Ultrasensitive disulfide scrambling analysis of mAbs by LC-MS with post-column reduction and glycine signal enhancement.

Explicitly confirming the complete disulfide bond linkage pattern of a monoclonal antibody (mAb) presents a challenge in the biopharmaceutical industry. Although proper native disulfide connections are in high abundance for analytical purposes within a peptide mapping digest under non-reducing conditions, disulfide scrambling can also exist but be difficult to detect, let alone characterize, particularly at low levels. Here, we developed an ultrasensitive high-confidence method for identifying explicit disulfide connectivity in mAbs. By applying a post-column addition of tris (2-carboxyethyl)phosphine hydrochloride (TCEP) to the liquid chromatography (LC) eluent of a non-reduced mAb digest, partial reduction of disulfide peptides is achieved after the initial peptide separation, allowing both the parent disulfide and its reduced daughter peptides to co-elute for simultaneous mass spectrometry (MS) detection. Combining this concept with the recently discovered ability of glycine to enhance MS signal when added to the LC eluent, we demonstrate a method for detecting, characterizing and quantifying low-abundance disulfide scrambling in mAbs.

Isobaric Tandem Mass Tag Multiplexed Post-Translational Modification Quantitation of Biopharmaceuticals by Targeted High-Resolution Mass Spectrometry.

Peptide mapping coupled with liquid chromatography-mass spectrometry (LC-MS) has become an essential analytical technique to quantify the quality attributes (e.g., post-translational modifications [PTMs]) of monoclonal antibodies (mAbs) during drug development. However, the traditional label-free approach for relative quantitation of PTMs requires a great amount of instrument time for LC-MS data acquisition of individual digested samples, which limits the efficiency of peptide mapping when there is an increasing demand for protein characterization. Here, we developed a tandem mass tag (TMT)-based approach in combination with targeted mass spectrometry for multiplexed site-specific PTM quantitation of monoclonal antibodies to overcome this limitation. This approach enables the simultaneous quantitation of quality attributes (e.g., PTMs) for multiple samples in a single LC-MS run. By adjusting higher-energy collision dissociation (HCD) normalized collisional energies (NCEs) from 35 to 90, different types of PTMs were quantified with percentages comparable to those obtained using the conventional approach. The TMT overlabeling on the off-target amino acid residues serine, threonine, and tyrosine was observed to pose a challenge for this targeted MS/MS-based PTM quantitation. However, we inhibited this off-target overlabeling by adding a small-molecule additive during the TMT labeling as a decoy reagent to deplete the excess amount of TMT reagent. The PTM quantitative performance of this approach demonstrated high sensitivity and reproducibility of PTM quantitation with levels as low as 1.0%. Finally, this approach has been utilized to quantify the PTMs for forced degradation samples, comparability samples, and trisulfide standards of monoclonal antibodies.

Simple Addition of Glycine in Trifluoroacetic Acid-Containing Mobile Phases Enhances the Sensitivity of Electrospray Ionization Mass Spectrometry for Biopharmaceutical Characterization.

Trifluoroacetic acid (TFA) is a commonly used mobile phase additive in liquid chromatography-mass spectrometry (LC-MS)-based biopharmaceutical characterization to enhance reversed-phase chromatographic performance of peptide separation; however, it leads to significant mass spectrometry signal suppression during electrospray ionization. Previous studies have shown that introducing large amounts of carboxylic acids or ammonium hydroxide to LC eluents postcolumn can improve MS sensitivity. In this study, we discovered glycine as a simple additive for TFA mobile phases, which mitigates ion suppression through a similar mechanism but in a more effective way than weak acid or weak base and boosts mass spectrometry responses (signal-to-noise ratio) of peptides by more than 1 order of magnitude on average after directly adding a small amount (e.g., 2 mM) into TFA mobile phases without compromising the chromatographic performance of peptide separation. Other small molecule additives in TFA mobile phases, such as amino acids containing extended side chains with different chemical properties and glycine-based variants, were also evaluated. The results demonstrated that glycine offered the best response boosting on peptides. The discovery of this glycine additive in TFA mobile phases provides a simple and conventional approach to achieve greater mass spectrometry detection sensitivity than TFA mobile phases for LC-MS-based characterization of biopharmaceuticals.

Kinetics of Trisulfide-to-Disulfide Conversion of Therapeutic IgG1 Monoclonal Antibodies Under Physiological Conditions: A Case Study of Casirivimab And Imdevimab.

The percentage of trisulfide variants is a product quality metric that is monitored during the manufacture of monoclonal antibody (mAb)-based therapeutics. Results from earlier preclinical studies revealed that trisulfide linkages in mAbs are rapidly converted to disulfides in circulation. In this study, casirivimab and imdevimab, which are both IgG1 subclass mAbs that target the non-overlapping epitopes in SARS-CoV2 Spike protein, are used as models to study the kinetics of trisulfide-to-disulfide conversion in vivo in human circulation. To determine the percentage of trisulfide variants in systemic circulation immediately after intravenous injection, both mAbs were immunoprecipitated from serum samples collected from COVID-19 patients that received this cocktail antibody treatment as part of a first-in-human study. The immunoprecipitated mAbs were then digested under non-reducing conditions and evaluated by liquid-chromatography-mass spectrometry (LC-MS). Significant reductions in the percentages of trisulfide variants were observed in serum samples as early as 1 hr after completion of the intravenous infusion. A flow-through dialysis model designed to mimic the redox potential of blood revealed a plausible chemical mechanism for the rapid trisulfide-to-disulfide conversion of IgG1 subclass mAbs under physiological conditions.

