In the field of recombinant production of therapeutic proteins, nonhuman cells such as the Chinese hamster ovary (CHO) or the mouse myeloma cell line (NS0) are routinely used. Genetic differences, however, between these cells and human cells can lead to the production of recombinant proteins with post-translational modifications that could cause immunogenic responses in human patients. Although in some cases these problems can be avoided through careful control of the manufacturing process, a safer solution is to completely eliminate the possibility of immunogenic variants by knocking out the genes that lead to the production of the problematic variants.
“As a major raw material supplier to the biopharmaceutical industry, our goal at SAFC is to ultimately develop a “Super CHO” expression system consisting of cell lines, vectors, media, and feeds that will provide maximum output of biotherapeutic proteins that meet all of the needs of the industry in terms of efficacy and safety. As a result, we are targeting genes in our CHOZN platform that have been identified as potentially problematic and are designing alternative cell lines stacked with knockouts for them,” notes Kevin Kayser, director at SAFC Commercial.
New dual knockout cell line
One of the newest cell lines from SAFC is a dual knockout line, which was created in the robust CHOZN GS-/- host. The CHOZN GS -/- host allows for rapid methionine sulfoximine (MSX)-free generation of stable producing clones. The new traits stacked into the host cell line are the dual knockouts of the CMAH and GGTA1 gene sequences. CMAH and GGTA1 encode proteins that are responsible for the presence of Neu5Gc sialic acid and α-gal moieties on the glycan structures, respectively. Since humans lack functional CMAH and GGTA1 genes, a patient may have an immunogenic response to glycoproteins containing these moieties. The knockout of the function of these two genes enables the expression of recombinant proteins that do not contain the antigenic α-gal and Neu5Gc moieties on the glycan structures.
“There is a growing body of literature on immunogenicity and a growing interest in this issue in the biopharmaceutical industry. We believe that by developing new cell lines with knockouts of gene sequences that lead to the formation of variants that have the potential to create immunogenic responses, we are at the forefront of a new wave of developments in biologics manufacturing,” observes Kayser. He expects that as more data continue to come in, there will be growing demand for cell lines specifically designed to avoid such problematic moieties as a-gal and Neu5GC. The trait-stacked cell line is now commercially available from SAFC as a custom product.
Importance of the CMAH gene sequence
The CMAH gene encodes cytidine monophosphate–sialic acid hydroxylase, an enzyme responsible for converting the terminal sialic acid group of a glycoprotein from the Neu5Ac form to the hydroxylated Neu5Gc form. Unlike CHO and mouse cell lines, humans lack a functional CMAH gene, and, therefore, do not produce glycoproteins containing Neu5Gc moieties. Humans may have circulating antibodies against Neu5Gc, however. Therefore Neu5Gc-containing recombinant proteins may have reduced efficacy and serum half-life in these patients compared to the Neu5Ac variants. Most importantly, Neu5Gc-containing therapeutics may elicit a dangerous immune response when administered to the patient.
Although CHO cells produce less of the Neu5Gc form of sialic acid than other mammalian cell types, such as mouse cell lines, the presence of the functional CMAH gene still poses an avoidable safety risk.
“While CHO cells do not produce a significant amount of this form of scialic acid, and often no immunogenicity is observed, we believe that since it is possible to eliminate the production of the Neu5GC variant, it is best to avoid the problem completely,” asserted Kayser. Thus, SAFC has developed a CHO cell line with the CMAH disrupted, thereby eliminating the possibility of Neu5Gc containing glycoproteins.
Why disrupt the GGTA1 gene sequence?
The GGTA1 gene encodes galactosyltransferase alpha 1,3, which is responsible for adding an additional galactose sugar in the 1,3 configuration. This particular variant is known as 1,3-α-gal, or α-gal, for short. The addition of this second galactose unit is strongly associated with immunogenicity. In fact, α-gal has been recognized as a problem in the transplantation field for many years. In terms of recombinant proteins, Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor used for the treatment of metastatic colorectal cancer and head and neck cancer, is also thought to have some immunogenicity associated with it as a result of the presence of α-gal moieties, according to Kayser.
“What is very interesting is that a review of clinical trial results for Cetuximab showed that many of the immunogenic responses occurred in people from Southern states, where the lone star tick is known to exist. This tick transmits the α-gal moiety from raccoons to humans, and while rare, there are people with antibodies to α-gal that experience an allergic (anaphylactic, in the most severe cases) response,” Kayser notes.
Although production of α-gal is far more prevalent in mouse myeloma cells, under certain batch manufacturing conditions, CHO cells can produce glycoproteins containing up to 4% α-gal, according to Kayser. In most cases, this level can be significantly reduced by properly controlling the nutrient feeds, but it is not known how much influence the oxygen consumption, pH, and other reaction conditions can affect the production of α-gal, or the precise levels of α-gal in any given therapeutic protein that will elicit the immune response.
“We have therefore again taken the approach that it is much better and safer to avoid the problem completely by knocking out the relevant gene,” Kayser says. “By knocking out the GGTA1 gene, we completely eliminate the possibility that the recombinant protein will contain α-gal moieties.”Future developments: working toward a “Super CHO” platform
SAFC is looking to improve the manufacturing performance of its CHOZN cell line by obtaining higher titers, better growth, and increased cell longevity, particularly in new perfusion systems. The company is also looking at sugar chemistry, including the alteration of glycan structures to achieve improved efficacy or to match the performance of a biosimilar to the branded drug. The development of a virus-resistant manufacturing CHO cell line is also a primary target.
“While infection of manufacturing systems does not happen often—and there are extensive controls in place to prevent such an event—it does occur, and once again, the best solution is to avoid the problem,” Kayser explained.
With respect to its host cell platform, SAFC is also looking to knock out genes that are common in CHO and human cells that can cause problems during manufacturing, such as interference with protein production or the production of proteins that are similar to and cannot be readily separated from the desired product.