Category: Formulation and Quality
Purpose: Oxidation in protein-based biopharmaceuticals is one of the key degradation pathways that needs monitoring and mitigation during production, purification, formulation, transportation, storage, and handling of biopharmaceuticals. The purpose of this study was to evaluate residue specific impact of stressors (i.e. metals, peroxide) and protectants (i.e. methionine, EDTA) on 3 protein candidates (mAbs and bispecifics) with respect to formulation variables such as buffer, pH, protein concentration, and levels of stressors and protectants.
Methods: A multivariate response surface DOE was used for formulation design and high throughput analytical technologies were employed to evaluate physical stability (SEC) and chemical stability (CEX and peptide map) for residue-specific oxidation modifications. A central composite design in six factors (metals, peroxide, methionine, EDTA, pH, and protein concentration) was used to establish a causal relationship between the design factors and relative oxidation levels at various sites of the molecules. The relative oxidation level of each oxidation sites was quantified by high throughput multi-attribute mass spectrometry method by calculating the ratio of XIC area of oxidized peptide species and total peptide species. The multi-dimensional data for physical stability and residue-specific oxidation modifications was analyzed by multivariate data analysis using JMP software for rational selection and optimization of protein formulations.
Results: High throughput multi-attribute mass spectrometry method was employed to quantify oxidation levels of methionine and tryptophan residues in different formulation matrixes. The central composite design allowed for a multivariate quadratic function in six factors - methionine, protein concentration, EDTA, pH, peroxide and iron - for the oxidation level of every methionine and tryptophan residue in each of the candidate proteins. Results suggest that modification of methionine and tryptophan residues are a function of formulation factors as well as levels of stressors (iron, peroxide), metal chelators (EDTA) and antioxidants (methionine). The stabilization provided by protectants (EDTA and methionine) in formulation was protein-dependent and residue-specific. For instance, EDTA increased the oxidation of one Trp residue versus decreased the oxidation levels at a different site within the same protein. The protective impact of methionine was retained in most of the modifications detected in this study.
Conclusion: These results indicate the need for systematic multi-variate experimental design, application of orthogonal analytics, and residue-specific understanding of oxidation for rational selection of excipients for preventing oxidation in protein formulations.
Danika Rodrigues– Malvern, Pennsylvania
Bo Zhai– Malvern, Pennsylvania
Andrew Mahan– Malvern, Pennsylvania
Dominick DeGrazio– Malvern, Pennsylvania
Dwaine Banton– Malvern, Pennsylvania
Lisa Hughes– Malvern, Pennsylvania
Hirsh Nanda– Malvern, Pennsylvania
Santosh Thakkar– Malvern, Pennsylvania