ZFN Technology Advances Biopharmaceutical Manufacturing - Applications of ZFN technology in biopharmaceutical cell-line engineering. - BioPharm International

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ZFN Technology Advances Biopharmaceutical Manufacturing
Applications of ZFN technology in biopharmaceutical cell-line engineering.


BioPharm International
Volume 26, Issue 4, pp. 64-66

Increased r-protein production

There are several other ways to boost the r-protein yield besides improving the selection process. Genes related to apoptosis can be targeted and knocked out, resulting in longer culture life. Genes that correlate with growth and productivity can be manipulated by changing existing elements that control gene expression.

Another potential method for boosting r-protein yield is a targeted integration approach. Traditionally, r-protein DNA integrates randomly into the genome. Several clones must be screened to isolate a stable, high-producing clone. If a desirable integration region is identified, ZFNs can be used to precisely integrate the transgene at that location, which can lead to higher-producing and consistently stable clones.

Managing post- translational modifications

Because of genetic differences between CHO and human cells, r-proteins that are manufactured in CHO cells may have different glycosylation patterns compared with proteins manufactured by human cells. These differences can cause an immunogenic response when the drug is administered to the patient. Two examples of glycosylation differences include Neu5Gc moieties and alpha 1, 3-galactose (alpha-gal) moieties. The genes responsible for these glycosylation patterns are functional in CHO cells, but not in humans. A r-protein produced in CHO cells may therefore contain Neu5Gc or alpha-gal moieties that could cause an immunogenic response when administered. Knocking out the genes responsible for these glyco-proteins can eliminate this risk.

Molecule efficacy can also be increased by engineering glycosylation patterns that increase the residence time of the drug in the bloodstream or by increasing the binding of the Fc region of the antibody to the Fc receptor. The circulating half-life of therapeutic recombinant glycoproteins can be improved by increasing the sialic acid concentration. Targeting genes that increase sialic acid concentrations can increase the residence time of the drug. Increased antibody-dependent cellular cytotocicity (ADCC) can be achieved by creating antibodies that have greater binding affinity to Fc receptors. Non-fucosylated glyco-proteins have greater binding affinity to Fc receptors, and knocking out genes responsible for fucosylation can result in more efficacious r-antibodies.

Management of post-translational modifications is also important in biosimilar manufacturing, when the glyco-profile of the original product must be matched. In these cases, ZFNs can be used to target genes that impact the glyco-profile to engineer a cell line that can produce a r-protein that matches the innovator material.

Improved downstream processing

ZFNs can be used to improve downstream processing by knocking out genes that encode interfering host-cell proteins. If the CHO cell line contains an endogenous protein that copurifies with the r-protein during chromatographic purification, additional and costly steps may be required to remove the endogenous protein. ZFNs can be used to knock out the gene that encodes this endogenous protein. Another potential target may be a protein within the CHO cell that binds the therapeutic r-protein. By knocking out the gene that encodes such a protein, growth and productivity can be improved. The CHO host cell may also produce proteolytic enzymes that could degrade the product before purification. Diminishing protease expression can minimize this effect.

Risk mitigation

The risk of prion or viral infection can be mitigated through genome editing. Retroviral titer in a cell could be reduced by targeting and removing retroviral elements. Additionally, viral uptake pathways can be targeted, conferring resistance to viral attack. Similarly, genes for prion proteins can be targeted and removed.

Combining ZFN modifications

Another benefit of ZFNs is that multiple modifications can be performed in the same clone. Desirable ZFN modifications can be trait stacked into the same cell line, enabling the potential development of a "super" CHO line precisely engineered to efficiently produce safe and effective therapeutic proteins.

GENOMIC CHANGES IMPROVE PRODUCTIVITY

Genome editing has vastly improved since the creation of the DUKXB11 cell line. Since 2009, SAFC has applied the ZFN technology to the development of robust CHO cell lines by introducing genomic changes that improve the productivity and processing characteristics of biopharmaceutical manufacturing cell lines. More than 30 specific modifications are available to the biopharmaceutical industry. Through microarray experiments, several key genes that impact cell growth and productivity have been identified and explored.

SAFC has several R&D scientists who identify and validate new genetic alterations that are relevant to the biopharmaceutical industry. They have created the CHOZN GS (GS-/-) and CHOZN DHFR (DHFR -/-) knock-out cell lines. Other available cell lines include knock-outs of GGTA (-/-) and CMAH (-/-), which result in cell lines that produce r-proteins without alpha-gal or Neu5Gc moieties, respectively.

Kate Achtien is an R&D scientist at SAFC,


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