Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals

Abstract

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a “hit-or-miss” strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody–drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

“Build Your Own” ADC Mimics: Identification of Nontoxic Linker/Payload Mimics for HIC-Based DAR Determination, High-Throughput Optimization, and Continuous Flow Conjugation

“…Transferring this principle to conjugation process development would require identifying LP mimics that (i) are nontoxic; (ii) show similar conjugation reactivity compared to actual linker payloads; and (iii) enable the use of established analytical methods for conjugation analysis. Since analytical hydrophobic interaction chromatography (HIC) is often considered the gold standard for determining an ADC’s drug-to-antibody ratio (DAR; a key quality attribute of ADCs), − we focused our efforts on identifying linkers that mimic HIC shifts of the commonly used monomethyl auristatin E (MMAE) payload . Herein, we report the identification of HIC-shifting LP mimics (Scheme ) that allow HIC detection of maleimide conjugation to both engineered − and interchain , cysteines.…”

“…6 μL of the resulting solution (corresponding to 10 μg) was injected onto a HIC column (Sepax Proteomix Phenyl NP-1.7 4.6 × 100 mm 2 , 1.7 μm); analysis was performed with a gradient of 85% mobile phase A (3.0 M NH 4 OAc with 50 mM K 2 HPO 4 , pH 7.0, 5% acetonitrile)/15% mobile phase B (5% acetonitrile) to 25% A/75% B (column temperature 35 °C, see the SI for further method details). These analytical conditions had previously been identified through an automated HIC screening workflow 16 for vcMMAE conjugates of the mAb bearing engineered cysteines. Peak detection was performed at 280 nm; high-throughput visualization and integration was performed using Virscidian Analytical Studio Professional.…”

“Build your own” ADC mimics: Identification of non-toxic linker/payload mimics for HIC-based DAR determination, high-throughput optimization, and continuous flow conjugation

“…Transferring this principle to conjugation process development would require identifying LP mimics that (i) are non-toxic; (ii) react similarly to actual linker-payloads in conjugation; and (iii) enable the use of established analytical methods for conjugation analysis. Since analytical hydrophobic interaction chromatography (HIC) is often considered the gold standard for determining an ADC's drug-to-antibody ratio (DAR; a key quality attribute of ADCs), [15][16][17][18][19][20] we focused our efforts on identifying linkers that mimic HIC shifts of the commonly used Monomethyl Auristatin E (MMAE) payload. 1 Herein, we report the identification of HICshifting LP mimics (Scheme 1) that allow HIC detection of maleimide conjugation to both engineered [21][22][23] and interchain 24,25 cysteines.…”