Improved Diffusion Interaction Parameter Measurement to Predict the Viscosity of Concentrated mAb Solutions.

During the developability assessment of therapeutic monoclonal antibody (mAb) candidates, utilization of robust high-throughput predictive assays enables rapid selection of top candidates with low risks for late-stage development. Predicting the viscosities of highly concentrated mAbs using limited materials is an important aspect of developability assessment because high viscosity can complicate manufacturability, stability, and administration. Here, we report a high-throughput assay measuring protein-protein interactions to predict mAb viscosity. The diffusion interaction parameter (kD) measures colloidal self-association in dilute solutions and has been reported to be predictive of the mAb viscosity at high concentrations. However, kD of Amgen early stage IgG1 mAb candidates measured in 10 mM acetate at pH 5.2 containing sucrose and polysorbate (denoted A52SuT) shows only weak correlation to their viscosities at 140 mg/mL in A52SuT. We hypothesize that kD measured in A52SuT reflects primarily long-range electrostatic repulsions because most of these mAb candidates carry strong net positive charges in this low ionic strength formulation with pH (5.2) well below pI values of mAb candidates. However, the viscosities of high concentration mAbs depend heavily on short-range molecular interactions. We propose an improved kD method in which salt is added to suppress charge repulsions and to allow for detection of key short-range interactions in dilute solutions. Salt types and salt concentrations were screened, and an optimal salt condition was identified. This optimized method was further validated using two test mAb sets. Overall, the method improves the Pearson R2 between kD and viscosity (6-230 cP) from 0.24 to 0.80 for a data set consisting of 37 mAbs.

High Pressure Light Scattering of Therapeutic Proteins To Probe Aggregation and Protein-Protein Interactions.

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.

High-Pressure, Low-Temperature Induced Unfolding and Aggregation of Monoclonal Antibodies: Role of the Fc and Fab Fragments.

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.

An Evaluation of the Glass Vial Hydrolytic Resistance Method.

The European Pharmacopoeia (Ph. Eur.) hydrolytic resistance method for glass vials is routinely used to screen pharmaceutical glass vial supply. In an effort to better understand and control the factors affecting method precision and robustness, several potential sources of variability in the Ph. Eur. alkalinity method have been studied for 3 cc glass vials. Method parameters including vial rinsing, vial covering, autoclave cycle execution, sample hold times, and titration procedure were evaluated in this study. The results of this study indicate the method parameters which require stringent control in order to achieve acceptable method precision and robustness.LAY ABSTRACT: The European Pharmacopoeia (Ph. Eur.) hydrolytic resistance method for glass vials is routinely used to screen biopharmaceutical glass vial supply. The method was studied to assess contributions to its variability and to potentially improve its reliability. The results of this study indicate which method parameters require stringent control in order to generate reliable data using the Ph. Eur. hydrolytic resistance method.

Comparison of Acid Titration, Conductivity, Flame Photometry, ICP-MS, and Accelerated Lamellae Formation Techniques in Determining Glass Vial Quality.

Certain types of glass vials used as primary containers for liquid formulations of biopharmaceutical drug products have been observed with delamination that produced small glass like flakes termed lamellae under certain conditions during storage. The cause of this delamination is in part related to the glass surface defects, which renders the vials susceptible to flaking, and lamellae are formed during the high-temperature melting and annealing used for vial fabrication and shaping. The current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. Four alternative techniques with improved throughput, convenience, and/or comprehension were examined by subjecting seven lots of vials to analysis by all techniques. The first three new techniques of conductivity, flame photometry, and inductively coupled plasma mass spectrometry measured the same sample pools as acid titration. All three showed good correlation with alkalinity: conductivity (R(2) = 0.9951), flame photometry sodium (R(2) = 0.9895), and several elements by inductively coupled plasma mass spectrometry [(sodium (R(2) = 0.9869), boron (R(2) = 0.9796), silicon (R(2) = 0.9426), total (R(2) = 0.9639)]. The fourth technique processed the vials under conditions that promote delamination, termed accelerated lamellae formation, and then inspected those vials visually for lamellae. The visual inspection results without the lot with different processing condition correlated well with alkalinity (R(2) = 0.9474). Due to vial processing differences affecting alkalinity measurements and delamination propensity differently, the ratio of silicon and sodium measurements from inductively coupled plasma mass spectrometry was the most informative technique to assess overall vial quality and vial propensity for lamellae formation. The other techniques of conductivity, flame photometry, and accelerated lamellae formation condition may still be suitable for routine screening of vial lots produced under consistent processes. LAY ABSTRACT: 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.

