An end-to-end automated platform process for high-throughput engineering of next-generation multi-specific antibody therapeutics

Abstract

Next-generation multi-specific antibody therapeutics (MSATs) are engineered to combine several functional activities into one molecule to provide higher efficacy compared to conventional, mono-specific antibody therapeutics. However, highly engineered MSATs frequently display poor yields and less favorable drug-like properties (DLPs), which can adversely affect their development. Systematic screening of a large panel of MSAT variants in very high throughput (HT) is thus critical to identify potent molecule candidates with good yield and DLPs early in the discovery process. Here we report on the establishment of a novel, format-agnostic platform process for the fast generation and multiparametric screening of tens of thousands of MSAT variants. To this end, we have introduced full automation across the entire value chain for MSAT engineering. Specifically, we have automated the in-silico design of very large MSAT panels such that it reflects precisely the wet-lab processes for MSAT DNA library generation. This includes mass saturation mutagenesis or bulk modular cloning technologies while, concomitantly, enabling library deconvolution approaches using HT Sanger DNA sequencing. These DNA workflows are tightly linked to fully automated downstream processes for compartmentalized mammalian cell transfection expression, and screening of multiple parameters. All sub-processes are seamlessly integrated with tailored workflow supporting bioinformatics. As described here, we used this platform to perform multifactor optimization of a next-generation bispecific, cross-over dual variable domain-Ig (CODV-Ig). Screening of more than 25,000 individual protein variants in mono- and bispecific format led to the identification of CODV-Ig variants with over 1,000-fold increased potency and significantly optimized production titers, demonstrating the power and versatility of the platform.

High-Throughput and Format-Agnostic Mispairing Assay for Multispecific Antibodies Using Intact Mass Spectrometry

“…46,57−59 Another advantage of the HT screening is the collection of large data sets which can be used for machine learning and AI (artificial intelligence)-based approaches to improve the individual lead and also the format platform. 46 Similarly, accumulated data are key for the development of appropriate algorithms for in silico prediction of molecular attributes that can be applied for early deselection of binders with inappropriate properties. 46 Despite the broad range of possible applications for the HT MS workflow, every process has its optimization potential.…”

“…46 Similarly, accumulated data are key for the development of appropriate algorithms for in silico prediction of molecular attributes that can be applied for early deselection of binders with inappropriate properties. 46 Despite the broad range of possible applications for the HT MS workflow, every process has its optimization potential. The measurement time of the described RP-HT-MS workflow of 10 min per sample could potentially be further reduced by applying different new, rapid separation methods such as RapidFire-MS or SampleStream-MS with injection times of 15 s per sample.…”

“…All protein production campaigns were realized via automated transient expression on the s.c Protein Science Station (PPS). 46 Freestyle HEK293 (Life) cells growing in F17 serum-free suspension culture (Life) were transfected with the expression plasmid. Transfections were performed using a polyethyleneimine-mediated, transient transfection method.…”

“…In both cases, selection or engineering, a high number of candidates needs to be produced and analyzed making high-throughput (HT) production and characterization workflows a key factor to success. Thus, HT analytical assays gain more and more importance especially in the pharmaceutical industry where thousands of molecules require screening in physicochemical and functional assays to select the desired characteristics and profiles. − Several analytical methods are available to characterize mispaired species including chromatographic techniques, electrophoretic methods, functional characterization, and liquid chromatography coupled with mass spectrometry (LC-MS). A review of these techniques can be found elsewhere .…”

Democratizing the rapid screening of protein expression for materials development

“…In comparison to many other existing high-throughput approaches, this system reflects the upstream batch production very closely. Most current high-throughput approaches generate new targets via synthetic biology 70,71 or screen large libraries, 72 which would be highly complementary to this new approach. By making this system widely available to the protein materials community with limited initial investment (a complete cost table for the platform is available in the ESI, † section J), this system represents a first step toward rapid protein materials design cycles.…”