Aqueous Stability of Recombinant Human Thrombopoietin as a Function of Processing Schemes

Abstract

Preformulation studies conducted with recombinant human thrombopoietin (rhTPO), a 332 amino acid glycoprotein which stimulates platelet production, show distinctions in degradation profiles as a function of processing schemes. The stability-limiting degradation pathways change as a function of purification stage and method and are dependent upon the presence of contaminating protease. The stability-limiting degradation pathway of affinity-purified and in-process rhTPO preparations is primarily attributed to proteolysis initiated by a protease present as a fermentation contaminant. The proteolysis increases with increasing pH as a function of temperature. The degradation profiles for these preparations show that bioactivity initially increases and then decreases with increasing pH as a function of temperature. This is consistent with proteolysis to active forms which ultimately undergo degradation to less active forms. Similar studies conducted with rhTPO preparations purified by a combination of more conventional chromatographic steps show different stability-limiting degradation pathways and a different pH-stability profile when compared to affinity purified or in-process preparations. In this case, degradation is accompanied by decreases in activity under all conditions, consistent with the conversion to less active forms. These results illustrate the importance of preformulation and stability characterization of protein pharmaceuticals in support of both process and formulation development. Issues related to storage and handling of inprocess preparations differ from those with formulated product since the stability-limiting degradation pathways change as a function of purification stage.

High Throughput Quantification of N-Glycans Using One-Pot Sialic Acid Modification and Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

“…Also, during process development and optimization, changes in bioreactor operating parameters and downstream harvesting and purification methods can have a dramatic effect on the N-glycan structures in the final product 10-14. Storage conditions of process intermediates and even final vialed product can also affect the glycan profile 1,15-17. Therefore, it is essential to ensure consistency of glycosylation, particularly sialylated glycans.…”

Impact of Residual Impurities and Contaminants on Protein Stability

“…In addition to the process‐related impurities, contaminants can be brought into the final DS preparations through any of the above processes. The difference in the type and level of impurities or contaminants in different protein preparations could explain the different extent and mechanism of degradation for recombinant human thrombopoietin (rhTPO), and different aggregation behaviors of three commercially holo‐α‐lactalbumin products—Sigmaα‐La, IEXα‐La, and Cα‐La, at different pHs …”

“…In contrast, presence of residual proteases in the DS can significantly influence the long‐term storage stability depending on their level in the preparations . Rapid protease‐catalyzed hydrolysis was observed for affinity‐purified rhTPO at pH 6–8, and a Chinese hamster ovary‐derived high‐purity human IgG1 mAb . The carboxypeptidase‐catalyzed C‐terminal lysine clipping in mAb's leads to charge heterogeneity, which may potentially impact the protein stability, as the number of charges in mAb's plays a role in protein aggregation .…”

“…To minimize the impact of residual proteases, the level of HCPs needs to be minimized in the purified DS to an acceptable level. Proteases can be removed by altering the purification scheme, or inhibited by use of a protease inhibitor …”

Consideration of Conformational Transitions And Racemization During Process Development of Recombinant Glucagon-Like Peptide-1

“…This complicates the recombinant production and purification of homogeneous protein preparations having the desired biological and physicochemical characteristics. 1,2 Our studies involving glucagon-like peptide-1 (GLP-1) address these issues and illustrate the importance of physicochemical characterization in support of process development (in addition to formulation and analytical development) of recombinant proteins and peptides.…”