Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

Abstract

(2014) Resolving selfassociation of a therapeutic antibody by formulation optimization and molecular approaches, mAbs, 6:6, 1533-1539, DOI: 10.4161/19420862.2014 To link to this article: https://doi.org/10. 4161/19420862.2014 A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of selfassociation, formation of a semi-solid gel or "gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6 C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1 C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.

Process optimization and protein engineering mitigated manufacturing challenges of a monoclonal antibody with liquid-liquid phase separation issue by disrupting inter-molecule electrostatic interactions

“…2 In recent years, LLPS challenges during the formulation development of multiple mAbs or mAb derivatives have been described in the literature. [1][2][3][4][5][6] Several of the reports propose that the progression to, and final equilibrium state of, LLPS are dictated by physicochemical conditions, such as temperature, pH, ionic strength, buffering agents, and the type and concentration of other excipients. [3][4][5] MAbs are effective therapeutics for numerous pathological conditions, including cancer, inflammation, infectious diseases, autoimmune diseases, and neurological disorders.…”

“…LLPS is often considered a manageable challenge for downstream processing with the majority of the reported LLPS cases today arising from formulation development, particularly for mAbs requiring high concentration formulation. [1][2][3][4][5][6] Luo et. al.…”

“…14,15 The driving forces that underlie antibody self-association are diverse, with both electrostatic and hydrophobic interactions likely playing key roles. 16,17 Mutational substitutions of hydrophobic residues (such as tryptophan 18 and phenylalanine 12 ) or charged residues (such as histidine 16 and lysine 6 ) have proven to be effective at reducing selfassociations. Self-associations leading to LLPS can often be dominated by electrostatic interactions.…”

“…al reported that the single substitution of a negatively charged glutamate residue with a positively charged lysine residue effectively mitigated the LLPS by reducing electrostatic interactions between molecules. 6 While there is a clear link between electrostatic interactions and RSA, a more detailed understanding that includes contributions of charged residues and differences due to amino acid type, especially those in the complementarity-determining regions (CDRs), is limited.…”

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

“…19,20 Multiple publications have demonstrated that the amino acid sequence of the constant and variable regions of antibodies can affect molecular stability, expression levels, downstream processing conditions, and formulation conditions required for stable longterm storage. [19][20][21][22][23][24][25][26][27] Clark et al 20 demonstrated that introduction of multiple amino acid substitutions into the Fv region of an IgG2 to alter the amount of exposed hydrophobic and hydrophilic surface areas resulted in an increased titer that correlated with an increase in the thermal stability of the variants. Pettit et al 22 inserted a missing Lys residue at position 148 of the light chain (LC) for an anti-ebola antibody and demonstrated a significant increase in titer, reduction in high-molecular-weight (HMW) species, and increase in thermal stability by differential scanning fluorimetry (DSF) from material produced by stable CHO cell lines as compared to the parental material demonstrating the importance of primary sequence in expressions and biophysical properties of an antibody.…”