Extensive Characterization of Polysorbate 80 Oxidative Degradation Under Stainless Steel Conditions.

Polysorbate-80 (PS-80) is a common surfactant used in biologics formulations. However, the tendency of oxidation to PS-80 when exposed to stainless steel surfaces brings various challenges during manufacturing processes, such as inconsistent shelf-life of PS-80 solutions, which can further impact the biologics and vaccines production. In this work, the root causes of PS-80 oxidation when in contact with stainless steel conditions were thoroughly investigated through the use of various complementary analytical techniques including U/HPLC-CAD, LC-MS, ICP-MS, peroxide assay, and EPR spectroscopy. The analytical tool kit used in this work successfully revealed a PS-80 degradation mechanism from the perspective of PS-80 content, PS-80 profile, iron content, peroxide production, and radical species. The combined datasets reveal that PS-80 oxidative degradation occurs in the presence of histidine and iron in addition to being combined with the hydroperoxides in PS-80 material. The oxidative pathway and potential degradants were identified by LC-MS. The PS-80 profile based on the U/HPLC-CAD assay provided an effective way to identify early-signs of PS-80 degradation. The results from a peroxide assay observed increased hydroperoxide along with PS-80 degradation. EPR spectra confirmed the presence of histidine-related radicals during PS-80 oxidation identifying how histidine is involved in the oxidation. All assays and findings introduced in this work will provide insight into how PS-80 oxidative degradation can be avoided, controlled, or detected. It will also provide valuable evaluations on techniques that can be used to identify PS-80 degradation related events that occur during the manufacturing process.

Selective Plate-Based Assay for Trace EDTA Analysis via Boron Trifluoride-methanol Derivatization UHPLC-QqQ-MS/MS Enabling Biologic and Vaccine Processes.

The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Derivatization of EDTA to its methyl ester (Me-EDTA) serves to significantly improve chromatographic performance (retention, peak shape, and selectivity), while also delivering a tremendous enhancement of sensitivity in the positive ion mode electrospray ionization (ESI+). This procedure, in contrast to previous EDTA methods based on complexation with metal ions, is not affected by high concentration of other metals, buffers, and related salts abundantly present in biopharmaceutical processes (e.g., iron, copper, citrate, etc.). Validation of this assay for the determination of ng·mL-1 level EDTA in monoclonal antibody and vaccine products demonstrated excellent performance (repeatability, precision, and linear range) with high recovery from small sample volumes while also providing an advantageous automation-friendly workflow for high-throughput analysis.

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control.

For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their Mw using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (Mw increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.

Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications.

Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) is a powerful analytical method used for the separation of molecular variants of therapeutic proteins. The method has been employed for monitoring various post-translational modifications, including proteolytic fragments and domain misfolding in etanercept (Enbrel®); tryptophan oxidation, aspartic acid isomerization, the formation of cyclic imide, and α amidated carboxy terminus in recombinant therapeutic monoclonal antibodies; and carboxy terminal heterogeneity and serine fucosylation in Fc and Fab fragments. HIC-HPLC is also a powerful analytical technique for the analysis of antibody-drug conjugates. Most current analytical columns, methods, and applications are described, and critical method parameters and suitability for operation in regulated environment are discussed, in this review.

Effect of pegylation on self-association of IFN-α2b.

Pegylation of therapeutic proteins is an established technology used to enhance the bioavailability of an active pharmaceutical ingredient in the body of patients. While the physiochemical properties of pegylated monomeric proteins have been extensively described, there is still limited information on the characterization of pegylated oligomeric proteins. In this study, we report the characterization of a pegylated interferon alpha2b (PEGIFN-α2b) concentration-dependent oligomerization by a series of orthogonal biochemical and biophysical methods. These methods include sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, matrix-assisted laser desorption ionization, and size exclusion chromatography of bissulfosuccinimidyl suberate cross-linked PEGIFN. We report here that PEGIFN-α2b self-associates in a concentration-dependent manner into mainly monomers, dimers, and trimers. In the presence of the chemical cross-linker, PEGIFN-α2b is primarily monomeric (57%) at concentration lower than 0.3 mg/mL and contains about equal amount of monomers and dimers (47.0% and 37.7%, respectively), about 15% of trimers, and up to 4% of higher molecular weight species at 0.7 mg/mL and above.

Fine-tuning the pH trigger of self-assembly.

The creation of smart, self-assembling materials that undergo morphological transitions in response to specific physiological environments can allow for the enhanced accumulation of imaging or drug delivery agents based on differences in diffusion kinetics. Here, we have developed a series of self-assembling peptide amphiphile molecules that transform either isolated from molecules or spherical micelles into nanofibers when the pH is slightly reduced from 7.4 to 6.6, in isotonic salt solutions that simulate the acidic extracellular microenvironment of malignant tumor tissue. This transition is rapid and reversible, indicating the system is in thermodynamic equilibrium. The self-assembly phase diagrams show a single-molecule-to-nanofiber transition with a highly concentration-dependent transition pH. However, addition of a sterically bulky Gd(DO3A) imaging tag on the exterior periphery shifts this self-assembly to more acidic pH values and also induces a spherical micellar morphology at high pH and concentration ranges. By balancing the attractive hydrophobic and hydrogen-bonding forces, and the repulsive electrostatic and steric forces, the self-assembly morphology and the pH of transition can be systematically shifted by tenths a pH unit.