Light Scattering for the Masses Monoclonal Antibodies and the Preparative Use of Flow Field-Flow Fractionation (AF4)
The Application Notebook
Proteins - especially monoclonal antibodies (MABs) - have become increasingly important in pharmaceutical work. However, there are some important differences between conventional, chemically-synthesized drugs and proteins. Because of the complex and weak structure of proteins, even a slight change in conditions, such as pH value, temperature, or mechanical stress, may lead to aggregation and a loss of activity or stability.
A.J. Freitag, K. Wittmann, G. Winter, and J. Myschik, Wyatt Technology Corporation
Proteins - especially monoclonal antibodies (MABs) - have become increasingly important in pharmaceutical work. However, there are some important differences between conventional, chemically-synthesized drugs and proteins. Because of the complex and weak structure of proteins, even a slight change in conditions, such as pH value, temperature, or mechanical stress, may lead to aggregation and a loss of activity or stability.
A separation of MABs before analysis is preferred because a detailed investigation of a fraction would be much easier and provide a better insight into the physical and chemical properties, compared to an analysis in the presence of a coexisting monomeric protein.
Asymmetrical flow field-flow fractionation (AF4) is a wellâestablished method for sizing and quantifying different aggregate species in protein formulations. A major advantage of AF4 is the use of the formulation buffer of the protein as the mobile phase. The separation is independent of the ionic strength of the buffer.
The capability of AF4 as a preparative tool for the separation of aggregates, fragments, and monomeric species was investigated in this study. The system consisted of an Eclipse (Wyatt Technology Europe GmbH, Dernbach, Germany), equipped with DAWN multiâangle light scattering (MALS), refractive index (RI) and ultraviolet (UV) detectors, and a fraction collector. The separation was achieved by using a semiâpreparative SP channel with 350 μm height.
A monoclonal antibody sample was exposed to light, resulting in the formation of fragments and aggregates (Figure 1). A sample of 1 mg of protein per run was injected into the channel and collected in tubes made of glass, using a Gilson FC-203B fraction collector. The collected fractions were concentrated and the final concentration was determined by UV absorbance at 280 nm using a Micro-BCA Assay.
Figure 1: UV 280 nm fractograms and MALS detection of light exposed MAb1 (red line) and native MAb1 (blue line).
AF4 is a useful method for analytical and preparative separation of protein aggregates since the separation can be performed in each buffer or even pure water, thus facilitating subsequent activities. Furthermore, an investigation of possible immune responses triggered by protein aggregates in comparison to monomeric proteins can be performed, which is currently of major interest in administrative organizations and the pharmaceutical industry at large.
The results presented in this application note have been published before in A.J. Freitag, K. Wittmann, G. Winter, and J. Myschik, LCGC Europe24(3), 134–140 (2011).
Wyatt Technology Corporation
6300 Hollister Ave., Santa Barbara, California 93117, USA
Tel: +1 (805) 681 9009 Fax: +1 (805) 681 0123
E-mail: info@wyatt.com
Website: www.wyatt.com
2024 EAS Awardees Showcase Innovative Research in Analytical Science
November 20th 2024Scientists from the Massachusetts Institute of Technology, the University of Washington, and other leading institutions took the stage at the Eastern Analytical Symposium to accept awards and share insights into their research.
Inside the Laboratory: The Richardson Group at the University of South Carolina
November 20th 2024In this edition of “Inside the Laboratory,” Susan Richardson of the University of South Carolina discusses her laboratory’s work with using electron ionization and chemical ionization with gas chromatography–mass spectrometry (GC–MS) to detect DBPs in complex environmental matrices, and how her work advances environmental analysis.
AI and GenAI Applications to Help Optimize Purification and Yield of Antibodies From Plasma
October 31st 2024Deriving antibodies from plasma products involves several steps, typically starting from the collection of plasma and ending with the purification of the desired antibodies. These are: plasma collection; plasma pooling; fractionation; antibody purification; concentration and formulation; quality control; and packaging and storage. This process results in a purified antibody product that can be used for therapeutic purposes, diagnostic tests, or research. Each step is critical to ensure the safety, efficacy, and quality of the final product. Applications of AI/GenAI in many of these steps can significantly help in the optimization of purification and yield of the desired antibodies. Some specific use-cases are: selecting and optimizing plasma units for optimized plasma pooling; GenAI solution for enterprise search on internal knowledge portal; analysing and optimizing production batch profitability, inventory, yields; monitoring production batch key performance indicators for outlier identification; monitoring production equipment to predict maintenance events; and reducing quality control laboratory testing turnaround time.
Infographic: Be confidently audit ready, at any time and reduce failures in pharma QC testing
November 20th 2024Discover how you can simplify the audit preparation process with data integrity dashboards that provide transparency to key actions, and seamlessly track long-term trends and patterns, helping to prevent system suitability failures before they occur with waters_connect Data Intelligence software.
Critical Role of Oligonucleotides in Drug Development Highlighted at EAS Session
November 19th 2024A Monday session at the Eastern Analytical Symposium, sponsored by the Chinese American Chromatography Association, explored key challenges and solutions for achieving more sensitive oligonucleotide analysis.
