Nailing Your Recombinant Antibody Project
By Dr. Leung How Wing and Dr. Luan Bowu
With the emergence of the COVID-19 pandemic, there has been more interest in antibodies and what they can be used for. So, what are antibodies? Antibodies are proteins that are made by the immune system to fight infections caused by viruses or bacteria. They work by recognizing and directly neutralizing antigens from viruses or bacteria, or tagging a microbe or infected cell for destruction by the immune system.
By being able to bind to biological targets with high specificity, antibodies are getting popular as a form of drug treatment for various diseases, such as cancer and autoimmune disorders. In addition, antibodies are valuable reagent tools in biomedical sciences and medicinal research. These purified antibodies are used to label cells in flow cytometry, identify proteins separated by electrophoresis in western blot analysis, separate proteins and their binding partners from cell lysate in the technique known as immunoprecipitation, and locating protein expression in tissue sections in immunohistochemistry and immunofluorescence staining.
Hybridoma technology was initially used to generate antibodies. This process involves the injection of an antigen into a mammalian organism to initiate an immune response. The antibody producing B cells from the organism’s spleen are then harvested and fused with myeloma to generate a hybrid cell line known as a hybridoma. While this technique produces highly consistent, specific, and sensitive monoclonal antibodies in large quantities, over time hybridoma cell lines can experience genetic drift, resulting in slight variations to the antibodies produced. Additionally, it is difficult to generate antibodies against targets such as toxins, nucleotides, and membrane-bound proteins. Therefore, to consistently produce antibodies for such targets, hybridoma technology may not be the best method.
With the advancement of cloning technologies and sequencing, the biotechnology industry has moved towards producing recombinant antibodies (rAb). rAb are monoclonal antibodies synthesized by cloning antibody genes into expression vectors, which are then introduced into suitable production cell lines. This modern method of producing rAb overcomes the limitations in hybridoma technology, by ensuring consistency and reproducibility between production batches and a guaranteed long-term supply.
Of course, there are many steps that come between designing the antibody gene expression constructs to obtaining the final antibody protein. At each step, various optimizations are required to ensure that the antibody is soluble, stable and can be expressed at high levels. The design of the gene construct to enhance antibody production is the very first step of this process.
In this article, we will be presenting several important considerations that researchers and bioentrepreneurs can take note of when designing the antibody DNA construct for optimal rAb production in a chosen cell line.
DNA expression vector elements
There are a variety of plasmid vectors that have been optimized for DNA cloning and rAb production. These vectors contain genetic elements with distinct functionalities (Table 1), that can be interchanged or added to improve the production of rAb.
The key genetic element that is used to initiate and control the genetic expression of rAb is the promoter. Some of the common constitutively active promoters that can be used in a variety of mammalian cell lines include the human cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), and cellular elongation factor 1-alpha promoter (EF1a)1,2 promoter. The performance of these promoters can be further enhanced by adding enhancers and splicing-competent introns at the downstream end. If you wish to improve the transcript stability and translation efficiency of the rAb mRNA, the SV40 or bovine growth hormone (BGH) polyadenylation signal sequence can be placed at the end of the gene construct.
Other key vector characteristics that you may need to consider when designing your rAb gene construct would be the Kozak consensus sequence for translation initiation, antibody selection markers, origin of replication and chromatin remodeling elements.
Table 1. Table illustrating vector characteristics, their respective functions, and examples of the corresponding plasmid features.
|Vector characteristics||Function||Plasmid features|
|Promoters||Transcriptional control and initiation||• Human cytomegalovirus (CMV)
• Simian vacuolating virus 40 (SV40)
• Elongation factor 1-alpha promoter (EF1a)
|Introns||Provide directionality to enhance transcription||• Splicing-competent introns|
|Polyadenylation signal||Promote stability of mRNA||• SV40 polyadenylation signal
• Bovine growth hormone (BGH) polyadenylation signal
|5' UTR||Enhances translation initiation||• Kozak sequence|
|3' UTR||Promote mRNA exportation to cytoplasm for translation||• Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element|
|Origin of replication||Allow for episomal amplification of plasmid||• Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1)
• SV40 large-T antigen (293E or 293T cells)
|Selection marker||To select for clones with gene of interest||• Antibiotic resistant gene|
Besides looking at the gene elements, we also need to look at the structure of antibodies and how they become a fully functional protein. Antibodies, such as IgG, are normally made up of paired heavy and light chains. Only a completely assembled antibody molecule can bind to antigens and carry out effector functions. It has been found that light chains are synthesized 15-25% faster than heavy chains, and the isotype of light chain is key in influencing the kinetics of intracellular IgG assembly2,3. Choosing to express the heavy and light chains with separate promoters (monocistronic) can cause an unbalanced amount of each Ig chain and result in inefficient antibody assembly.
