John P. Gabrielson

Flow cytometry: a promising technique for the study of silicone oil-induced particulate formation in protein formulations.

Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also used to investigate the effects of silicone oil emulsions on the stability of BSA, lysozyme, abatacept, and trastuzumab formulations containing surfactant, sodium chloride, or sucrose. To aid in particle characterization, the fluorescence detection capabilities of flow cytometry were exploited by staining silicone oil with BODIPY 493/503 and model proteins with Alexa Fluor 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. The addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations.

Qualification of FTIR spectroscopic method for protein secondary structural analysis

Fourier transform infrared (FTIR) spectroscopy is widely used to study protein secondary structure both in solution and in the solid state. The FTIR spectroscopic method has also been employed as a characterization method by the biopharmaceutical industry to determine the higher order structure of protein therapeutics, and to determine if any changes in protein conformation have occurred as a result of changes to process, formulation, manufacture, and storage conditions. The results of these studies are often included in regulatory filings; when comparability is assessed, the comparison is often qualitative. To demonstrate that the method can be quantitative, and is suitable for these intended purposes, the precision and sensitivity of the FTIR method were evaluated. The results show that FTIR spectroscopic analysis is reproducible with suitable method precision, that is, spectral similarity of replicate measurements is greater than 90%. The method can detect secondary structural changes caused by pH and denaturant. The sensitivity of the method in detecting structural changes depends on the extent of the changes and their impact on the resulting spectral similarity and characteristic FTIR bands. The results of these assessments are described in this paper.

Applications of differential scanning calorimetry for thermal stability analysis of proteins: Qualification of DSC

Differential scanning calorimetry (DSC) has been used to characterize protein thermal stability, overall conformation, and domain folding integrity by the biopharmaceutical industry. Recently, there have been increased requests from regulatory agencies for the qualification of characterization methods including DSC. Understanding the method precision can help determine what differences between samples are significant and also establish the acceptance criteria for comparability and other characterization studies. In this study, we identify the parameters for the qualification of DSC for thermal stability analysis of proteins. We use these parameters to assess the precision and sensitivity of DSC and demonstrate that DSC is suitable for protein thermal stability analysis for these purposes. Several molecules from different structural families were studied. The experiments and data analyses were performed by different analysts using different instruments at different sites. The results show that the (apparent) thermal transition midpoint (T(m)) values obtained for the same protein by same and different instruments and/or analysts are quite reproducible, and the profile similarity values obtained for the same protein from the same instrument are also high. DSC is an appropriate method for assessing protein thermal stability and conformational changes.

Applications of circular dichroism (CD) for structural analysis of proteins: qualification of near‐ and far‐UV CD for protein higher order structural analysis

Circular dichroism (CD) spectroscopy is routinely used in the biopharmaceutical industry to study the effects of manufacturing, formulation, and storage conditions on protein conformation and stability, and these results are often included in regulatory filings. In this context, the purpose of CD spectroscopy is often to verify that a change in the formulation or manufacturing process of a product has not produced a change in the conformation of a protein. A comparison of two or more spectra is often required to confirm that the proteins structure has been maintained. Traditionally, such comparisons have been qualitative in nature, based on visually inspecting the overlaid spectra. However, visual assessment is inherently subjective and therefore prone to error. Furthermore, recent requests from regulatory agencies to demonstrate the suitability of the CD spectroscopic method for the purpose of comparing spectra have highlighted the need to appropriately qualify CD spectroscopy for characterization of biopharmaceutical protein products. In this study, we use a numerical spectral comparison approach to establish the precision of the CD spectroscopic method and to demonstrate that it is suitable for protein structural characterization in numerous biopharmaceutical applications.

Measuring low levels of protein aggregation by sedimentation velocity

The required performance of an analytical method depends on the purpose for which it will be used. As a methodology matures, it may find new application, and the performance demands placed on the method can increase. Sedimentation velocity analytical ultracentrifugation (SV-AUC) has a long and distinguished history with important contributions to molecular biology. Now the technique is transitioning into industrial settings, and among them, SV-AUC is now used to quantify the amount of protein aggregation in biopharmaceutical protein products, often at levels less than 1% of the total protein mass. In this paper, we review recent advances to SV methodology which have been shown to improve quantitation of protein aggregation. Then we discuss the performance of the SV method in its current state, with emphasis on the precision and quantitation limit of the method, in the context of existing industrial guidance on analytical method performance targets for quantitative methods.

