Quality risk management and data integrity in R&D laboratories supporting CMC lifecycle of biological products.

The development of pharmaceutical products is the critical bridge that moves a potential new medicine from academic discovery to applied treatment of patients. It translates an idea for a new drug to bench-level research on how it can be manufactured, formulated, characterized and controlled for use in non-clinical and early clinical trials. From pre-clinical R&D discovery work through the commercial launch, substantial R&D CMC data is generated to develop and optimize cGMP manufacturing and testing operations, while also supporting product comparability, elucidating product / impurity structures, assessing critical quality attributes, developing the drug delivery mode, and developing the product formulation for long-term stability.

Developing Lyophilized Formulations for Protein Biopharmaceuticals Containing Salt that Produce Placebos of Corresponding Appearance.

Obtaining an elegant finished pharmaceutical product remains a problem when salt concentration is high, protein concentration is low, and particularly when both conditions are combined. We propose a simple approach to develop a robust lyophilized formulation in the presence of salt and at low protein concentration. We combine this with a commercially viable lyophilization cycle that can serve as a starting point for many protein-based pharmaceutical products.

Universal Qualification of Analytical Procedures for Characterization and Control of Biologics.

A diverse set of analytical tools is required to characterize the complex structural properties of biopharmaceutical products and to ensure their quality, stability, safety, and efficacy. It is generally necessary to demonstrate that such tools are capable of measuring one or more intended attribute(s) of the product with a desired degree of precision, accuracy, linearity, specificity and sensitivity. Here we present a general framework upon which experiments may be designed to establish analytical procedure performance, predicated on the hypothesis that many analytical procedures have universal performance characteristics - that is, the validity of the measured result is a function of the measurement system and data characteristics and is not a function of the specific analyte being measured.

Determining Spectroscopic Quantitation Limits for Misfolded Structures.

Protein secondary structures are frequently assessed using infrared and circular dichroism spectroscopies during drug development (e.g., during product comparability and biosimilarity studies, reference standard characterization, etc.

Technical Decision Making With Higher Order Structure Data: Perspectives on Higher Order Structure Characterization From the Biopharmaceutical Industry.

Characterization of the higher order structure (HOS) of protein-based biopharmaceutical products is an important aspect of their development. Opinions vary about how best to apply biophysical methods, in which contexts to use these methods, and how to use the resulting data to make technical decisions as drug candidates are commercialized [Gabrielson JP, Weiss WF IV. J Pharm Sci.

Rapid Identification and Characterization of Formulated Protein Products by Raman Spectroscopy Coupled with Discriminant Analysis.

Unlabelled: The rapid identification of protein drug products for packaging and receiving can significantly reduce disposition cycle time, and thereby improve the efficiency and productivity of the supply chain to better meet the needs of patients. In this feasibility study, we demonstrate a novel methodology that combines Raman spectroscopy with discriminant analysis that can be used for rapid identification or verification of finished products. With this methodology, Raman spectra of formulated therapeutic proteins were collected non-invasively with the samples either in a quartz cuvette or in the original glass vials, and analyzed without subtraction of buffer or placebo solutions.

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.

Technical decision making with higher order structure data: higher order structure characterization during protein therapeutic candidate screening.

Protein therapeutics differ considerably from small molecule drugs because of the presence of higher order structure (HOS), post-translational modifications, inherent molecular heterogeneity, and unique stability profiles. At early stages of development, multiple molecular candidates are often produced for the same biological target. In order to select the most promising molecule for further development, studies are carried out to compare and rank order the candidates in terms of their manufacturability, purity, and stability profiles.

Technical decision-making with higher order structure data: starting a new dialogue.

Characterization of the higher order structure (HOS) of biological products has been growing in importance in recent years. Scientists in the biopharmaceutical industry, academic researchers, and regulators are all increasingly aware of the critical role that HOS plays in maintaining the stability and intended biological function of biopharmaceutical products. We organized a consortium of scientists and researchers from industry and academic institutions to address how HOS data can be used most effectively to drive decisions during product development.

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.

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).

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 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.

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.

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.

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.

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.

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.

Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged in the biopharmaceutical industry as a technique to detect small quantities of protein aggregates. However, the limits of detection and quantitation of these aggregates are not yet well understood. Although diverse factors (molecule, instrument, technique, and software dependent) preclude an all-encompassing measurement of these limits for the complete system, it is possible to use simulated data to determine the quantitation limits of the data analysis software aspect.

Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity.

Size-exclusion high-performance liquid chromatography (SE-HPLC, SEC) is the long-standing biopharmaceutical industry standard for quantitation of soluble protein aggregates. Recently, sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged as a possible orthogonal technique to SEC for soluble aggregate quantitation. Moreover, asymmetrical flow field flow fractionation (AF4) has shown early promise in quantifying protein aggregates, both soluble and insoluble.