Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.

The observed CEX pre-peak increase was caused by multiple degradations, especially deamidation and clipping. This elucidation of degradants in CEX peaks may apply to other therapeutic IgG1 monoclonal antibodies.

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The effects of high pressure and low temperature on the stability of two different monoclonal antibodies (MAbs) were examined in this work. Fluorescence and small-angle neutron scattering were used to monitor the in situ effects of pressure to infer shifts in tertiary structure and characterize aggregation prone intermediates. Partial unfolding was observed for both MAbs, to different extents, under a range of pressure/temperature conditions. Fourier transform infrared spectroscopy was also used to monitor ex situ changes in secondary structure. Preservation of native secondary structure after incubation at elevated pressures and subzero ° C temperatures was independent of the extent of tertiary unfolding and reversibility. Several combinations of pressure and temperature were also used to discern the respective contributions of the isolated Ab fragments (Fab and Fc) to unfolding and aggregation. The fragments for each antibody showed significantly different partial unfolding profiles and reversibility. There was not a simple correlation between stability of the full MAb and either the Fc or Fab fragment stabilities across all cases, demonstrating a complex relationship to full MAb unfolding and aggregation behavior. That notwithstanding, the combined use of spectroscopic and scattering techniques provides insights into MAb conformational stability and hysteresis in high-pressure, low-temperature environments.

Glass flakes can sometimes appear in liquid pharmaceutical drugs contained in glass vials. These glass flakes are a result of several factors related to the glass vial production process, glass vial sterilization procedures, and the formulation of the liquid pharmaceutical drug. Vial testing is routinely done in order to select glass vials that are less likely to form glass flakes. The factors leading to the formation of glass flakes were studied and applied to a method designed to directly screen vials for their propensity to form glass flakes. The washing of vials followed immediately by sterilization at high temperatures was determined to be a critical factor in the formation of glass flakes. As a result, a laboratory mimic of this procedure was incorporated into the newly developed method for screening vials. This mimic procedure as well as robust accelerated incubation conditions and a sensitive visual inspection procedure are key aspects of this vial screening method.

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There is interest in the direct in situ measurement of protein aggregation and reversible protein–protein interactions at high pressure as a means to assess protein stability. This is currently limited by the availability of in-house analytical methods. High-pressure (HP) scattering instrumentation (using either neutrons, X-rays, or light sources) are relatively rare, due to extensive development hurdles and lack of standardization. This report focuses on design, operation, and application of a new HP light scattering apparatus based on commercially available equipment with a view to wider applications. HP static light scattering results were obtained for two monoclonal antibodies (MAbs) that exhibit different extents of unfolding and aggregation at these conditions. Aggregation that was observed during in situ pressure incubations varied by MAb and total ionic strength of solution. This was conducted in tandem with ex situ measurements on MAb solutions that were incubated under pressure, where monomer loss was measured with size exclusion chromatography. Pressure cycling was also used to assess the extent of pressure-induced reversible and irreversible aggregation. Finally, the ability of the HP light scattering apparatus to assess the influence of pressure on reversible protein–protein interactions in the canonical sense of second osmotic virial coefficients was assessed using lysozyme, a relatively well-characterized protein under hydrostatic pressure. The method offers a convenient and reproducible capability that complements current small angle neutron/X-ray instrumentation, providing measurements that can be used to optimize the planning and interpretation of scattering data from synchrotron or neutron research facilities. Our results address a growing demand to characterize protein aggregates and aggregation-prone partially unfolded intermediates.

Recently, delamination that produced small glass like flakes termed lamellae has been observed in glass vials that are commonly used as primary containers for pharmaceutical drug products under certain conditions during storage. The main cause of these lamellae was the quality of the glass itself related to the manufacturing process. Current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. As alternative to the European Pharmacopoeia method, four other techniques were assessed. Three new techniques of conductivity, flame photometry, and inductively coupled plasma mass spectrometry measured the vial extract pool as acid titration to quantify quality, and they demonstrated good correlation with original alkalinity. The fourth technique processed the vials under conditions that promote delamination, termed accelerated lamellae formation, and the vials were then inspected visually for lamellae. The accelerated lamellae formation technique also showed good correlation with alkalinity. Of the new four techniques, inductively coupled plasma mass spectrometry was the most informative technique to assess overall vial quality even with differences in processing between vial lots. Other three techniques were still suitable for routine screening of vial lots produced under consistent processes.