Recombinant antibodies have emerged as dominant players in the biopharma industry despite the copious quantities required for treatment. Although nonantibody biologics are typically produced at much smaller scale, are less complex, and have limited secondary modifications, their production is also fraught with challenges. Not surprisingly overcoming protein-production hurdles was a key theme at IBC’s “Bioprocessing Meeting”, held recently in La Costa CA.
“There are a host of issues to consider when looking at various expression options,” says David Robinson, Ph.D., Merck & Co. “This includes any possible impact on patient safety and the presence of adventious agents, live organisms, and other immunogenic material.”
Other factors that weigh into the decision are the specific biology of the product, since the ability to carry out secondary modifications to expressed proteins varies tremendously from one system to another. Regulatory concerns and intellectual property issues always looms in the background.
Despite the interest in alternatives, mammalian cell expression systems still dominate the industry—out of 23 blockbuster biologic drugs produced in 2006, only eight of these were generated through other means. There are a number of reasons for this dominance, but perhaps the most cogent is the vast store of experience gained over the course of four decades. This highly standardized technological base allows producers to rapidly generate large quantities of antibody with a low risk of failure.
Today, manufacturers rely on well-known stirred-tank bioreactors, which have a proven track record. With so many advantages to staying the course, why would companies want to change? The tedious process of cell-line construction is one reason. When a new gene transcript is being readied for expression, it must work its way through the transformation process, and it must be forced through layers of selection, cloning, and expansion.
Even with the process largely automated, this can take weeks of effort. Dr. Robinson estimated the entire cycle time at between four and eight months. If there is a failure or a breakdown in the process, weeks or months more will be required to bring the task to successful fruition, and the commitment continues, since with the slow generation time of the mammalian cell, the culturing process itself will take from 10 to 30 days to expand the volume of material appropriately.
But one of the most difficult issues to deal with is the transient and unstable process of glycosylation in mammalian cells, which frequently proves difficult for the investigator to control.
Animal cells also produce nonhuman varieties of glycans, and while these are generally well tolerated, there are exceptions. So alternatives to the mammalian cell must possess the ability to express multiple human proteins with the correct post-translational modifications, as well as perform appropriate glycosylations with ease, all in the context of existing physical plant hardware.
Dr. Robinson and his team compared proteins expressed in Pichia pastoris with those expressed in mammalian cells and have observed that IgG1 molecules expressed in the severely engineered GlycoFi Pichia strain have similar antigen binding ability and in vitro efficacy as that expressed in CHO cells. Moreover, erythropoetin, expressed in the same yeast strain has the appropriate mass and activity compared to molecules produced in the CHO cell line. Because the yeast cell cycle is much shorter than that of a mammalian cell, combining its biological properties with robotic automated systems for cloning and selection makes the process of developing new transformed cell lines more convenient.
“Indeed, the whole process of moving from transformants to high-producing cell lines was completed in weeks in the Merck facilities, rather than the months it would have taken with traditional mammalian cell technology,” stated Dr. Robinson. “I believe that our platform provides an opportunity to produce high-quality homogeneous therapeutics that will enable novel biologic therapies.”
Since wild type yeast proteins are heavily mannosylated, it was necessary to develop cell lines that were humanized with respect to their glycosylation ability. The Merck research facility boasts an extensive GMP manufacturing suite with nine units, which allows for the simultaneous processing of mammalian cells, microbial, and viral products. With these facilities in place Dr. Robinson and his team are able to rapidly scale up to grams per liter titers.