Biologic products encounter various types of interfacial stress during development, manufacturing, and clinical administration. When proteins come in contact with vapor–liquid, solid–liquid, and liquid–liquid surfaces, these interfaces can significantly impact the protein drug product quality attributes, including formation of visible particles, subvisible particles, or soluble aggregates, or changes in target protein concentration due to adsorption of the molecule to various interfaces. Protein aggregation at interfaces is often accompanied by changes in conformation, as proteins modify their higher order structure in response to interfacial stresses such as hydrophobicity, charge, and mechanical stress. Formation of aggregates may elicit immunogenicity concerns; therefore, it is important to minimize opportunities for aggregation by performing a systematic evaluation of interfacial stress throughout the product development cycle and to develop appropriate mitigation strategies. The purpose of this white paper is to provide an understanding of protein interfacial stability, explore methods to understand interfacial behavior of proteins, then describe current industry approaches to address interfacial stability concerns. Specifically, we will discuss interfacial stresses to which proteins are exposed from drug substance manufacture through clinical administration, as well as the analytical techniques used to evaluate the resulting impact on the stability of the protein. A high-level mechanistic understanding of the relationship between interfacial stress and aggregation will be introduced, as well as some novel techniques for measuring and better understanding the interfacial behavior of proteins. Finally, some best practices in the evaluation and minimization of interfacial stress will be recommended.

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Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective.

Freezing-induced protein aggregation - Role of pH shift and potential mitigation strategies.

Excipients for Room Temperature Stable Freeze-Dried Monoclonal Antibody Formulations.

Enzymatic polysorbate (PS) degradation and resulting free fatty acid (FFA) particles are detrimental to biopharmaceutical drug product (DP) stability. Different types and grades of polysorbate have varying propensity to form FFA particles. This work evaluates the homogenous all-oleate (AO) PS80 alongside heterogeneous PS20 and PS80 grades in terms its propensity to form FFA particles and other important attributes like interfacial protection and oxidation susceptibility. FFA particle formation rates were compared by degrading PS using non-immobilized hydrolases and fast degrading DP formulations. Interfacial protection of monoclonal antibodies (mAbs) was assessed by agitation studies in saline using non-degraded and degraded PS. Several antioxidants were assessed for their ability to mitigate AO PS80 oxidation and subsequent mAb oxidation by a 40°C placebo stability study and a 2, 2′-Azobis (2-amidinopropane) dihydrochloride stress model, respectively. Visible and subvisible particles were significantly delayed in AO PS80 formulations compared with heterogeneous PS20 and PS80 formulations. Non-degraded AO PS80 was less protective of mAbs against the air-water interface compared with heterogeneous PS20. Interfacial protection by AO PS80 improved upon degradation owing to high surface activity of FFAs. Diethylenetriaminepentaacetic acid (DTPA) completely mitigated AO PS80 oxidation unlike L-methionine and N-Acetyl-DL-Tryptophan. However, DTPA did not mitigate radical mediated mAb oxidation. AO PS80 is a promising alternative to reduce FFA particle formation compared with other PS types and grades. However, limitations observed here---such as lower protection against interfacial stresses and higher propensity for oxidation---need to be considered in assessing the risk/benefit ratio in using AO PS80.

A Comprehensive Assessment of All-Oleate Polysorbate 80: Free Fatty Acid Particle Formation, Interfacial Protection and Oxidative Degradation.

A Multicompany Assessment of Submicron Particle Levels by NTA and RMM in a Wide Range of Late-Phase Clinical and Commercial Biotechnology-Derived Protein Products.

Strategies for determining particulate matter in therapeutic protein injections, including extrinsic and intrinsic particles, are reviewed. Special attention is devoted to the advantages and limitations of various methods used for these purposes, each of which enables different particle characteristics to be determined. The source of particles (extrinsic, intrinsic, or inherent) can be understood better and particle-size distribution and other characteristics can be studied and used to differentiate them if methods based on different measurement principles are used. Protein aggregates in drugs have broad particle-size distributions, from oligomers to particles reaching hundreds of microns. The particle properties can be used to assess the risk associated with protein aggregates in the drug and to study their possible formation mechanisms. Such information could be useful during drug development and manufacturing to reduce the particulate matter content.

Subvisible Particulate Matter in Therapeutic Protein Injections

The importance of using isolation technologies, e.g., an isolator, for sterility testing of medicines is demonstrated. The advantages and disadvantages for the Sterility parameter of using isolators in manufacturing processes and pharmaceutical analysis during examination and quality control stages are discussed. The goal of the work was to optimize the chamber biological cleaning mode with a working load in a GPPI isolator (ESCO, Indonesia), which enables this equipment to be used for sterility testing of medicines. The parameters on which the success of the biodecontamination cycle of the isolator chamber depends are analyzed. Potential biological cleaning modes are studied. The optimum mode for the working load using the GPPI isolator as an example is found. The isolator system is proven to provide sterile working conditions corresponding to cleanroom class A, which makes the equipment suitable for sterility testing of medicines.

Use of Isolation Technologies for Sterility Testing of Medicinal Products

Issues of Determining Particle Sizes of Various Drugs in Evaluating Their Quality

The article presents materials on processing and interpretation of the results of microbiological analysis of the quality of medicines in accordance with the State Pharmacopoeia of the Russian Federation and the Pharmacopoeia of the Eurasian Economic Union. It exemplifies the use of the rules for processing test results and counting aerobic microorganisms and microscopic fungi on agar culture media in Petri dishes and on membrane filters. The author discusses the possibility of and prospects for applying various sample inoculation methods, as well as using calculation factors when interpreting their results. The text covers approaches to repeating microbiological testing of medicines to verify the reliability of a non-compliant first result.

O. V. Gunar

Papers

Subvisible Particulate Matter in Therapeutic Protein Injections

Use of Isolation Technologies for Sterility Testing of Medicinal Products

Issues of Determining Particle Sizes of Various Drugs in Evaluating Their Quality

Practical Aspects of Processing and Interpretation of the Results of Microbiological Analysis of the Quality of Non-Sterile Medicines

Verification of the Membrane Filtration Method for Determining Sterility of Drugs under Medical Isolator Conditions