Replacement of the Mouse LD50 Assay for Determination of the Potency of AbobotulinumtoxinA with a Cell-Based Method in Both Powder and Liquid Formulations.

Botulinum neurotoxins (BoNTs) are important therapeutic agents. The in vivo median lethal dose (LD50) assay has been commonly used to measure the potency of BoNT commercial preparations. As an alternative, we developed cell-based assays for abobotulinumtoxinA in both powder (Dysport®, Azzalure®) and liquid (Alluzience®) formulations using the in vitro BoCell® system. The assays demonstrated linearity over 50-130% of the expected relative potency, with a correlation coefficient of 0.98. Mean recoveries of 90-108% of the stated potency were observed over this range. The coefficients of variation for powder and liquid formulations, respectively, were 3.6% and 4.0% for repeatability and 8.3% and 5.0% for intermediate precision. A statistically powered comparability assessment of the BoCell® and LD50 assays was performed. Equivalence was demonstrated between the assays for the liquid formulation at release and end of shelf life using a paired equivalence test with predefined equivalence margins. For the powder formulation, the assays were also shown to be equivalent for release samples and when determining loss of potency following thermal degradation. The BoCell® assay was approved for establishing the potency of abobotulinumtoxinA for both powder and liquid formulations in Europe and for the powder formulation only in the USA.

Rate of Asparagine Deamidation in a Monoclonal Antibody Correlating with Hydrogen Exchange Rate at Adjacent Downstream Residues.

Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development.

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.