Discovery of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1: optimization of kinase selectivity and pharmacokinetics.

The kinase selectivity and pharmacokinetic optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. The intersection of insights from molecular modeling, computational prediction of metabolic sites, and in vitro metabolite identification studies resulted in a simple and unique solution to both of these problems. These efforts culminated in the discovery of compound 13a, a potent, relatively selective inhibitor of TAK1 with good pharmacokinetic properties in mice, which was active in an in vivo model of ovarian cancer.

Discovery of potent, selective and orally bioavailable imidazo[1,5-a]pyrazine derived ACK1 inhibitors.

This Letter describes the medicinal chemistry effort towards a series of novel imidazo[1,5-a]pyrazine derived inhibitors of ACK1. Virtual screening led to the discovery of the initial hit, and subsequent exploration of structure-activity relationships and optimization of drug metabolism and pharmacokinetic properties led to the identification of potent, selective and orally bioavailable ACK1 inhibitors.

Discovery and optimization of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1.

The discovery and potency optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. Micromolar hits taken from high-throughput screening were optimized for biochemical and cellular mechanistic potency to ~10nM, as exemplified by compound 12az. Application of structure-based drug design aided by co-crystal structures of TAK1 with inhibitors significantly shortened the number of iterations required for the optimization.

Novel 6-aminofuro[3,2-c]pyridines as potent, orally efficacious inhibitors of cMET and RON kinases.

A series of novel 6-aminofuro[3,2-c]pyridines as kinase inhibitors is described, most notably, OSI-296 (6). We discuss our exploration of structure-activity relationships and optimization leading to OSI-296 and disclose its pharmacological activity against cMET and RON in cellular assays. OSI-296 is a potent and selective inhibitor of cMET and RON kinases that shows in vivo efficacy in tumor xenografts models upon oral dosing and is well tolerated.

Potent and selective cyclohexyl-derived imidazopyrazine insulin-like growth factor 1 receptor inhibitors with in vivo efficacy.

Preclinical and emerging clinical evidence suggests that inhibiting insulin-like growth factor 1 receptor (IGF-1R) signaling may offer a promising therapeutic strategy for the treatment of several types of cancer. This Letter describes the medicinal chemistry effort towards a series of 8-amino-imidazo[1,5-a]pyrazine derived inhibitors of IGF-1R which features a substituted quinoline moiety at the C1 position and a cyclohexyl linking moiety at the C3 position. Lead optimization efforts which included the optimization of structure-activity relationships and drug metabolism and pharmacokinetic properties led to the identification of compound 9m, a potent, selective and orally bioavailable inhibitor of IGF-1R with in vivo efficacy in an IGF-driven mouse xenograft model.

Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray.

Growth in the pharmaceutical industry has led to an increasing demand for rapid characterization of therapeutic monoclonal antibodies. The current methods for antibody sequence confirmation (e.g., N-terminal Edman sequencing and traditional peptide mapping methods) are not sufficient; thus, we developed a fast method for sequencing recombinant monoclonal antibodies using a novel digestion-on-emitter technology. Using this method, a monoclonal antibody can be denatured, reduced, digested, and sequenced in less than an hour. High throughput and satisfactory protein sequence coverage were achieved by using a non-specific protease from Aspergillus saitoi, protease XIII, to digest the denatured and reduced monoclonal antibody on an electrospray emitter, while electrospray high voltage was applied to the digestion mixture through the emitter. Tandem mass spectrometry data was acquired over the course of enzyme digestion, generating similar information compared to standard peptide mapping experiments in much less time. We demonstrated that this fast protein sequencing method provided sufficient sequence information for bovine serum albumin and two commercially available monoclonal antibodies, mouse IgG1 MOPC21 and humanized IgG1 NISTmAb. For two monoclonal antibodies, we obtained sequence coverage of 90.5-95.1% for the heavy chains and 98.6-99.1% for the light chains. We found that on-emitter digestion by protease XIII generated peptides of various lengths during the digestion process, which was critical for achieving sufficient sequence coverage. Moreover, we discovered that the enzyme-to-substrate ratio was an important parameter that affects protein sequence coverage. Due to its highly automatable and efficient design, our method offers a major advantage over N-terminal Edman sequencing and traditional peptide mapping methods in the identification of protein sequence, and is capable of meeting an ever-increasing demand for monoclonal antibody sequence confirmation in the biopharmaceutical industry.

Discovery of novel insulin-like growth factor-1 receptor inhibitors with unique time-dependent binding kinetics.

This letter describes a series of small molecule inhibitors of IGF-1R with unique time-dependent binding kinetics and slow off-rates. Structure-activity and structure-kinetic relationships were elucidated and guided further optimizations within the series, culminating in compound 2. With an IGF-1R dissociative half-life (t 1/2) of >100 h, compound 2 demonstrated significant and extended PD effects in conjunction with tumor growth inhibition in xenograft models at a remarkably low and intermittent dose, which correlated with the observed in vitro slow off-rate properties.

Discovery of an Orally Efficacious Imidazo[5,1-f][1,2,4]triazine Dual Inhibitor of IGF-1R and IR.

This report describes the investigation of a series of 5,7-disubstituted imidazo[5,1-f][1,2,4]triazine inhibitors of insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (IR). Structure-activity relationship exploration and optimization leading to the identification, characterization, and pharmacological activity of compound 9b, a potent, selective, well-tolerated, and orally bioavailable dual inhibitor of IGF-1R and IR with in vivo efficacy in tumor xenograft models, is discussed.