Analytical QbD: Development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates

Abstract

This paper presents a quality by design (QbD) based development of a novel native PAGE (N-PAGE) method as a low-cost analytical tool for analysis of aggregates of monoclonal antibodies. Comparability to the present gold standard of SEC has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making N-PAGE relevant to those academicians and other small companies involved in early-stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Furthermore, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and analyte resulting in higher elution time and therefore underestimation of the analyte size. The proposed N-PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A QbD-based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration, and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments. The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N-PAGE has been in use for protein analysis for several decades, use of N-PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel.

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…QbD is a manufacturing principle in which product quality is integrated into the manufacturing process. The QbD method has been implemented for the process development and analytical characterization of recombinant proteins, including mAbs (Pathak et al, 2014;Yu et al, 2014;Kumar et al, 2019;Narayanan et al, 2019a;Shekhawat et al, 2019). Using the QbD approach, the effect of various media components and process parameters in Fab production was studied, resulting in a 5-fold enhancement of the target protein titer as compared to the basal medium, thus demonstrating the efficacy of QbD (Kumar et al, 2019).…”

Trends in Upstream and Downstream Process Development for Antibody Manufacturing

“…Furthermore, concepts of Quality by Design (QbD) in combination with HTP methods or DoE [33,34] are encouraged to be applied in process [35][36][37][38] and analytical development [33,34]. QbD is a manufacturing principle in which product quality is built into the manufacturing process by understanding the associated risks and including strategies to mitigate those risks during manufacture [35][36][37][38].…”

“…Harms et al (2008) [40] as well as Abu-Absi et al [41] published case studies on mapping design space for fermentation and cell culture. In DSP, Jiang et al (2010) [35] described a case study on the application of QbD principles for hydrophobic interaction chromatography and Pathak et al (2014) [34] as well as Michels et al (2012) [33] published examples for QbD-based development of analytical methods for antibody aggregates and size heterogeneity.…”

Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review

“…Furthermore, for an efficient development of manufacturing processes, the concept of QbD amalgamated with high throughput methods or DoE (Michels et al, 2012; Pathak et al, 2014) are highly recommended to be applied in both upstream, downstream as well analytical processes (Rathore, 2009; del Val et al, 2010; Jiang et al, 2010; Martin-Moe et al, 2011). The QbD method is implemented for the development of more robust and proficient production processes for recombinant proteins including mAbs with augmented clinical efficacy (del Val et al, 2010).…”

“…Harms et al (2008) and Abu-Absi et al (2010) studied and published the mapping design space for upstream cell culture and fermentation processes. Jiang et al (2010) published important findings on the application of QbD principles for hydrophobic interaction chromatography (HIC) and Pathak et al (2014) as well as Michels et al (2012) published article on QbD-based analytical method development for mAb aggregates and size heterogeneity analysis.…”