Detection of Protein Aggregates by Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC): Sources of Variability and Their Relative Importance

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has found application in the biopharmaceutical industry as a method of detecting and quantifying protein aggregates. While the technique offers several advantages (i.e., matrix-free separation and minimal sample handling), its results exhibit a high degree of variability relative to orthogonal size-sensitive separation techniques such as size exclusion chromatography (SEC). The goal of this work is to characterize and quantify the sources of variability that affect SV-AUC results, particularly size distributions for a monoclonal antibody monomer/dimer system. Contributions of individual factors to the overall variability are examined. Results demonstrate that alignment of sample cells to the center of rotation is the most significant contributing factor to overall variability. The relative importance of other factors (e.g., temperature equilibration, time-invariant noise, meniscus misplacement, etc.) are quantified and discussed.

Common Excipients Impair Detection of Protein Aggregates During Sedimentation Velocity Analytical Ultracentrifugation

The final formulations of modern pharmaceutical protein products typically contain sugars or sugar alcohols as stabilizers. Migration of these sugars under the influence of an applied gravitational field during sedimentation velocity analytical ultracentrifugation (SV-AUC) produces dynamic density and viscosity gradients. If the formation of such gradients is not taken into account during data analysis, the capability of the SV-AUC technique to detect protein oligomers/aggregates may be dramatically impacted. In the example described here, the limit of quantitation (LOQ) of a simulated monoclonal antibody (mAb) dimer increases from 0.8% to 2.4% upon addition of 5% sorbitol to the formulation. This study uses simulated and experimental SV-AUC data to demonstrate the detrimental effect of dynamic gradients; it further explores how sophisticated data analysis techniques, including SEDFITs inhomogeneous solvent options, may be used to mitigate the detection problems caused by the sedimentation of excipients.

Precision of protein aggregation measurements by sedimentation velocity analytical ultracentrifugation in biopharmaceutical applications.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is routinely applied in biopharmaceutical development to measure levels of protein aggregation in protein products. SV-AUC is free from many limitations intrinsic to size exclusion chromatography (SEC) such as mobile phase and column interaction effects on protein self-association. Despite these clear advantages, SV-AUC exhibits lower precision measurements than corresponding measurements by SEC. The precision of SV-AUC is influenced by numerous factors, including sample characteristics, cell alignment, centerpiece quality, and data analysis approaches. In this study, we evaluate the precision of SV-AUC in its current practice utilizing a multilaboratory, multiproduct intermediate precision study. We then explore experimental approaches to improve SV-AUC measurement precision, with emphasis on utilization of high quality centerpieces.

Comparison of quantitative spectral similarity analysis methods for protein higher-order structure confirmation.

Optical and vibrational spectroscopic techniques are important tools for evaluating secondary and tertiary structures of proteins. These spectroscopic techniques are routinely applied in biopharmaceutical development to elucidate structural characteristics of protein products, to evaluate the impact of processing and storage conditions on product quality, and to assess comparability of a protein product before and after manufacturing changes. Conventionally, the degree of similarity between two spectra has been determined visually. In addition to requiring a significant amount of analyst training and experience, visual inspection of spectra is inherently subjective, and any determination of comparability based on visual analysis of spectra is therefore arbitrary. Here, we discuss a general methodology for evaluating the suitability of numerical methods to calculate spectral similarity, and then we apply the methodology to compare four quantitative spectral similarity methods: the correlation coefficient, area of spectral overlap, derivative correlation algorithm, and spectral difference methods. While the most effective spectral similarity method may depend on the particular application, all four approaches are superior to visual evaluation, and each is suitable for assessing the degree of similarity between spectra.

In Vitro Stoichiometry of Complexes between the Soluble RANK Ligand and the Monoclonal Antibody Denosumab

The in vitro binding stoichiometry of denosumab, an IgG2 fully human monoclonal therapeutic antibody, to RANK ligand was determined by multiple complementary size separation techniques with mass measuring detectors, including two solution-based techniques (size-exclusion chromatography with static light scattering detection and sedimentation velocity analytical ultracentrifugation) and a gas-phase analysis by electrospray ionization time-of-flight mass spectrometry from aqueous nondenaturing solutions. The stoichiometry was determined under defined conditions ranging from small excess RANK ligand to large excess denosumab (up to 40:1). High concentrations of denosumab relative to RANK ligand were studied because of their physiological relevance; a large excess of denosumab is anticipated in circulation for extended periods relative to much lower concentrations of free soluble RANKL. The studies revealed that an assembly including 3 denosumab antibody molecules bound to 2 RANKL trimers (3D2R) is the most stable complex in DPBS at 37 °C. This differs from the 1:1 binding stoichiometry reported for RANKL and osteoprotegerin (OPG), a soluble homodimeric decoy receptor which binds RANKL with high affinity. Denosumab and RANKL also formed smaller assemblies including 1 denosumab and 2 RANKL trimer molecules (1D2R) under conditions of excess RANKL, 3 denosumab molecules and 1 RANKL trimer (3D1R) under conditions of excess denosumab, and larger assemblies, but these intermediate species were only present at lower temperatures (4 °C), shortly after mixing denosumab and RANKL, and converted over time to the more stable 3D2R assembly.