Evaluation of dynamic light scattering as a technique for the routine monitoring of therapeutic proteins on stability
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Date
2023
Authors
O'Kennedy, Laura
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
Throughout the course of biotherapeutic development and manufacturing, the detection and quantification of protein aggregates and particles is critical to ensure patient safety. Size Exclusion Chromatography (SEC) is one of the most widely used techniques for the separation of proteins and allows for the detection and quantification of aggregates. In terms of aggregate detection at drug substance (DS) and drug product (DP) release, as well as for stability testing, the ICH guidelines reference SEC as an example. However, it is acknowledged that new analytical techniques are continuously being developed and can be used where appropriate. Dynamic Light Scattering (DLS) is one such technique, that has been described on several occasions as an early indicator of protein aggregation. Some benefits of DLS over other aggregation-monitoring techniques, is its ability to analyse proteins in their native environment, its fast analysis time, and its ability to detect a wide particle size range. Additionally, for high molecular weight species (HMWS) detection, DLS is an exceptionally sensitive technique. This sensitivity and level of detection are particularly important in the biopharmaceutical industry as the presence of even a small number of aggregates can significantly increase upon long-term storage. However, this sensitivity can also be a weakness, as scattering from traces of other large particulates can interfere with results, and overall, the quantification of results can be difficult. Consequently, the technique is typically used in a complementary fashion with more widely used techniques such as SEC. The capabilities and applications of DLS have been improved over time with the invention and incorporation of e.g. multi angle DLS (MADLS), extended size range analysis and diffusion interaction parameter (kD) analysis, into the one instrument and software.
The aim of this research was to explore the possible benefits and limitations associated with DLS, in order to determine if the technique has the potential to be used more widely for testing protein therapeutics on stability. A range of biotherapeutics including monoclonal, bispecific and trispecific antibodies were tested, that varied in concentration from 2 mg/mL to 160 mg/mL. Multiple stressed samples were included in the test panel to explore the techniques’ ability to detect large particles and aggregates. The particle size and size distribution results were compared to those generated from SEC, to explore whether DLS could give additional insight into the molecules stability. It was found that the technique showed great sensitivity and, in some cases, detected an increase in large particles where SEC failed to do so. These results were then substantiated by looking at data from sub-visible particle analysis via light obscuration. The zetasizer instrument, used to measure DLS, can also perform various other measurements that are indicators of colloidal and/or thermal stability. These include electrophoretic light scattering (ELS), extended size range analysis, particle concentration, kD determination, osmotic second virial coefficient (B22) determination and molecular weight (Mw) measurements. These measurements were performed to determine their respective abilities to provide additional insight into therapeutic protein stability. For most of the measurements however, the results and trends weren’t as consistent and as reliable as the particle size and particle distribution measurements. Further evaluation of these measurements, protein concentration optimisation and additional knowledge of the sample properties would be needed to provide more accurate results. However, overall, DLS is seen to be a highly sensitive and fast analysis tool for the determination of particle size and can provide additional insight into molecule stability when used in conjunction with SEC.
Description
Controlled Access
Keywords
Dynamic light scattering , Protein therapeutics , Protein stability , Protein aggregation , Biotherapeutics
Citation
O'Kennedy, L. 2023. Evaluation of dynamic light scattering as a technique for the routine monitoring of therapeutic proteins on stability. MSc Thesis, University College Cork.