant antibody preparation. According to the subsequent purification needs, different tags can be selected, such as adding His tags, FLAG tags, etc., for affinity chromatography purification. Different host cells also have varying effects on the efficiency of IgG antibody preparation. Common mammalian expression cells for antibodies include CHO cells and HEK293 cells, which can provide correct post-translational modifications to ensure antibody activity.

Fab antibodies retain the antigen binding fragment (Fab) of IgG antibodies, but do not have a constant C-terminal region, resulting in poor stability of Fab antibodies. However, their stability can be improved by integrating the IgG CH3 domain into the Fab fragment. The commonly used expression vectors for designing Fab antibodies include PET series prokaryotic expression vectors and eukaryotic expression vectors such as pCMV, which can insert promoters, transcription termination signals, etc. into the expression vectors to ensure efficient transcription and translation of genes. Perform affinity purification of antibodies by adding His tags, FLAG tags, etc. Different host cells also have different effects on the efficiency of Fab antibody preparation. The commonly used host is Escherichia coli, which has a fast growth rate and can be used for large-scale recombinant antibody production, but cannot correctly fold and modify eukaryotic proteins. Antibody expressing mammalian cells such as HEK293 cells and CHO cells can utilize the advantages of mammalian protein expression systems to produce highly stable Fab recombinant antibodies.

Single chain antibodies are the smallest binding units with antibody activity composed of light chain variable regions (VL) and heavy chain variable regions (VH) that are unlinked by short flexible peptides. This design removes the Fc segment while maintaining the antigen binding ability of the antibody, reducing immunogenicity and molecular weight, and improving its stability and penetration in vivo. The commonly used expression vectors for designing single chain antibodies include PET series prokaryotic expression vectors and eukaryotic expression vectors such as pCMV, which can insert promoters, transcription termination signals, etc. into the expression vectors to ensure efficient transcription and translation of genes. By adding His tags for affinity purification, adding GST tags can increase protein solubility, but the tags need to be removed in subsequent applications. Large scale production of single chain recombinant antibodies can choose Escherichia coli as the host. HEK293 cells and CHO cells, among other antibody expressing mammalian cells, can produce highly stable single chain antibodies.

Fig. 2 Western Blot identification results of polyclonal antibodies

KMD Bioscience has a comprehensive recombinant antibody expression production platform and rich experience in recombinant protein and antibody expression. We can provide customers with full-length and scFv, Fab, VHH nanobodies, chimeric antibodies, bispecific antibodies, Fc fusion antibody expression services for various species including human, mouse, rabbit, sheep, dog, camel, cow, etc. Customers only need to provide antibody sequence information, hybridoma cells or cDNA. The company's technical support can design solutions based on customer needs, combined with the company's independently designed lactation system high expression vector and large-scale recombinant antibody fermentation platform, to provide customers with high-quality one-stop recombinant antibody preparation services including sequence analysis, linker design, fusion tag design, high expression vector design and construction, expression scheme design optimization, recombinant expression condition optimization, antibody purification, etc.

Reference

[1] Tiller KE, Tessier PM. Advances in Antibody Design. Annu Rev Biomed Eng. 2015;17:191-216.

[2] O'Flaherty R, Bergin A, Flampouri E, et al. Mammalian cell culture for production of recombinant proteins: A review of the critical steps in their biomanufacturing. Biotechnol Adv. 2020;43:107552.