The Effect of Downstream Processing Steps on Process Yield and Protein Quality

Abstract

Proteinaceous drug substances produced in or recovered from a biological source are called biologics or biopharmaceuticals. This class of drugs has grown exponentially with the advances made in genetic engineering and recombinant DNA technology in the 1970s and 80s. Many previously untreatable conditions and autoimmune diseases now have treatment options thanks to biologics produced by genetically modified mammalian cell cultures. These proteinaceous therapies are often large subunit or multi-subunit substances requiring specific folding mechanisms to take place for an active version of the product. Mammalian cell machinery allows for proper post-translational modification and folding.

The biologics industry brings in an annual $275 billion in revenue and more than a quarter of all FDA approved drugs are in this category. Biologics are more complex drug substances than the traditional small-molecule drugs produced by chemical synthesis and their cost of manufacture follows suit: biologics range from $100- 1000/g while synthesized drugs can be made as cheaply as $5/g. The high cost is reflected in the finely-tuned growth media and operating parameter control required for cultivation, and the optimized multi-step purification scheme. The steep cost to manufacture necessitates high yield purification steps for economic feasibility.

Careful consideration for each purification step is required to meet high recovery demands. Investigation of buffer chases after depth filtration for primary clarification with mAb producing CHO cultures brought insight to host cell protein and dsDNA removal as well as mAb recovery. While performing a high ionic strength buffer chase could recover more mAb, the tradeoff of dsDNA released was unfavorable.

Affinity purification of two COVID-19 spike protein variants (S-2P and HexaPro) produced in HEK 293 cell cultures was also investigated. The spike variants were designed with cleavable purification tags enabling affinity purification and subsequent tag removal. While investigating various desalting steps (gel filtration, normal flow filtration with spin tubes, and TFF) of affinity purified spike proteins a visible turbidity was observed in TFF-processed samples. The presence of turbidity (presumably due to protein aggregation) prompted further investigation of the origin of turbidity. Performing SEC on affinity purified spike protein samples confirmed the presence of spike protein aggregates. Once tags were cleaved from the sample, SEC analysis estimated the trimeric spike protein primarily in the non-aggregated form and concluded that the very hydrophobic nature of the amino acid residues that make up the tails encouraged aggregation of the trimers in an aqueous environment.