The Application Notebook
There has been a significant resurgence in the development of antibody-drug conjugates (ADC) as target-directed therapeutic agents for cancer treatment. Among the factors critical to effective ADC design is the Drug Antibody Ratio (DAR). The DAR describes the degree of drug addition that directly impacts both potency and potential toxicity of the therapeutic, and can have significant effects on properties such as stability and aggregation. Determination of DAR is, therefore, of critical importance in the development of novel ADC therapeutics.
Wyatt Technology Corporation
There has been a significant resurgence in the development of antibody-drug conjugates (ADC) as target-directed therapeutic agents for cancer treatment. Among the factors critical to effective ADC design is the Drug Antibody Ratio (DAR). The DAR describes the degree of drug addition that directly impacts both potency and potential toxicity of the therapeutic, and can have significant effects on properties such as stability and aggregation. Determination of DAR is, therefore, of critical importance in the development of novel ADC therapeutics.
DAR is typically assessed by mass spectrometry (MALDI–TOF or ESI–MS) or UV spectroscopy. Calculations based on UV absorption are often complicated by similarities in extinction coefficients of the antibody and small molecule. Mass spectrometry, though a powerful tool for Mw determination, depends on uniform ionization and recovery between compounds - which is not always the case for ADCs.
Here we present a method for DAR determination based on size-exclusion chromatography combined with multi-angle light scattering (SEC–MALS) in conjunction with UV absorption and differential refractive index detection. Figure 1 shows UV traces for two model ADCs; molecular weights of the entire ADC complexes are determined directly from light scattering data.
Figure 1: Molar masses for two distinct ADC formulations are determined using SEC-MALS analysis.
Component analysis is automated within the ASTRA 6 software package by using the differential refractive index increments (dn/dc) and extinction coefficients, which are empirically determined for each species or mined from the literature, to calculate the molar mass of the entire complex as well as for each component of the complex.
In this example an antibody has been alkylated with a compound having a nominal molecular weight of 1250 Da (Figure 2). Molar masses of the antibody fractions are similar, which indicates that the overall differences between the two formulations reflect distinct average DARs that are consistent with values obtained by orthogonal techniques. Note that the molar mass traces for the conjugated moiety represent the total amount of attached pendant groups; the horizontal trends indicate that modification is uniform throughout the population eluting in that peak.
Figure 2: Molar masses for the antibody and total appended drug are calculated in the ASTRA software package based on prior knowledge of each components extinction coefficent and dn/dc, allowing determination of DAR based on a nominal Mw of 1250 Da for an individual drug.
Wyatt Technology Corporation
6300 Hollister Avenue, Santa Barbara, California 93117, USA
Tel: +1 (805) 681 9009 Fax: +1 (805) 681 0123
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.