Therefore, we recommend the use of bicistronic vectors to co-express both the heavy and light chain antibody genes from the same RNA transcript via an internal ribosome entry site (IRES)2 (Fig. 1). The use of the IRES in bicistronic vectors allow translational initiation independent of the mRNA’s 5’cap. This permits the balanced expression of both the IgH and IgL from a single mRNA.
Fig. 1. Bicistronic vectors co-expressing heavy chain (IgH) and light chain (IgL) of antibodies from the same mRNA using IRES.
Due to the degeneracy of the genetic code, 18 out of 20 amino acids can be encoded by multiple three-base pair codon combinations. This codon redundancy is what accounts for the existence of a synonymous mutation, where the change in the DNA sequence that codes for the amino acid in a protein sequence does not change the final translated amino acid4.
However, synonymous codons can still affect protein expression levels because in a cell, certain codons are translated more efficiently than others, creating the phenomenon known as codon bias4,5 (Fig. 2). A synonymous mutation in a codon with limited availability of corresponding tRNA anticodons can result in ribosome stalling. When ribosomes are stalled on non-optimal codons or truncated mRNA, the cell receives signals to degrade the incomplete protein and mRNA. This results in significantly lower protein expression with non-optimal codons6.
We recommend using bioinformatics tools that can optimize codon usage within the target rAb expression system and reduce codon bias7,8. Using the GenSmartTM Codon Optimization tool, you will be able to identify and avoid low frequency codons in the human expression system and generate optimized antibody sequences for your antibody gene construct (Fig. 2).
Fig. 2. Codon usage frequency table for in the human expression host system. Red box indicates the low frequency of the codons in the human expression system which could result in low expression of the amino acids in proteins.
Cell Line Engineering
In addition to optimizing rAb expression plasmids, cell lines have also been genetically engineered to enhance rAb production across all gene expression systems and conditions. With the advancement in cell engineering technology, researchers are spoilt for choice with the different cell lines available for antibody production. We have made a summary (Table 2), highlighting the common cell lines used and their respective advantages. We hope that this may give you a better understanding of each cell line and help you choose the most suitable cell line for your needs.
Table 2. Table indicating advantages and disadvantages of each type of cell line commonly used in rAb production.
|Chinese Hamster Ovary (CHO)||i) Resilience to growth conditions|
ii) Resistant to viral infections
iii) High protein capacity
iv) Most frequently used for biologics production
vi) Post-translational modifications and folding most resemble that of human cells
|i) More challenging to transiently transfect
ii) Lack of certain glycosylating enzymes important for production and therapeutic efficacy
iii) Produce additional alpha-gal and NGNA glycans that could increase risks of adverse immunological reactions
|CHO/dhFr-||i) Both alleles of DHFR are deleted to provide ease for screening for successfully transfected plasmids containing both rAb gene and DHFR|
ii) Increase number of antibody gene copies in genome through gene amplification during selection, resulting in high antibody yield
|CHO-DG44||i) CHO/dhFr- but with the overexpression of anti-apoptotic protein Bcl-xL to increase longevity and production yield|
|Human Embryonic Kidney (HEK) 293||i) Transfected at high efficiency|
ii) Highly productive cell suspensions
|i) Greater risk for viral contamination and their subsequent transmission|
|Protease-deficient baculovirus strain for insect cell lines||i) Complex glycosylation system closely resembling mammalian systems for effective bioactivity and proper rAb clearance||i) Use of virus for expression|
While we have provided just a few examples of cell lines engineered to improve rAb production in stably and transiently transfected calls, new cell lines are constantly being developed and tested to improve rAb expression and production. It is also possible to tailor cell lines for the specific production of your rAb using revolutionary CRISPR/Cas9 gene editing technology. You can consider outsourcing the effort to generate high rAb producing customized cell lines (see further reading).
