A rational approach to improving titer in Escherichia coli‐based cell‐free protein synthesis reactions

Abstract

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement. K E Y W O R D S cell-free protein synthesis, multivariate data analysis, process development 1 | INTRODUCTION Cell-free protein synthesis (CFPS) first emerged in the 1960s as part of research uncovering the genetic code. 1 Subsequently, this in vitro production method has been adapted to produce a variety of products including antibodies, biotherapeutic peptides, fusion proteins, vaccine candidates, membrane proteins, toxic proteins, and bacteriophages. 2 There are broadly two types of CFPS: reconstituted CFPS and crude lysate CFPS. In reconstituted CFPS, also known as the Protein synthesis Using Recombinant Elements (PURE) system, purified recombinant proteins and ribosomes are added to the reaction in a bottom-up approach. 3 In crude lysate CFPS, a top-down approach is used in which

Biotechnology Applications of Cell-Free Expression Systems

“…However, concerning the optimization of CFPS yields, it must be mentioned that selected parameters could be specific for the target protein, and this represents a form of overfitting that can be solved only by performing more experiments using other target proteins [141]. Concerning the optimization of CFPS yields in a multidimensional experimental space, there are also examples where the multivariate statistical analysis was applied, requiring less computational resources [144][145][146]. For instance, multivariate statistical data analysis (MSDA) was applied to predict the yield of monoclonal antibodies (MAbs) using a scalable cell-free expression system developed by Sutro Biopharma [144].…”

Escherichia coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles

“…This means that multiple products can be produced at the same time. There is no need to fundamentally change the other reaction components, namely, the cell extract and the concentrated reaction mixture, although some minor modifications to other process parameters may allow for dramatic improvements in titer [19].…”

“…The cell extract was prepared using BL21 Star TM (DE3) (Thermo Fisher Scientific, Paisley, UK) E. coli cells according to the protocol in Colant et al, 2020 [19]. When OD 600 ≈ 0.6 was achieved, 500 µL of 1M IPTG was used to induced T7 RNA polymerase expression.…”

Multivariate statistical data analysis of cell‐free protein synthesis toward monitoring and control

“…24,25 Additionally, the present work aims to provide a generic methodology that allows rapid process characterization and optimization to the manufacturing of other therapeutic proteins and biopolymers by developing data-based relationships between process parameters and process outputs such as yield, which are likely to be highly dependent on the cell extract type, target protein, construct design, and so forth. 26 Section 2 describes the methodology that was followed when applying the MSDA techniques, with Section 3 providing the methodology for operating the laboratory equipment and collecting data. Section 4 presents the results and provides a discussion and finally, Section 5 provides the conclusions and the authors' perspective on the use of MSDA techniques as tools for optimizing the performance of CFPS processes.…”

“…The specific case study for the analyses presented in this article is the scalable cell‐free synthesis of monoclonal antibodies (mAb), using the cell‐free lysate system developed by Sutro Biopharma 24,25 . Additionally, the present work aims to provide a generic methodology that allows rapid process characterization and optimization to the manufacturing of other therapeutic proteins and biopolymers by developing data‐based relationships between process parameters and process outputs such as yield, which are likely to be highly dependent on the cell extract type, target protein, construct design, and so forth 26 …”