Compendial Methods: Suitability Verification, Challenges and Recommendations for Proteins.

Compendial testing methods are not required to be fully validated, but their suitability for testing should be verified under actual conditions of use. This requirement is established in 21 CFR 211.194(a)(2) of the current Good Manufacturing Practice regulations in the United States. ANVISA (Agência Nacional de Vigilância Sanitária) also requires that compendial analytical methods shall have their suitability demonstrated for the intended use by a partial validation study. Suitability verifications or partial validation can be divided into two major categories: visual and instrumental methods. For visual methods, the color and opalescence of interferences should be checked. If the color or clarity/opalescence of the sample is outside of the range of the Pharmacopeia standards/reference solutions, the validity of the test results should be evaluated. Specificity is usually waived because the methods are not specific to products, and accuracy/precision can be addressed by comparing results from analyst to analyst. For instrument methods, specificity can also be waived for certain assays. Accuracy is addressed by implementation of instrument calibration and/or method control. Precision is required either in suitability verification or when testing the samples. Here, we present approaches for suitability verification and the scientific rationale supporting compendial methods: visible particulates, subvisible particles, pH, osmolality, color and clarity/opalescence. Current challenges and recommendations are also discussed specifically for the analysis of protein products.

Characterization of particles in protein solutions: reaching the limits of current technologies.

Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 µm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2-10 µm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5-150 mg/ml for particles <10 µm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.