Jason K. Cheung

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.

Improving monoclonal antibody selection and engineering using measurements of colloidal protein interactions.

A limitation of using mAbs as therapeutic molecules is their propensity to associate with themselves and/or with other molecules via nonaffinity (colloidal) interactions. This can lead to a variety of problems ranging from low solubility and high viscosity to off-target binding and fast antibody clearance. Measuring such colloidal interactions is challenging given that they are weak and potentially involve diverse target molecules. Nevertheless, assessing these weak interactions-especially during early antibody discovery and lead candidate optimization-is critical to preventing problems that can arise later in the development process. Here we review advances in developing and implementing sensitive methods for measuring antibody colloidal interactions as well as using these measurements for guiding antibody selection and engineering. These systematic efforts to minimize nonaffinity interactions are expected to yield more effective and stable mAbs for diverse therapeutic applications.

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability

ABSTRACT With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (kD) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25°C and 40°C) and long-term storage conditions (2–8°C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2–8°C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the CH2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.

DSF method optimization and its application in predicting protein thermal aggregation kinetics

Differential scanning fluorimetry (DSF) has gained wide acceptance in the therapeutic protein development. However, the effects of dyes and surfactants that may affect structural transitions have not been studied thoroughly to date. We therefore first optimized the DSF method by studying surfactant-containing formulations and found that the presence of surfactants generally required medium-to-high protein concentrations and that high SYPRO® Orange concentration in a DSF experiment may lower protein thermal transitions. We also benchmarked DSF against differential scanning calorimetry (DSC) and evaluated the capability of thermal parameters (from DSF/DSC) to predict real-time thermal aggregation kinetics monitored by size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) in different scenarios. For monoclonal antibody (MAb) fragment, both DSF and DSC were predictive of thermal aggregation rate. For MAb3, a good correlation was observed between DSF and DSC, none of which was, however, indicative of protein aggregation kinetics. In a surfactant ranging study, DSF did not agree with DSC and was not predictive of the aggregation kinetics of the MAb fragment. The concentration-dependent thermal behavior was also studied by DSF. Although higher concentration, in general, tends to lower protein transition temperature, case where it was independent of protein concentration was also presented.

Analysis of monoclonal antibody oxidation by simple mixed mode chromatography.

Analysis of oxidation of monoclonal antibodies (mAbs) in most cases relies on peptide mapping and LC-MS, which is time consuming and labor-intensive. A robust chromatography based method that is able to resolve and quantitate mAb oxidation variants due to oxidized methionine or tryptophan is highly desired. Here we developed a novel mixed mode chromatography method using the unique property of Sepax Zenix SEC-300MK column to analyze mAb oxidation levels. The separation of oxidized species relied upon the mixed mode of size exclusion and hydrophobic interaction between the resin and antibodies. The chromatography was performed in a regular SEC mobile phase, PBS, containing NaCl at a concentration (0-2.4M) specific for individual antibodies. This method was able to resolve and quantitate the oxidized antibodies as prepeaks, of either methionine-oxidized species induced by the common oxidants TBHP, tryptophan-oxidized species triggered by AAPH, or oxidized species by UV photo-irradiation. The prepeaks were further characterized by SEC-MALLS as monomers and confirmed by LC-MS as oxidized antibody variants with a mass increase of 16 or 32Da. This method has been successfully applied to monitor multiple monoclonal antibodies of IgG1, IgG2, and IgG4 subclasses.

Forced degradation of recombinant monoclonal antibodies: A practical guide

ABSTRACT Forced degradation studies have become integral to the development of recombinant monoclonal antibody therapeutics by serving a variety of objectives from early stage manufacturability evaluation to supporting comparability assessments both pre- and post- marketing approval. This review summarizes the regulatory guidance scattered throughout different documents to highlight the expectations from various agencies such as the Food and Drug Administration and European Medicines Agency. The various purposes for forced degradation studies, commonly used conditions and the major degradation pathways under each condition are also discussed.

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.

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.

Intravenous Admixture Compatibility for Sterile Products: Challenges and Regulatory Guidance

Intravenous (IV) administration of many sterile drug products requires admixture preparation using a diluent, brief storage in an IV container, and dosing through an infusion device. To ensure patient safety and drug efficacy, regulatory agencies require that the sterile drug product is compatible with the diluents and the infusion devices. Therefore, admixture compatibility and stability studies are key components of the pharmaceutical development process. On the surface these studies may seem straightforward, but in practice they require detailed planning, meticulous execution, and appropriate data analysis. The purpose of this chapter is to discuss various requirements and challenges associated with conducting IV admixture studies and the related regulatory guidance.