High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Optimization and automation of an end-to-end high throughput microscale transient protein production process.

High throughput protein production from transient transfection of mammalian cells is used in multiple facets of research and development studies. Commonly used formats for these high number expressions are 12-, 24- and 96-well plates at various volumes. However there are no published examples of a 96-deep well plate microscale (1,000 µL) suspension process for mammalian transient expression. For this reason, we aimed to determine the optimal operating conditions for a high producing, microscale HEK293 transient system. We evaluated the hydrodynamic flow and measured the oxygen transfer rate (OTR) and transient protein expression for 96-deep well plates of different well geometries filled at 600-1,000 µL working volumes and agitated at various speeds and orbital diameters. Ultimately, a round well-round bottom (RR) 96-deep well plate with a working volume of 1,000 µL agitated at 1,000 RPM and a 3 mm orbital diameter yielded the highest and most consistent total transient protein production. As plate cultures are subject to evaporation, water loss from different plate seals was measured to identify an optimal plate sealing method. Finally, to enable higher capacity protein production, both expression and purification processes were automated. Functionality of this end-to-end automation workflow was demonstrated with the generation of high levels of human IgG1 antibodies (≥360 µg/mL) with reproducible productivity, product quality and ≥78% purification recovery.

Development of a semi-automated high throughput transient transfection system.

Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.