Visible and Sub-visible Particle Formation for a Model Bioconjugate.

The time-course and extent of visible particle (VP) and sub-visible particle (SVP) formation was monitored as a function of interfacial area (IA) for a model bioconjugate. To facilitate particle formation, the bioconjugate was agitated in a glass vial and exposed to IAs up to 478 mm2. Since vials had equal fill and headspace volumes, the area of the air-water interface was varied by placing vials on angled blocks at 0°, 30°, 60°, or 90° from the horizontal. A significant increase in visible and sub-visible particle formation was observed with increasing air-water IA. Exposure to IAs below ∼305 mm2 resulted in the formation of very few particles, while IAs > ∼305 mm2 resulted in substantial particle formation. Visible and sub-visible particle morphology varied with interfacial area and time. The sub-visible particles initially increased with time but did not reach steady state; instead the initial increase was followed by complete depletion. These phenomena indicate that visible particle formation likely increased at the expense of the sub-visible particle population and demonstrate a potential link between the two particle populations for this model bioconjugate. Initiation of particle formation did not result in corresponding decreases in protein concentration or increases in soluble aggregates. However, extended agitation time resulted in a significant decrease in protein concentration.

Characterizing the freeze-drying behavior of model protein formulations.

The freeze-drying behavior of three model proteins, namely, lysozyme, BSA, and IgG, has been studied using a variety of techniques under two different primary drying conditions (shelf temperatures of -25°C and +25°C, respectively) in an amorphous formulation. Manometric temperature measurements were used to characterize product temperature (T (pr)), sublimation rates, and product resistance (R (p)) during primary drying. Biophysical techniques such as circular dichroism, fluorescence, and Fourier transform infrared spectroscopy were used to study protein conformation. Size exclusion chromatography was used to monitor the formation of high-molecular-weight species (HMWS) over time on storage, and cake morphology was studied using scanning electron microscopy. The differences in the freeze-drying behavior of the three proteins were more evident at higher protein concentrations, where the protein significantly influences the behavior of the formulation matrix. However, these differences were minimized in the aggressive mode and were insignificant at lower protein concentrations where excipients dominated the freeze-drying behavior. Differences in cake morphology were observed between the two drying conditions employed as well as between the three proteins studied. The stability and the protein structure, however, were equivalent for the protein cakes generated using the two different primary drying conditions.

Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody.

The effects of secondary structure on asparagine (N) deamidation in a 22 amino acid sequence (369-GFYPSDIAVEWESNGQPENNYK-390) of the crystallizable (Fc) fragment of a human monoclonal antibody (Fc IgG1) were investigated using high-resolution ultra performance liquid chromatography with tandem mass spectrometry (UPLC/MS). Samples containing either the intact Fc IgG (approximately 50 kD) ("intact protein"), or corresponding synthetic peptides ("peptide") were stored in Tris buffer at 37 degrees C and pH 7.5 for up to forty days, then subjected to UPLC/MS analysis with high energy MS1 fragmentation. The peptide deamidated only at N(382) to form the isoaspartate (isoD(382)) and aspartate (D(382)) products in the ratio of approximately 4:1, with a half-life of approximately 3.4 days. The succinimide intermediate (Su(382)) was also detected; deamidation was not observed for the other two sites (N(387) and N(388)) in peptide samples. The intact protein showed a 30-fold slower overall deamidation half-life of approximately 108 days to produce the isoD(382) and D(387) products, together with minor amounts of D(382). Surprisingly, the D(382) and isoD(387) products were not detected in intact protein samples and, as in the peptide samples, deamidation was not detected at N(388). The results indicate that higher order structure influences both the rate of N-deamidation and the product distribution.

Protein conformation in amorphous solids by FTIR and by hydrogen/deuterium exchange with mass spectrometry.

Solid-state hydrogen/deuterium exchange (ssHDX) with electrospray ionization mass spectrometry (ESI-MS) and Fourier transform infrared (FTIR) spectroscopy were used to assess protein conformation in amorphous solids. Myoglobin, lysozyme, beta-lactoglobulin, ribonuclease A, E-cadherin 5, and concanavalin A were co-lyophilized with carbohydrates (trehalose, raffinose, and dextran 5000), linear polymers (polyvinyl alcohol and polyvinyl pyrrolidone) or guanidine hydrochloride (negative control). For ssHDX, samples were exposed to D2O vapor at 33% relative humidity and room temperature, and then reconstituted at low temperature (4 degrees C) and pH 2.5 and analyzed by ESI-MS. Peptic digestion of selected proteins was used to provide region-specific information on exchange. FTIR spectra were acquired using attenuated total reflectance. FTIR and ssHDX of intact proteins showed preservation of structure by raffinose and trehalose, as indicated by FTIR band intensity and protection from exchange. ssHDX of peptic digests further indicated that these protective effects were not exerted uniformly along the protein sequence but were observed primarily in alpha-helical regions, a level of structural resolution not afforded by FTIR. The results thus demonstrate the utility of HDX with ESI-MS for analyzing protein conformation in amorphous solid samples.

Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs.

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6).