C-Terminal Lysine Processing of IgG in Human Suction Blister Fluid: Implications for Subcutaneous Administration.

C-terminal lysine (CTK) is often classified as a potential critical quality attribute for therapeutic antibodies being developed for subcutaneous (SC) administration because of its potential to impact antibody SC bioavailability/pharmacokinetics (PK). This classification both inflates development costs and increases comparability risks for SC administration of antibodies. However, prior risk assessments of CTK have not fully considered biotransformation of CTK in the SC space, which may play an important role given that circulating carboxypeptidases in humans rapidly process CTK on intravenously administered antibodies. Here, CTK biotransformation in biofluid derived from human SC space was investigated. The representative fluid from the human SC space was sampled from 10 healthy human subjects using the suction blister method. Glycosylated antibody containing high levels of CTK (expressed using a carboxypeptidase D CRISPR/Cas9 knockout CHO cell line) was incubated in the collected suction blister fluids (SBFs), recovered using cognate antigen pulldowns, and characterized for remaining CTK levels using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analysis. CTK processing (i.e., carboxypeptidase activity) was evident in all SBF and exhibited first-order kinetics with rate constants of 2.18 ± 0.57 d-1 (at 37 °C). PK simulations that integrated CTK processing pathways and their associated rate constants were subsequently performed using a range of clinically observed PK parameters for therapeutic antibodies, including atezolizumab- and pertuzumab-specific parameters. The impact of CTK content (even up to 100%) on SC PK outcomes such as bioavailability and Ctrough were modest (<14%) for all combinations of PK parameters tested in the sensitivity analysis. This study forms the cornerstone data package for derisking CTK as a PK liability for antibody SC programs and highlights the usefulness of fully considering biotransformation during product quality criticality assessments.

Using differential scanning calorimetry for the development of non-reduced capillary electrophoresis sodium dodecyl sulfate methods for monoclonal antibodies.

Analysis of non-reduced and reduced monoclonal antibodies (mAbs) by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is routinely used to detect product size variants and process-related impurities. Levels of high molecular weight (HMW) forms obtained from this method usually trend comparably to those obtained by orthogonal methods such as size-exclusion ultra-high performance liquid chromatography (SE-UHPLC). However, in the presented case study, comparison of CE-SDS data for three IgG1 mAbs (trastuzumab, mAb1, and mAb2) showed a discrepancy between amounts of observed HMW forms in mAb2 compared with its native forms determined by SE-UHPLC (~17% vs. ~0.5%, respectively). SDS chemical denaturation, as measured by differential scanning calorimetry, demonstrated that the high thermal stability of mAb2 caused an unidentified HMW peak observed by non-reduced (NR)-CE-SDS, which was the result of improper denaturing, resulting in a partially folded species. More so, this strategy enabled the rapid identification of optimal SDS concentration and temperature conditions needed for suitable denaturation for mAb2. This case study presents an alternative option for quick optimization of NR-CE-SDS methods when characterizing mAbs or other thermally stable proteins. Also, this strategy can be used to understand basic biophysical mechanisms of protein unfolding and investigate the higher-order structure imparted by specific sequences and understand how these sequences might affect the results of an analytical method such as CE-SDS.

Epitope mapping of anti-drug antibodies to a clinical candidate bispecific antibody.

Anti-drug antibodies (ADA) can limit the efficacy and safety of therapeutic antibodies. However, determining the exact nature of ADA interactions with the target drug via epitope mapping is challenging due to the polyclonal nature of the IgG response. Here, we demonstrate successful proof-of-concept for the application of hydroxyl radical footprinting (HRF)-mass spectrometry for epitope mapping of ADAs obtained from goats that were administered a knob-into-hole bispecific antibody (BsAb1). Subsequently, we performed epitope mapping of ADAs obtained from cynomolgus (cyno) monkeys that were administered BsAb1 as we described in a recently published paper. Herein, we provide the first data to demonstrate the feasibility of using HRF for ADA epitope mapping, and show that both goat and cyno-derived ADAs specifically target the complementary-determining regions in both arms of BsAb1, suggesting that the ADA epitopes on BsAb1 may be species-independent.

Understanding Loss of Soluble High Molecular Weight Species during Filtration of Low Concentration Therapeutic Monoclonal Antibodies.

Sterile filtration is an integral step in the manufacturing process of biological therapeutics. Protein adsorption to the surface of the filter is an unfortunate, common occurrence that can result in manufacturing difficulties, such as filter fouling or product loss. Although many filters have surface modifications to minimize adsorption, under certain conditions binding can still occur. We observed the loss of high molecular weight species (HMWS) during sterile filtration of eight different therapeutic monoclonal antibodies formulated at low protein concentrations across a commonly used hydrophilic polyvinylidene fluoride or polyvinylidene difluoride (PVDF) filter membrane. The protein absorption was specific to HMWS, and each antibody exhibited different degrees of filter adsorption. Debye screening length parameters of the solution (e.g. ionic strength) were adjusted, and influenced the amount of HMWS lost during filtration. Additionally, HMWS of a representative antibody (mAb1) were observed to be more positively charged than other size variants by ion-exchange chromatography. From these results, it is concluded that this HMWS loss is due to electrostatic interactions between HMWS and the filter surface. This adsorption can be reduced by increasing the ionic strength of the buffer matrix, demonstrating the influence of the Debye screening length in the filtration of low concentration proteins.

Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.

An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aß peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aß residues 33-42 in CDR3. Interestingly, co-selection for improved Aß binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aß binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments.