Large-scale Expression & Bacterial Disruption

The emergence of recombinant antibody production represents an unprecedented technology for the industrialization of monoclonal antibody. The unique antibody production method needs not the immunization and screening process, antibody gene sequence can be directly & stably used to manufacture large quantities of antibodies. Creative Biolabs has a complete recombinant antibody expression & production platform and much experience on the production of recombinant antibodies. We offer large-scale expression & bacterial disruption service for the rapid production of recombinant antibodies ranging from milligrams to kilograms, meeting the high throughput and large-scale production requirements of customers.

Large-scale Expression for Antibody Production

Large-scale expression of recombinant antibodies can used different expression systems such as bacteria, yeast, or mammalian cell lines. To date, Escherichia coli remains one of the most commonly used host organisms for recombinant protein production, not only for analytical and biotechnological purposes but also for pharmaceutical industry. It is one of the most comprehensively studied organisms and is used for recombinant protein production since the first recombinant peptide was produced in late 1970s. For therapeutic purposes, the production of recombinant antibodies is mainly used the mammalian cell lines to decrease the risk of immunogenicity caused by non-human post-translational modifications, in particular glycosylation.

For the large-scale expression of recombinant antibodies using mammalian cell lines such as HEK293/CHO cells, mainly steps include gene synthesis and codon optimization, vector construction, expression & purification and QC analysis.

For large-scale expression of recombinant antibodies using bacteria (Escherichia coli), process are similar to the expression in mammalian cell lines. However, bacterial cell disruption is a key step in antibody production.

Fig.1. Recombinant antibody formats for different applications compared to IgG. (Frenzel, 2013)

Bacterial Cell Disruption: A Crucial Step in Antibody Protein Production

Various methods have been used for bacterial cell disruption. Chemical and enzymatic lyses are often used on the laboratory scale and the industrial scale mechanical methods are more suitable. For chemical methods, it is not a suitable method for isolation of non-classical IBs inclusion bodies (ncIBs) that are soluble in the detergents thus a significant amount of the protein is lost from them. The method has been optimized and suitable for use in mammalian cell cultures and other biological interfaces. Enzymatic lysis with enzyme lysozyme is another method often used for bacterial cell disruption. The method was found suitable for the isolation of most soluble proteins. Mechanical methods were found to be suitable for isolation of recombinant proteins, and the isolation is very efficient also for larger samples. The method is often used for the isolation of soluble proteins and classical inclusion bodies (IBs). However, the method can damage the soluble recombinant protein due to the sonication can damage the protein structure. The combination with other methods to allow lower sonication power outputs for bacterial cell disruption would probably be the safest option. Therefore, the bacterial cell disruption method should be chosen regarding the solubility of the produced recombinant protein as well as the intended use of the isolated product.

Steps of Large-scale Expression for Antibody Production

Large-scale Expression Using Mammalian Cell Lines
Gene synthesis and codon optimization
Vector construction:
Cloning the heave chain and light chain gene into optimized expression vectors, respectively
Plasmid sequencing
Large scale plasmid production
Expression & purification:
Transient transfection of HEK293/CHO cells
Antibody purification
Polishing if needed
QC analysis:
Analysis by SDS-PAGE and UV
Purity analysis by HPLC-SEC and endotoxin detection

Large-scale Expression Using E. coli

Harvest the bacterial cells by centrifugation at 3000 g for 15 min at 4°C.
Wash the cells with bacterial cell lysis buffer to remove the residual culture medium. Harvest the washed cells by centrifugation at 3000 g for 15 min at 4°C.
Decant the supernatant. Weigh the wet pellet.
Resuspend the washed E. coli cells in ~3mL of lysis buffer per gram of cell pellet. Stir the suspension for 30 min at 4°C.
Add lysozyme to a concentration of 0.1% (w/v). Incubate for 35 min at 4°C, shaking gently.
Stir the suspension for 30 min at 4°C to remove the viscous nucleic acid.
Centrifuge the suspension at 23,000 g for 30 min at 4°C. Reserve the supernatant.
Resuspend a small portion of the pellet in 1X Laemmli sample buffer containing 100 mM DTT.
Analyze aliquots of both the soluble protein fraction and pellet fraction for the presence of the target protein using analytical SDS-PAGE.

Fig.2 A comparison of a conventional in vivo system and a CFPS system. (Khambhati, 2019)

What Can We Do For You?

Large-scale expression for antibody production in gram amounts is guaranteed with systematic optimization of sequence design, culture protocol, transfection method, and cell lines. Creative Biolabs has vast experience for antibody development and our optimized technology to maximize successful antibody production. We believe our large-scale expression & bacterial disruption service can meet your specific needs and guarantee the quantity and quality of our delivered antibody. For more information about our services, please do not hesitate to contact us.

Work flow of rAb production includes:

References

Frenzel, A.; et al. Expression of recombinant antibodies. Frontiers in immunology. 2013, 4: 217.
Khambhati, K.; et al. Exploring the potential of cell-free protein synthesis for extending the abilities of biological systems. Frontiers in bioengineering and biotechnology. 2019, 7.