Vikas K. Sharma

In silico selection of therapeutic antibodies for development: Viscosity, clearance, and chemical stability

Significance mAbs are increasingly being used for treatment of chronic diseases wherein the subcutaneous delivery route is preferred to enable self-administration and at-home use. To deliver high doses (several hundred milligrams) through a small volume (∼1 mL) into the subcutaneous space, mAb solutions need to have low viscosity. Concomitantly, acceptable chemical stability is required for adequate shelf life, and normal in vivo clearance is needed for less frequent dosing. We propose in silico tools that provide rapid assessment of atypical behavior of mAbs (high viscosity, chemical degradation, and fast plasma clearance), which are simply predicted from sequence and/or structure-derived parameters. Such analysis will greatly improve the probability of success to move mAb-based therapeutics efficiently into clinical development and ultimately benefit patients. For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery.

Investigation into temperature-induced aggregation of an antibody drug conjugate.

Conjugation of an antibody to a drug can produce heterogeneous species that may have different physical stabilities and safety profiles. We explored the effect of thermal stress on the physical stability, specifically aggregation, of an antibody drug conjugate (ADC), ADC 1, wherein the antibody was linked to the val-cit-Monomethyl Auristatin E (vc-MMAE) linker drug through the reduction of interchain disulfides. We also explored the effects of conjugation on the secondary and tertiary structures of ADC 1. Circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry showed that for species with high drug loading, conjugation does not measurably alter the secondary structure, but it does render the CH2 domain less stable to thermal stress such that ADC 1 rapidly forms high molecular weight species (HMWS) at 40 °C. Characterization of the HMWS using chromatographic and electrophoretic methods showed that it is an irreversible, noncovalent, and structurally altered form of ADC 1 primarily composed of molecules with six or eight drugs. Furthermore, the variable domain of the antibody may contribute to the extent of aggregation, since eight ADCs with over 90% sequence homology exhibited monthly rates of HMWS formation that differ by up to a factor of 2.

Effect of polyols on polyethylene glycol (PEG)-induced precipitation of proteins: Impact on solubility, stability and conformation.

Effect of polyols on the solubility of bovine serum albumin (BSA) in the presence of polyethylene glycols (PEGs) was investigated in order to strengthen the understanding of the observed effects of polyols and PEGs on protein properties in solution. Effect of polyols and/or PEGs on the thermodynamic (conformational) stability of BSA was measured using DSC and circular dichroism (CD). Glucose, sucrose, raffinose, glycerol and sorbitol, all reduced the extent of protein precipitation. Solubility of BSA in the presence of ethylene glycol increased in the case of PEG 1450 and PEG 8000, but was unaffected in the case of PEG 400. DSC studies indicated that smaller PEGs have destabilizing influence on protein structure. CD studies showed that smaller PEGs (ethylene glycol) induce subtle unfolding while stabilizing polyols induce subtle compaction. Results show that, effect of polyols on the apparent solubility of the protein correlates with their effect on the thermodynamic stability of the protein, smaller PEGs are not appropriate for estimating the activity of proteins in saturated solutions, and subtle changes in protein conformation can significantly affect protein precipitation. Though smaller PEGs have weak attractive interactions with protein molecules, perturbation of protein structure by PEGs can be balanced by utilizing appropriate stabilizing solutes.

Culture temperature modulates aggregation of recombinant antibody in cho cells.

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤5% aggregates at harvest. Aggregate levels increased 4–12‐fold in 33°C cultures when compared to 37°C, with a corresponding 2–4‐fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25–75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2–5‐fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery. Biotechnol. Bioeng. 2012;109: 125–136.

Evaluation of a multimodal resin for selective capture of CHO-derived monoclonal antibodies directly from harvested cell culture fluid.

This proof-of-concept study examines the applicability of using multimodal chromatography to selectively capture recombinantly produced monoclonal antibodies (mAb) directly from harvested mammalian cell culture fluid (HCCF) with minimal optimization. Capto MMC is a multimodal resin that contains a ligand with the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. Twelve mAb HCCF feedstocks were examined for dynamic binding capacity (DBC) and then two representative feedstocks were selected to develop a systematic approach for elution buffer development. A range of dynamic binding capacities was observed for 10 feedstocks (24-53g/L) and two feedstocks had poor binding properties (<10g/L) despite load conditioning towards a more favorable pH. Analysis of the DBC versus molecular properties showed that the mAb-ligand binding interaction was predominantly charge based. Four separate elution strategies were identified to selectively recover the mAb and could be applied with minimal optimization to other mAb feedstocks. Downstream processing of the Capto MMC pools showed that it is feasible to produce material with comparable purity to a process with affinity capture after two chromatography steps.

