(566a) Manufacturing of Aglycosylated Monoclonal Antibodies in Yeast

The COVID-19 pandemic revealed insufficient global manufacturing capacity for biologic medicines including vaccines, antiviral drugs, and monoclonal antibodies (mAbs). Before the pandemic, mAbs were the fastest growing class of biologic medicines by number of new approved products and biosimilars, number of patients, and total revenue. This rapid growth is in part due to platform manufacturing processes based on expression in Chinese hamster ovary (CHO) cells and purification with Protein A chromatography. While reliable, CHO cells require complex culture media, stringent sterilization, and viral filtration of the secreted mAb product, all of which contribute to high manufacturing costs. Additionally, development of a stable CHO cell line for manufacturing may take six months or more, due to the slow growth rate. Indeed, development of new mAb treatments for COVID-19 has been outpaced by evolving variants of SARS-CoV-2. New platforms for manufacturing mAbs with shorter development timelines, lower production costs, and larger scales are needed to increase global access and improve pandemic response.

Single-celled eukaryotic hosts such as yeast may be promising alternatives to CHO cells for mAb manufacturing. Yeasts grow quickly to high cell densities on inexpensive media, which enables low-cost fermentation processes. The yeast Komagataella phaffii (Pichia pastoris) is particularly suited for mAb manufacturing because it contains an advanced secretory pathway, secretes few native host-cell proteins, and is routinely used to manufacture therapeutic proteins like insulin and subunit vaccines. In 2020, the FDA approved eptinezumab (Vyepti), an aglycosylated mAb manufactured by Alder/Lundbeck in K. phaffii for treatment of migraine. Aglycosylated mAbs are a growing product class, and could be developed, in theory, for any indication that does not require a particular effector function, such as antiviral therapy. Further engineering and characterization are needed to encourage the widespread adoption of K. phaffii and other microbial hosts over traditional CHO cell processes.

Here, we report a platform for reliable manufacturing of aglycosylated mAbs in K. phaffii. We developed a modular vector for simple integration and expression of both protein chains, and identified an effective signal peptide for secretion. We assessed the quality of mAbs secreted from K. phaffii, and engineered a human IgG1 mAb backbone sequence with minimal modifications that eliminate most product-related variants. We integrated five different aglycosylated mAbs into a strain of K. phaffii that we previously engineered to have enhanced secreted productivity, and demonstrated consistent production of high-quality drug substance at lab scale. Finally, we demonstrated fed batch and perfusion manufacturing of mAbs using both K. phaffii and CHO platforms. We observed comparable product quality between the two hosts. While the total titer was higher for CHO-based processes, the space-time yield (g/L/day) was comparable between CHO and the microbial host.

These results demonstrate a reliable platform for expression of multiple mAbs in yeast. Microbial hosts, generally, will enable manufacturing of mAbs and other therapeutic proteins at low costs and large scales that are not achievable with mammalian expression systems. The speed of lab scale expression and cell line development, additionally, will enable rapid development and preclinical testing of new antibody products to reduce development costs and assist with pandemic response.