Common Problems and Solutions for Recombinant Antibody Expression Experiments in lactation-Special topic on antibody modification-Tekbiotech-Yeast Display Service,Phage display technology

Common Problems and Solutions for Recombinant Antibody Expression Experiments in lactation

I. What are the main forms of recombinant antibodies?

Single-chain antibodies and Fab are the most widely used antibody fragments produced in prokaryotes. Other antibody formats have been produced in prokaryotes and eukaryotic cells, such as disulfide-stabilized scFv (ds-scFv), single-chain Fab fragments (scFab) that combine the properties of scFv and Fab, and bi- and multimeric antibody formats, such as diabodies, triabodies, transporters or mini-antibodies (miniAbs) include different formats consisting of scFv connected to an oligomerization domain, such as the immunoglobulin CH3 domain, leucine zipper, helix-turn-helix motif streptavidin or scFv-scFv tandem. Bispecific antibody formats combine two different antigen-binding domains in one molecule. The smallest antibody fragments are camel heavy chain antibodies and VHH of single domain antibodies (dAb).

For most therapeutic applications, the Fc part of the immunoglobulin is crucial for the method of action because it mediates effector functions, such as cell-dependent cytotoxicity or activation of the complement system. Therefore, antibody fragments have been fused to the Fc domain to restore effector function and affinity.

Different forms of recombinant antibodies-tekbiotech.png

Figure 1 Different forms of recombinant antibodies

II. How to increase the expression of recombinant antibodies in HEK293 cell lines?

The human embryonic kidney (HEK) 293 cell line is widely used for transient protein expression because it can efficiently transfect plasmid DNA. Some derivatives are further transformed with simian virus 40 (SV40) large T antigen (called HEK293T) or with Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) (called HEK293E) to mediate semistable episomal propagation of vectors containing SV40 or EBV replication origins (ori), respectively. Transient transfection of plasmid DNA in HEK293 cells can also be performed on a large scale by calcium phosphate transfection (180), cationic liposomes, and polymers such as polyethyleneimine (PEI).

Transient production of IgG-like scFv-Fc antibodies in the HEK293-6E cell line, a genetically modified variant with an unblocked version of EBNA1 grown in suspension and chemically refined serum-free medium, achieved volumetric yields of up to 0.6 g/L using simple shake flask cultures. Improved production media, fed-batch replenishment, and well-controlled bioreactor processes allowed higher cell densities and longer production times, both of which increased yields.

III. Transfection strategies for recombinant antibodies? How to promote transfection stability?

(1) Both genes are expressed under the control of a single promoter on the same vector; (2) Two separate vectors encoding HC (heavy chain) or LC (light chain) genes are co-transfected. In the second case, different selectable markers may be associated with the HC or LC plasmids, such as HC expression selected by co-expression of DHFR and LC by neomycin selection.

The variability in transgene expression experienced by stable transfections is often attributed to the number of transgene copies integrated, as well as to the specific integration site in the host chromatin structure. Chromatin can be subdivided into two forms, decondensed euchromatin that is transcriptionally active and condensed heterochromatin that exists in a state of transcriptional silence. Considering that heterochromatin constitutes a major part of the genome, the probability that transgenes will integrate in this region and thus be repressed is high. Furthermore, it can be assumed that recombinant DNA preferentially inserts into regions of genomic DNA that are susceptible to genomic deletion rates. There are currently two main approaches used to overcome these expression problems: (1) site-specific integration - integrating the transgene directly into known hotspots of transcriptional activity; and (2) flanking the transgene with genomic DNA elements to prevent heterochromatin repression and promote high transcriptional activity.

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