Modulation of the thermodynamic stability of proteins by polyols: Significance of polyol hydrophobicity and impact on the chemical potential of water

The exact mechanism of the modulation of chemical potential of proteins by polyols is not yet well understood. Present study investigates the role of hydrophobicity of polyols, and their impact on water activity and/or surface tension, in determining their stabilization/destabilization potential. Results with ribose and methyl-glucose show that the enhanced stability of proteins is not mediated via the effect on interfacial tension, a hypothesis that has so far been restricted to glycerol. An exemplary correlation between thermodynamic stabilization (ΔG(f-uf)), and polyol osmolality, confirms/generalizes the prominent role of water activity in the observed stabilization effects. Results show that even seemingly hydrophilic sugars such as deoxy-ribose can interact favorably with proteins, suggesting that properties other than the presence of hydroxyl groups also contribute to the net effect of polyols. We demonstrate that the hydrophobicity index of polyols and the net stabilization effect afforded to proteins have an excellent inverse correlation. These studies show that the weak hydrophobicity of polyols is critical for promoting their interactions with proteins, weakening of the hydrophobic forces within the protein interior and counteracting the polyol induced-solvent mediated stabilization effect.

Triple light chain antibodies: factors that influence its formation in cell culture.

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species—Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression—evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)—correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production—evaluated as the ratio of the cell‐specific production rate of GSH (qGSH) to the cell‐specific production rate of THIOMAB (qp)—corresponded to decreased 3LC levels. In time‐lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and qGSH/qp ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high qGSH/qp ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity. Biotechnol. Bioeng. 2010. 105: 748–760.

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. Tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from -5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔGu, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. The Gibbs-Helmholtz expression fit to the ΔGu versus temperature data from fluorescence gave a ΔCp of 8.0 kcal mol-1 K-1, a maximum apparent stability of 23.7 kcal mol-1 at 18°C, and an apparent cold denaturation temperature (TCD) of -23°C. ΔGu values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative TCD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. Thus, mAbs have a potential to undergo cold denaturation at storage temperatures near -20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.

Isomerization of Asp–Asp Motif in Model Peptides and a Monoclonal Antibody Fab Fragment

Isomerization of aspartyl (Asp or D) residues is a critical degradation route to consider for stable monoclonal antibody formulations. Among the known hotspot sequences, the DD motif is relatively understudied. To gain mechanistic insights, we used model hexapeptides, YADXFK, YADDXK, and DIDDDM, as surrogates for the hotspots in a Fab protein (YADDFK and DIDDDM), to characterize the rate-pH profile of Asp isomerization. Compared with the YADGFK peptide, isomerization of D3 (the first D in the DD pair) in YADDFK was highly pH dependent. Comparison of rate-pH profiles of YADDFK, YADNFK, and YADHFK revealed a charge effect of the n + 1 residue-isomerization rate is accelerated by the positive side chain and reduced by negative side chain at n + 1 residue. Studies on YADDFK, YADDAK, and YADDGK indicated a mutual impact of D3 and D4 on their respective isomerization rates through charge effect. Comparison of rate-pH profile of DIDDDM sequence in peptide models with that in the complementary determining region of the Fab showed a faster rate in the Fab than in peptides, presumably because of contribution from structural factors in the former.

Role of benzyl alcohol in the prevention of heat-induced aggregation and inactivation of hen egg white lysozyme.

The aim of the study was to investigate the stability of a model protein, lysozyme, in the presence of the commonly used preservative benzyl alcohol. Techniques including lytic assay, size exclusion chromatography, circular dichroism, differential scanning calorimetry, native polyacrylamide gel electrophoresis and dynamic light scattering were used to study the overall stability of lysozyme in the presence of benzyl alcohol. The stability of lysozyme against thermal stress was higher in the presence of benzyl alcohol. In the presence of 0.5%, 0.9% and 2% v/v benzyl alcohol, the enzyme showed 33%, 42% and 75% residual activity, respectively, when exposed to 75 degrees C for 2 h, as compared to the 22% activity of control sample. A gradual increase in the size of aggregates was observed for the control sample relative to the samples containing benzyl alcohol, as a result of loss of monomer concentration. The effect was found to be concentration-dependent with 2% benzyl alcohol showing maximum prevention of heat-induced unfolding and aggregation. This effect is remarkable since the thermal transition temperature of the enzyme decreases in the presence of benzyl alcohol. Benzyl alcohol favours the thermal denaturation of lysozyme but stabilizes the lysozyme against the heat-induced aggregation.