High‐throughput screening techniques for rapid PEG‐based precipitation of IgG₄ mAb from clarified cell culture supernatant

Abstract

Locating optimal protein precipitation conditions for complex biological feed materials is problematic. This article describes the application of a series of high-throughput platforms for the rapid identification and selection of conditions for the precipitation of an IgG(4) monoclonal antibody (mAb) from a complex feedstock using only microliter quantities of material. The approach uses 96-microwell filter plates combined with high-throughput analytical methods and a method for well volume determination for product quantification. The low material, time and resource requirements facilitated the use of a full factorial Design of Experiments (DoE) for the rapid investigation into how critical parameters impact the IgG(4) precipitation. To aid the DoE, a set of preliminary range-finding studies were conducted first. Data collected through this approach describing Polyethylene Glycol (PEG) precipitation of the IgG(4) as a function of mAb concentration, precipitant concentration, and pH are presented. Response surface diagrams were used to explore interactions between parameters and to inform selection of the most favorable conditions for maximum yield and purification. PEG concentrations required for maximum yield and purity were dependant on the IgG(4) concentration; however, concentrations of 14 to 20% w/v, pH 6.5, gave optimal levels of yield and purity. Application of the high-throughput approach enabled 1,155 conditions to be examined with less than 1 g of material. The level of insights gained over such a short time frame is indicative of the power of microwell experimentation in allowing the rapid identification of appropriate processing conditions for key bioprocess operations.

Thermodynamic parameters for salt‐induced reversible protein precipitation from automated microscale experiments

“…Selective precipitation offers a highly efficient primary recovery step in the bioprocessing of therapeutic proteins, provided conditions can be found where the process is reversible (Knevelman et al, 2010;Richardson et al, 1990). Extensive thermodynamic characterization of protein precipitation and solubility over a range of bioprocess, salt, and pH conditions would refine our understanding of such processes and provide a better molecular understanding of the interaction of proteins with chemical precipitation agents.…”

Intelligent Automation Platform for Bioprocess Development

“…A large number of researches focus on the high throughput screening (HTS) which is an essential part of drug discovery and development (Gribbon & Andreas 2005;Mishra et al 2008). Many studies have contributed to microbial fermentation (Kostov et al 2001;Rios-Solis et al 2013 (Saitoh & Yoshimori 2008), precipitation (Knevelman et al 2010;Titchener-Hooker et al 2008) and chromatography (Chhatre et al 2010;Wiendahl et al 2008). …”

“…Therefore, large amount of experiments need to be executed in order to establish an empirical mathematical model (Knevelman et al 2010). High throughput automated system is well suited for precipitation studies.…”

“…The combination of microwell plates and liquid handling system was proved to largely improve the efficiency of research process. For example, the optimal condition of antibody precipitation using PEG on Tecan liquid handling system was reported (Knevelman et al 2010). …”

Establishment of a Design Space for Biopharmaceutical Purification Processes Using DoE

“…DoE has been used in many fields of application besides protein purification [8,9], such as development of culture media either for prokaryotic or eukaryotic cells [10], recombinant protein and virus productivity optimization [11,12].…”