Chemical modifications of therapeutic proteins induced by residual ethylene oxide.

Ethylene oxide (EtO) is widely used in sterilization of drug product primary containers and medical devices. The impact of residual EtO on protein therapeutics is of significant interest in the biopharmaceutical industry. The potential for EtO to modify individual amino acids in proteins has been previously reported. However, specific identification of EtO adducts in proteins and the effect of residual EtO on the stability of therapeutic proteins has not been reported to date. This paper describes studies of residual EtO with two therapeutic proteins, a PEGylated form of the recombinant human granulocyte colony-stimulating factor (Peg-GCSF) and recombinant human erythropoietin (EPO) formulated with human serum albumin (HSA). Peg-GCSF was filled in an EtO sterilized delivery device and incubated at accelerated stress conditions. Glu-C peptide mapping and LC-MS analyses revealed residual EtO reacted with Peg-GCSF and resulted in EtO modifications at two methionine residues (Met-127 and Met-138). In addition, tryptic peptide mapping and LC-MS analyses revealed residual EtO in plastic vials reacted with HSA in EPO formulation at Met-328 and Cys-34. This paper details the work conducted to understand the effects of residual EtO on the chemical stability of protein therapeutics.

Determining the delamination propensity of pharmaceutical glass vials using a direct stress method.

An accelerated lamellae formation (ALF) methodology has been developed to determine the delamination propensity and susceptibility of pharmaceutical glass vials. The ALF process consists of a vial wash and depyrogenation mimic procedure followed by stressing glass vials with 20 mM glycine pH 10.0 solution at 50 °C for 24 h and analyzing the resulting solutions by visual inspection for glass lamellae. ALF results demonstrate that while vial delamination propensity generally correlates with glass hydrolytic resistance, ALF is a more direct test of glass delamination propensity and is not affected by post-production vial washing that can affect results obtained using hydrolytic resistance tests. ALF can potentially be used by pharmaceutical companies to evaluate and screen incoming vial lots to minimize the risk of delamination during the shelf life of parenteral therapeutics, and by glass vial manufacturers to monitor and improve their vial manufacturing processes. LAY ABSTRACT: 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.

Nondestructive detection of glass vial inner surface morphology with differential interference contrast microscopy.

Glass particles generated by glass dissolution and delamination of the glass container for pharmaceutical products have become a major issue in the pharmaceutical industry. The observation of glass particles in certain injectable drugs, including several protein therapeutics, has recently resulted in a number of product recalls. Glass vial surface properties have been suggested to play a critical role in glass dissolution and delamination. Surface characterization of glass container, therefore, is important to evaluate the quality of the glass container. In this work, we demonstrate that differential interference contrast (DIC) microscopy is a powerful, effective, and convenient technique to examine the inner surface morphology of glass vials nondestructively. DIC microscopy does not require the cutting of the glass vial for scanning the inner surface and has sufficient spatial resolution to reveal glass pitting, phase separation, delamination scars, and other defects. Typical surface morphology of pharmaceutical glass vials with different alkalinity are compared and discussed.

Elucidation of degradants in acidic peak of cation exchange chromatography in an IgG1 monoclonal antibody formed on long-term storage in a liquid formulation.

PURPOSE: An IgG1 therapeutic monoclonal antibody showed an increase in acidic or pre-peak by cation exchange chromatography (CEX) at elevated temperatures, though stable at 2-8°C long-term storage in a liquid formulation. Characterization effort was undertaken to elucidate the degradants in CEX pre-peak and effect on biological activity. METHODS: Purified CEX fractions were collected and analyzed by peptide mapping, size exclusion, intact and reduced-alkylated reversed phase techniques. Biophysical characterization, isoelectric focusing and Isoquant analysis were also performed to determine nature of degradants. Bioassay and surface plasmon resonance experiments were performed to determine the impact on biological activity of the degradants. RESULTS: No major degradation due to oxidation, clipping or aggregation was detected; conformational differences between purified fractions observed were not significant. Sialic acid, N-terminal glutamine cyclization and glycation differences contributed to the CEX pre-peak in the mAb control sample; increase in CEX pre-peak at 25°C and higher was caused by additive degradation pathways of deamidation, related isomerization and clipping. CONCLUSIONS: 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.

Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study.

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.