A big picture approach to recombinant protein production

An increasing number of blockbuster drugs are recombinant mammalian proteins. In fact, the FDA has approved over 100 for therapeutic use. Production of recombinant proteins in mammalian hosts allows scientists to generate proteins with the post-translational modifications that contribute to the overall function of the protein. In other words, these proteins more closely mimic in vivo functions compared to those produced in other hosts such as bacteria, yeast, or insect cells. Let us take a brief look into the main elements of protein production in mammalian hosts to get a better picture of what it entails. 

Host Cell Lines

CHO
These are hardy, reliable cells that allow for adaptability to serum-free conditions and growth at very high densities. Many human pathogens do not replicate in CHO, which is advantageous from a regulatory standpoint. It is also worth noting that CHO lines facilitate glycosylation of recombinant proteins, which is important for overall function. 

HEK293
Proteins produced in this line are actually a closer match to in vivo human proteins. Scientists have also developed the HKB11 cell line (hybrid between HEK293S cell line and human B cell line) which is easy to transfect and secretes very large quantities of protein.  

Methods of delivery

Transient transfection 

Plasmid DNA is expressed using reagent. There is no need for clonal selection because the gene/s are not incorporated into the genome of the host. Downside is large quantities of plasmid are required for effective delivery. 

Stable transfection 

Gene of interest is expressed using editing technology such as CRISPR. Requires a clonal selection step. 

Recombinase mediated cassette exchange 

Procedure in which a preexisting gene cassette is exchanged for an analogous cassette carrying the gene of interested. For protein production, the recombinant target is directed to a genomic “hotspot” to increase yield.  

Viral delivery 

Allows for delivery of nucleic acid at large volume, produces high yield of secreted protein without the need for clonal selection.  

Clonal selection 

Methotrexate (MTX) 

Inhibits dihydrofolate reductase (DHFR). Used in combination with DHFR deficient lines in which the DHFR clonal selection gene is supplied in the expression cassette containing the gene of interest.  

Glutamine Synthetase (GS) 

Methionine sulfoximine (MSX) inhibits GS. Used in combination with GS-deficient cell lines in which the GS clonal selection gene is supplied in the expression cassette containing the gene of interest. 
Benefit to this approach: The drug concentration can be increased to “fine tune” and generate the strongest cell clones that produce the most protein. 

Expansion methods

Batch

All nutrients are supplied in the initial base medium

Fed-batch

Nutrients are added as they are depleted 

Perfusion

Medium is circulated through a growing culture, while waste is removed, additional nutrients are supplied, and the product is harvested.

Written by Angela

Scientific Support Specialist