Universal Qualification of Near Ultraviolet Circular Dichroism Spectroscopy: Method Performance Parameters and Limits of Detection for Structural Differences.

Analytical methods must be qualified as part of the method development lifecycle for product characterization of biotherapeutics. For higher order structure characterization methods, such as near ultraviolet circular dichroism spectroscopy, qualification is performed to determine the expected variability of the method and to establish criteria for analytical product comparability, reference standard qualification, and analytical similarity evaluations. Typical method qualifications require a single product to be tested across several days with multiple replicates, essential to establish a quantitative limit for future product evaluation studies, which may be burdensome with respect to time, instrumentation, and material requirements. In this note, a methodology is proposed to expedite the qualification process for the near ultraviolet circular dichroism spectroscopy method, decreasing the number of required qualification runs, in many cases, to just one for each product. The significant reduction in the number of assays for qualification is achieved by utilizing historical data that applies universally across products of variable classification, size, and test date. Despite their differences, the products exhibit comparable method performance when compared to a product-specific reference standard, and a universal detection threshold is established for application to future product evaluations that meet pre-determined method suitability criteria following a single verification run.

Guidance to Achieve Accurate Aggregate Quantitation in Biopharmaceuticals by SV-AUC.

The levels and types of aggregates present in protein biopharmaceuticals must be assessed during all stages of product development, manufacturing, and storage of the finished product. Routine monitoring of aggregate levels in biopharmaceuticals is typically achieved by size exclusion chromatography (SEC) due to its high precision, speed, robustness, and simplicity to operate. However, SEC is error prone and requires careful method development to ensure accuracy of reported aggregate levels. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an orthogonal technique that can be used to measure protein aggregation without many of the potential inaccuracies of SEC. In this chapter, we discuss applications of SV-AUC during biopharmaceutical development and how characteristics of the technique make it better suited for some applications than others. We then discuss the elements of a comprehensive analytical control strategy for SV-AUC. Successful implementation of these analytical control elements ensures that SV-AUC provides continued value over the long time frames necessary to bring biopharmaceuticals to market.

Technical decision making with higher order structure data: utilization of differential scanning calorimetry to elucidate critical protein structural changes resulting from oxidation.

Differential scanning calorimetry (DSC) is a useful tool for monitoring thermal stability of the molecular conformation of proteins. Here, we present an example of the sensitivity of DSC to changes in stability arising from a common chemical degradation pathway, oxidation. This Note is part of a series of industry case studies demonstrating the application of higher order structure data for technical decision making. For this study, six protein products from three structural classes were evaluated at multiple levels of oxidation. For each protein, the melting temperature (Tm ) decreased linearly as a function of oxidation; however, differences in the rate of change in Tm , as well as differences in domain Tm stability were observed across and within structural classes. For one protein, analysis of the impact of oxidation on protein function was also performed. For this protein, DSC was shown to be a leading indicator of decreased antigen binding suggesting a subtle conformation change may be underway that can be detected using DSC prior to any observable impact on product potency. Detectable changes in oxidized methionine by mass spectrometry (MS) occurred at oxidation levels below those with a detectable conformational or functional impact. Therefore, by using MS, DSC, and relative potency methods in concert, the intricate relationship between a primary structural modification, changes in conformational stability, and functional impact can be elucidated.

Quantitative spectral comparison by weighted spectral difference for protein higher order structure confirmation.

Previously, different approaches of spectral comparison were evaluated, and the spectral difference (SD) method was shown to be valuable for its linearity with spectral changes and its independence on data spacing (Anal. Biochem. 434 (2013) 153-165). In this note, we present an enhancement of the SD calculation, referred to as the "weighted spectral difference" (WSD), by implementing a weighting function based on relative signal magnitude. While maintaining the advantages of the SD method, WSD improves the method sensitivity to spectral changes and tolerance for baseline inclusion. Furthermore, a generalized formula is presented to unify further development of approaches to quantify spectral difference.

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.

The role of adjuvant in mediating antigen structure and stability.

The purpose of this study was to probe the fate of a model antigen, a cysteine-free mutant of bacteriophage T4 lysozyme, to the level of fine structural detail, as a consequence of its interaction with an aluminum (Al)-containing adjuvant. Fluorescence spectroscopy and differential scanning calorimetry were used to compare the thermal stability of the protein in solution versus adsorbed onto an Al-containing adjuvant. Differences in accessible hydrophobic surface areas were investigated using an extrinsic fluorescence probe, 8-Anilino-1-naphthalenesulfonic acid (ANS). As has been observed with other model antigens, the apparent thermal stability of the protein decreased following adsorption onto the adjuvant. ANS spectra suggested that adsorption onto the adjuvant caused an increase in exposure of hydrophobic regions of the protein. Electrostatic interactions drove the adsorption, and disruption of these interactions with high ionic strength buffers facilitated the collection of two-dimensional (15) N heteronuclear single quantum coherence nuclear magnetic resonance data of protein released from the adjuvant. Although the altered stability of the adsorbed protein suggested changes to the protein's structure, the fine structure of the desorbed protein was nearly identical to the protein's structure in the adjuvant-free formulation. Thus, the adjuvant-induced changes to the protein that were responsible for the reduced thermal stability were not observed upon desorption.

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.

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.

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 SEDFIT's inhomogeneous solvent options, may be used to mitigate the detection problems caused by the sedimentation of excipients.