Chaperone proteins take part in the folding of over half of all mammalian proteins. They are co- or overexpressed with the rAb to ensure that the rAb protein will not misfold and form non-functional aggregates. There are several chaperones commonly used to improve mammalian proteins expression (Table 3).
Table 3. Chaperones proteins used in different expression systems.
|Expression Systems||Chaperones||Functions||Good For|
ii) Peptidyl prolyl cis-trans isomerase, FkpA
|i) Increase solubility and folding||i) scFv
|Insect||i) Hsp70 with Flsp40||i) Increase solubility|
ii) Reduce aggregation
|Bacteria/ Yeast/ mammalian||i) BiP (Hsp70 member)||i) Bind stably to free, non-secrete IgG heavy chain to increase the secretion of IgG|
|Yeast/ insect/ CHO cells||i) Protein disulphide isomerase (PDI) (can be used with BiP)||i) Increase scFv titers|
ii) To assist in the disulphide bond formation to stabilize IgG structure
iii) To increase secretion
|Insect||i) coexpression of calreticulin (ER chaperone) with translation initiation factor eIF4E||i) Increase rAb secretion|
This chapter only highlights some of the tips to optimize recombinant antibody expression. In the next chapter, we will discuss on the downstream processes such as transfection, culture conditions, purification, and characterization.
We understand that there is no one size fits all strategy when it comes to recombinant antibody generation, therefore we have expert scientists and dedicated technical account managers who can help to maximize the success of your research project and will guide you in every step of your experiment.
Click the link below to read up on GenScript’s recombinant antibody services:
- GenSmart™ Codon Optimization – MAXIMIZE the chance to obtain functional proteins in one click (https://www.genscript.com/gensmart-free-gene-codon-optimization.html)
- GenCRISPR Cell Line Service – Outsource your research and development effort to generate high rAb producing customized cell lines. ((https://www.genscript.com/CRISPR-genome-edited-mammalian-cell-lines.html)
- HD Transient Expression Service – Enhance transient expression yield up to 100 fold and achieve transient expression antibody titers up to 3 g/L (https://www.genscript.com/high-density-transient-expression.html?src=leftbar)
- MamPilot Guaranteed Antibody Expression – Guaranteed recombinant antibody expression service offers flexible expression scale, rapid production time (from gene synthesis to purified antibodies, as soon as 20 BD). (https://www.genscript.com/guaranteed-antibody-pilot-expression.html)
- High Throughput Gene to Antibody Production – Fast delivery of purified antibodies up to 1 mg each (https://www.genscript.com/high-throughput-antibody-purification.html).
- New and improved CHO expression platform for higher yield and fast turnaround time in 2 weeks (https://www.genscript.com/transient-expression-overview.html?src=leftbar).
GenScript Biotech Corporation (Stock Code: 1548.HK) is a global biotechnology group. Built upon its leading gene synthesis technology, GenScript is divided into four major platforms including the life science contract research organization (CRO) platform, the biologics contract development and manufacturing organization (CDMO) platform, the global cell therapy platform, and the industrial synthesis biological product platform. Driven by the corporate mission of “making people and nature healthier through biotechnology”, GenScript strives to become the most trustworthy biotech company in the world. As of December 31, 2020, GenScript’s products or services have been cited by 52,500 peer-reviewed journal articles worldwide.
About the authors:
- Leung How Wing
Dr. Leung How Wing is the Field Application Scientist, supporting the protein expression and antibody production services at GenScript. Dr. Leung graduated from the National University of Singapore in 2011, majoring in Pharmacology and Neuroscience. She has more than 10 years of research experience in molecular biology techniques and extensive knowledge in protein structure modelling, protein expression and its purification. Contact Dr. Leung here: email@example.com
- Luan Bowu
Dr. Luan Bowu received his PhD in Biochemistry from Stony Brook University in 2014. After joining GenScript USA Inc. in 2016, Dr Luan currently holds the Product Manager role, and he has been managing portfolios including antibody and protein expression.
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