Valentyn Antochshuk

Developability studies before initiation of process development Improving manufacturability of monoclonal antibodies

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2−8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.

Solution pH that minimizes self-association of three monoclonal antibodies is strongly dependent on ionic strength

Monoclonal antibodies display highly variable solution properties such as solubility and viscosity at elevated concentrations (>50 mg/mL), which complicates antibody formulation and delivery. To understand this complex behavior, it is critical to measure the underlying protein self-interactions that govern the solution properties of antibody suspensions. We have evaluated the pH-dependent self-association behavior of three monoclonal antibodies using self-interaction chromatography for a range of pH values commonly used in antibody formulations (pH 4.4-6). At low ionic strength (<25 mM), we find that each antibody is more associative at near-neutral pH (pH 6) than at low pH (pH 4.4). At high ionic strength (>100 mM), we observe the opposite pH-dependent pattern of antibody self-association. Importantly, this inversion in self-association behavior is not unique to multidomain antibodies, as similar pH-dependent behavior is observed for some small globular proteins (e.g., ribonuclease A and α-chymotrypsinogen). We also find that the opalescence of concentrated antibody solutions (90 mg/mL) is minimized at low ionic strength at pH 4.4 and high ionic strength at pH 6, in agreement with the self-interaction measurements conducted at low antibody concentrations (5 mg/mL). Our results highlight the complexity of antibody self-association and emphasize the need for systematic approaches to optimize the solution properties of concentrated antibody formulations.

Application of a High-Throughput Relative Chemical Stability Assay to Screen Therapeutic Protein Formulations by Assessment of Conformational Stability and Correlation to Aggregation Propensity

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions.

Method qualification and application of diffusion interaction parameter and virial coefficient

This research focused on evaluation and application of two methods in studying weak protein-protein interactions, i.e. diffusion interaction parameter (KD) and second virial coefficient (B22), both of which are first-order coefficients of protein interactions. Although the plate-based KD method successfully distinguished KD values with relatively large difference in a pH ranging study, it failed to make a consistent statistical decision to determine close interactions as shown by the comprehensive ANOVA analysis. We also validated the DLS-based B22 method by using a model protein lysozyme. The dramatic change of solution appearance for lysozyme as a function of NaCl concentration highlighted the importance of B22 in understanding protein interactions. Moreover, B22 measurement for a MAb fragment suggested a more repulsive protein interaction in histidine buffer than in citrate buffer. The coefficient of variation was <10% when B22 was on an order of magnitude of 10(-4) L mmol/g(2) in contrast to >30% when it approached 10(-5) L mmol/g(2). In this research, we also made an attempt to study protein-protein interactions in concentrated MAb fragment solutions (e.g. >50 mg/mL). Our data suggested that such interactions could be empirically modeled by high-order virial expansions.

Engineering Glucose Responsiveness Into Insulin

Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a “smart” insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).

A Highly Sensitive Method for the Quantitation of Polysorbate 20 and 80 to Study the Compatibility between Polysorbates and m-Cresol in the Peptide Formulation

A highly sensitive method has been developed for the quantitation of polysorbate 20 (PS20) and 80 (PS80) in therapeutic peptide formulations. A mixed-mode HPLC column was used to separate polysorbates from the peptide and other excipients, and a charged aerosol detector (CAD) was used for the detection. The method was capable of reporting polysorbates as low as 5 ppm, and the sensitivity could be further improved on a needed basis. The method has been used to study the compatibility between polysorbates and m-cresol in the peptide formulation. It was found that both PS20 and PS80 are compatible with m-cresol (at 2.8 mg/ml) when their levels were not greater than 20 ppm. Significant losses of polysorbates were observed when PS20 and PS80 concentrations were above 50 ppm. Furthermore, the agitation study demonstrated that even trace levels of PS20 and PS80 (e.g., 20 ppm) could stabilize the peptide against fibrillation and aggregation.

Closing the Gap: Counting and Sizing of Particles Across Submicron Range by Flow Cytometry in Therapeutic Protein Products

Quantification and size distribution characterization of subvisible particles in parenteral biopharmaceutics, present as both proteinaceous and nonproteinaceous particles in the size range from 0.1 to 100 μm, are important for biopharmaceutical industry due to their potential safety and efficacy implications. Although a number of analytical techniques are available to count and size subvisible particles, characterization of particles ≤2 μm remains a significant challenge due to technical limitations of existing particle counting instruments. In this article, we demonstrate the ability of an optimized flow cytometry system to detect and quantify size distribution of subvisible particles without additional labeling that includes the critical submicron range in biopharmaceutical formulations. In addition, these qualitative and quantitative determinations are performed in a high-throughput manner using sample volumes that allow statistically significant evaluations. This approach can be used not only to ascertain the quality of therapeutic protein products but also to evaluate numerous conditions during the screening of drug candidates and their prospective formulations.

Erratum. Engineering Glucose Responsiveness Into Insulin. Diabetes 2018;67:299–308

In the above-mentioned article, several scientists were erroneously omitted from the acknowledgments section. The authors wish to acknowledge Hsuan-shen Chen, Huaibing He, Tina …