Abstract
Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire-time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.
Highlights
C Protein A magnetic beadsImmunosorbent assay (ELISA) were typically employed as a primary screen
Platform fedbatch production was performed in shake flasks with proprietary chemically defined media together with bolus feeds on days 3, 7, and 10 with a temperature to 35 °C on day 3
RapidFire pump 1 supplied loading buffer (0.1% formic acid, 10% acetonitrile) at 200 μL/min
Summary
Immunosorbent assay (ELISA) were typically employed as a primary screen. Using these assays, each BsIgG campaign requires significant assay development and validation effort up front when using these traditional analytical methods. While MS is routinely used to characterize the quality of the final purified bispecific antibody [23,24,25], throughput remains a limitation for screening applications. Using the RF-MS assay we can rapidly screen hundreds, and in principle, thousands of cell line clones through characterizing and quantitating bispecific antibodies to identify the clone that generates the desired BsIgG with minimal mispaired side products. Unlike with the ELISA detection traditionally used for BsIgG screening, the multiplexed RF-MS detection capability affords comprehensive identification of multiple species in a single label-free experiment
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