High‐throughput downstream process development for cell‐based products using aqueous two‐phase systems (ATPS) – A case study

Incubation at pH 4.0 or blanching at ∼65 • C facilitates the purification of biopharmaceutical proteins from plants by precipitating most of the host cell proteins (HCPs) before chromatography. However, both methods are compatible only with pH or thermostable target proteins whereas many target proteins may irreversibly denature, e.g., at pH < 4.0. Here, we developed a combined pH/temperature treatment for clarified tobacco extracts and intact leaves. The latter were subjected to a blanching procedure, i.e., the submersion into a hot buffer. Using a design of experiments approach we identified conditions that remove ∼70% of HCPs at ∼55 • C, using the thermosensitive antibody 2G12 and the pH-sensitive DsRed as model proteins. We found that pH and temperature exerted a combined effect during the precipitation of HCPs in the pH range 5.0-7.0 at 35 • C-60 • C. For clarified extracts, the temperature required to achieve a DsRed purity threshold of 20% total soluble protein (TSP) increased from 54 • C to 63 • C when the pH was increased from 6.4 to 7.3. The pH-stable antibody 2G12 was less responsive to the combined treatment, but the purity of 1% TSP was achieved at 35 • C instead of 44 • C when the pH was reduced from 6.3 to 5.8. When blanching intact leaves, product losses were not exacerbated at pH 4.0. Indeed, the highest DsRed purity (58% TSP) was achieved at this pH, combined with a temperature of 60 • C and an incubation time of 30 min. In contrast, the highest 2G12 purity (0.7% TSP) was achieved at pH 5.1 and 40 • C with an incubation time of 20 min. Our data suggest that a combined pH/temperature regime can avoid extreme values of either parameter; therefore, broadening the applicability of these simple purification techniques to other recombinant proteins. K E Y W O R D S blanching, design of experiments, downstream processing, plant-made pharmaceuticals, tobacco host cell proteins Abbreviations: DoE, design of experiments; DSP, downstream processing; HCPs, host cell proteins; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; TSP, total soluble protein This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Section: Introductionmentioning

confidence: 99%

A combined pH and temperature precipitation step facilitates the purification of tobacco‐derived recombinant proteins that are sensitive to extremes of either parameter

Opdensteinen

Lobanov

Buyel

2021

“…Based on the data shown in Figure , CCD‐curves were calculated by applying Equation :

F true(r true) = \frac{n!}{r! true(n - r true)!} P^{r} {(1 - P)}^{n - r}

where F represents the fraction of the total cell population appearing in each theoretical cavity ( r ) of the CCD‐apparatus, and n the total number of transfers. The experimental validation of the model was reported in previously work . We calculated CCD‐curves with 60 transfers for each cell line.…”

Section: Resultsmentioning

confidence: 99%

Cell Separation in Aqueous Two‐Phase Systems − Influence of Polymer Molecular Weight and Tie‐Line Length on the Resolution of Five Model Cell Lines

Zimmermann

Gretzinger

Zimmermann

et al. 2017

Self Cite

The availability of clinical-scale downstream processing strategies for cell-based products presents a critical juncture between basic research and clinical development. Aqueous two-phase systems (ATPS) facilitate the label-free, scalable, and cost-effective separation of cells, and are a versatile tool for downstream processing of cell-based therapeutics. Here, we report the application of a previously developed robotic screening platform, here extended to enable a multiplexed high-throughput cell partitioning analysis in ATPS. We investigated the influence of polymer molecular weight and tie-line length on the resolution of five model cell lines in "charge-sensitive" polyethylene-glycol (PEG)-dextran ATPS. We show, how these factors influence cell partitioning, and that the combination of low molecular weight PEGs and high molecular weight dextrans enable the highest resolution of the five cell lines. Furthermore, we demonstrate that the separability of each cell line from the mixture is highly dependent on the polymer molecular weight composition and tie-line length. Using a countercurrent distribution model we demonstrate that our screenings yielded conditions that theoretically enable the isolation of four of the five cell lines with high purity (>99.9%) and yield.

“…These experiments were performed on the recovery of lysozyme and separation of monoclonal antibodies and host cell proteins. Furthermore, HTS has been used for optimization studies of cell‐based products, where the separation of a promyelocytic cell line HL‐60 and optimization of its recovery was achieved . This research was able to conduct a complete development of an optimal ATPS for recovery of the cells by elaborating binodal curves and performing the characterization and optimization of the systems employed.…”

Section: Potential Trends and Current Challengesmentioning

confidence: 99%

Aqueous Two‐Phase Systems at Large Scale: Challenges and Opportunities

Torres‐Acosta

Mayolo‐Deloisa

González‐Valdez

et al. 2018

Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enhance recovery of industrially relevant bioproducts. After ATPS discovery, a variety of works have been published regarding their scaling from 10 to 1000 L. Although ATPS have achieved high recovery and purity yields, there is still a gap between their bench-scale use and potential industrial applications. In this context, this review paper critically analyzes ATPS scale-up strategies to enhance the potential industrial adoption. In particular, large-scale operation considerations, different phase separation procedures, the available optimization techniques (univariate, response surface methodology, and genetic algorithms) to maximize recovery and purity and economic modeling to predict large-scale costs, are discussed. ATPS intensification to increase the amount of sample to process at each system, developing recycling strategies and creating highly efficient predictive models, are still areas of great significance that can be further exploited with the use of high-throughput techniques. Moreover, the development of novel ATPS can maximize their specificity increasing the possibilities for the future industry adoption of ATPS. This review work attempts to present the areas of opportunity to increase ATPS attractiveness at industrial levels.