(T1430-01-03) Val-His-Ser (VHS) N-terminal Leader Sequence and Implications for mAb Subcutaneous Pharmacokinetics

Purpose: Elevated levels of Val-His-Ser (VHS), a N-terminal leader sequence resulting from incomplete cleavage of a signaling peptide, were observed in monoclonal antibody mAb1 after manufacturing process changes and led to out-of-trend results for mAb1 basic region. While VHS is not expected to impact efficacy/safety of mAbs dosed intravenously, for mAbs dosed subcutaneously (i.e. mAb1), VHS and its positive charge could interact with the negatively-charged subcutaneous space and potentially impact subcutaneous bioavailability; in this case, VHS would be important to control and/or monitor. In this work, we interrogated VHS and assessed its likelihood to impact subcutaneous pharmacokinetics by using a combination of physicochemical characterization, in vitro biological assays, clinical sample testing, and computational modeling.

Methods: In order to determine site-specific histidine pKas on mAb1 VHS, histidine hydrogen deuterium exchange – mass spectrometry (Miyagi 2008) was performed using mAb1 samples. In brief, mAb1 was incubated in deuterated solutions ranging in pH buffered with either acetate (pH 3.5-5), MES (pH 5.5-6.5), or Tris (pH 7-9) for 70 hours at 37C before being subjected to reduction, alkylation, and tryptic digestion followed by LC-MS/MS analysis. Deuterium uptake was quantified using HDExaminer (Sierra Analytics). Next, clinical serum retains were interrogated for either depletion or enrichment of VHS peptides relative to other mAb1 peptides, which would suggest lower or higher subcutaneous bioavailability of VHS, respectively. From the subcutaneous mAb1 arm of a clinical study, a subset of PK serum samples was selected, antigen affinity captured using magnetic beads, denatured with RapiGest, reduced, alkylated, on-bead trypsin digested, and analyzed by LC-MS/MS. Furthermore, mAb1 clearance rates as a function of VHS level were predicted by testing mAb1 samples with varying VHS using a kidney-huFcRN cell transcytosis assay described previously (Chung 2019). Monoclonal Ab1 with elevated VHS was enriched by fraction collecting the basic peak adjacent to main peak off of a semi-preparative ion exchange column (YMC Biopro SP-F). Finally, histidine pKas of VHS on heavy chains of IgG1/2/4 molecules were computationally calculated in GPU-accelerated AMBER20 based on the pKa-ANI method described previously (Gokcan 2022).

Results: Histidine hydrogen deuterium exchange – mass spectrometry elegantly revealed that the VHS histidine pKa’s in the mAb1 heavy chain and light chain microenvironments were 5.7 and 6.3, respectively, suggesting that VHS is predominantly uncharged at physiological pH, which should not impact its bioavailability in its subcutaneous administration. To validate this finding biologically, serum retains from a clinical study where mAb1 was dosed subcutaneously were affinity purified and analyzed by tryptic peptide monitoring. Ratios between VHS-containing mAb1 peptides and other mAb1 peptides were consistent across timepoints, as well as against the reference material, implying no differences in subcutaneous bioavailability attributable to VHS. Monoclonal Ab1 samples highly enriched in VHS ( >70%) also showed similar clearance propensities to wildtype mAb1 as predicted by an in vitro kidney-huFcRN cell transcytosis assay. Finally, molecular dynamics simulations and pre-trained machine learning algorithms predicted acidic pKa’s for VHS histidines on various IgG1/2/4 molecules, which helped extend the findings with mAb1 and more generally de-risk VHS as a pharmacokinetic liability for mAbs dosed subcutaneously.

Conclusion: A robust data package for mAb1 - consisting of physicochemical characterization, in vitro biological assays and clinical sample testing – supported de-risking VHS modification as a pharmacokinetic liability for subcutaneous administration. Computational experiments further indicated that our findings with mAb1 would likely generalize to other IgG1/2/4, primarily driven by VHS actually being neutrally-charged (rather than positively-charged presumably) at physiological pH.

References: Miyagi M and Nakazawa T. "Determination of pKa values of individual histidine residues in proteins using mass spectrometry". Anal Chem. 2008, 80, 6481-6487
Chung S et al. "An in vitro FcRN- dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans" mAbs. 2019, 11, 942-955
Gokcan H and Isayev O. "Prediction of protein pKa with representation learning" Chem Sci. 2022, 13, 2462

Acknowledgements: Authors are employees of Hoffman-La Roche

Histidine HDX-MS deuterium uptake plots of mAb1 VHS heavy chain (A, left) and VHS light chain (B, right) showing histidine pKa (m1) of 5.7 and 6.3 respectively

Ratios of mAb1 VHS peptide to other mAb1 peptides in clinical PK serum samples by affinity capture LC-MS/MS. “C” denotes administration cycle and “D” denotes timepoint in days.

Predicted clearance by kidney-huFcRN cell transcytosis assay of mAb1 with VHS enriched to varying degrees