A Micro–Polyethylene Glycol Precipitation Assay as a Relative Solubility Screening Tool for Monoclonal Antibody Design and Formulation Development

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a b s t r a c tAlthough live attenuated Rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low-to middle-income settings leading to interest in alternative vaccines. One promising candidate is a trivalent nonreplicating RV vaccine, comprising 3 truncated RV VP8 subunit proteins fused to the P2 CD4 þ epitope from tetanus toxin (P2-VP8-P[4/6/8]). A wide variety of analytical techniques were used to compare the physicochemical properties of these 3 recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate degradation pathways. P2-VP8-P[4] and P2-VP8-P[6] displayed similar physical stability profiles as function of pH and temperature while P2-VP8-P[8] was relatively more stable. Forced degradation studies revealed similar chemical stability profiles with Met 1 most susceptible to oxidation, the single Cys residue (at position 173/172) forming intermolecular disulfide bonds (P2-VP8-P[6] was most susceptible), and Asn 7 undergoing the highest levels of deamidation. These results are visualized in a structural model of the nonreplicating RV antigens. The establishment of key structural attributes of each antigen, along with corresponding stability-indicating methods, have been applied to vaccine formulation development efforts (see companion paper), and will be utilized in future analytical comparability assessments.

Section: Discussionmentioning

confidence: 99%

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens

Hickey

Sahni

Toth

et al. 2020

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“…As shown in Figure 5e, one sharp transition was observed in all samples that began (T onset ) at 45 C. The relative apparent solubility (thermodynamic activity) profiles of the 5 CRM 197 molecules were assessed using PEG precipitation assay as described previously. 18 As shown in Figure 5f, the shape of the PEG curves (concentration of protein, [mg/mL] versus percentage [w/v] PEG-6000) of Vaxform CRM appeared to have a shifted profile compared to the 4 other CRM 197 samples (C7 CRM, rCRM, EcoCRM™, and Pfenex CRM). A possible reason for the more rapid precipitation of Vaxform CRM as a function of PEG concentration is the relatively higher levels of aggregates present in the sample as observed by SEC and SV-AUC.…”

Section: Conformational Stability Aggregation Propensity and Relatimentioning

confidence: 90%

“…The protocol for the high-throughput version of the polyethylene glycol (PEG) assay for assessing apparent solubility of CRM molecules was adopted from the study by Gibson et al 17 and Toprani et al 18 Stock solutions of PBS pH 7.2 and PBS containing 40% w/ v PEG-10,000 at pH 7.2 were mixed to prepare various concentrations of PEG solutions ranging from 0% to 40% w/v PEG. A volume of 200 mL of the various PEG-10,000 solutions was added to wells of a 96-well polystyrene filter plate (Corning Life Sciences, Corning, NY).…”

Section: Polyethylene Glycol Precipitation Assaymentioning

confidence: 99%

Analytical Comparability Assessments of 5 Recombinant CRM 197 Proteins From Different Manufacturers and Expression Systems

Hickey

Toprani

Kaur

et al. 2018

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Cross-reacting material 197 (CRM), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM molecules demonstrate that recombinant CRM's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM expressed in alternative sources have the potential to replace CRM expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM carrier proteins for use in various conjugate vaccines.

“…Solubility was assessed according to the method previously described. 38 Analysis was performed in PBS and a final polyethylene glycol (PEG) 10,000 concentration of 7.9%. Protein at 1 mg/mL was diluted into the PEG solution at a 1:4 ratio and incubated at room temperature overnight in a 96-well 0.22-mm filter plate.…”

Section: Relative Solubilitymentioning

confidence: 99%

Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity

Kerwin

Bennett

Brodsky

et al. 2020

The broadly neutralizing anti-HIV antibody, 10-1074, is a highly somatically hypermutated IgG1 being developed for prophylaxis in sub-Saharan Africa. A series of algorithms were applied to identify potentially destabilizing residues in the framework of the Fv region. Of 17 residues defined, a variant was identified encompassing 1 light and 3 heavy chain residues, with significantly increased conformational stability while maintaining full neutralization activity. Central to the stabilization was the replacement of the heavy chain residue T108 with R108 at the base of the CDR3 loop which allowed for the formation of a nascent salt bridge with heavy chain residue D137. Three additional mutations were necessary to confer increased conformational stability as evidenced by differential scanning fluorimetry and isothermal chemical unfolding. In addition, we observed increased stability during low pH incubation in which 40% of the parental monomer aggregated while the combinatorial variant showed no increase in aggregation. Incubation of the variant at 100 mg/mL for 6 weeks at 40 C showed a 9-fold decrease in subvisible particles !2 mm relative to the parental molecule. Stability-based designs have also translated to improved pharmacokinetics. Together, these data show that increasing conformational stability of the Fab can have profound effects on the manufacturability and long-term stability of a monoclonal antibody.