Process intensification and integration for pneumococcal surface protein A production and purification

• Grant number: 15/10291-8
• Support type: Regular Research Grants
• Duration: April 01, 2016 - January 31, 2019
• Field of knowledge: Engineering - Chemical Engineering
• Principal researcher: Teresa Cristina Zangirolami
• Grantee: Teresa Cristina Zangirolami
• Home Institution: Centro de Ciências Exatas e de Tecnologia (CCET). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Assoc. researchers: Antonio Carlos Luperni Horta ; Felipe Fernando Furlan ; Joaquin Cabrera-Crespo ; Marcelo Perencin de Arruda Ribeiro ; Roberto de Campos Giordano ; Viviane Maimoni Gonçalves

Abstract

The bacterium Escherichia coli is one of the main hosts employed for recombinant protein production, with a turnover of E50-60 billion in the biopharmaceutical world market. Its choice as microbial cell factorial is due to the widespread knowledge accumulated about the genetics and metabolism of E. coli, the availability of fast and precise methods to modify its genome and the ease for obtaining high quantities of biomass by employing well-established cultivation protocols. Expression vectors with widespread application for recombinant protein production in E. coli, based on the lac operon, where lactose or its analogous, isopropyl beta-D-1-thiogalactopyranoside, are employed to induce the synthesis of the target protein, which normally accumulates inside the cell. Usually the target protein is produced in fusion with elements (histidine tags, among others), which provides affinity for transition metal ions (Ni+2, Co+2, Zn+2) and facilitates its purification by affinity chromatography. However, for the sake of final product specifications, the removal of these fusion elements is required, what increases the cost due to the high prices of enzymes as well as of the subsequent steps for the separation of the free fusion molecule. For proteins synthesized without fusion, the purification strategy to be applied is case specific and its development is based on the differences among the physical-chemical characteristics of the target protein and contaminants (such as proteins, DNA and lipopolysaccharides or endotoxins), with several possibilities for purification protocols. The presence of contaminants in the raw product obtained at the end of the production stage is directly related to the cultivation and induction strategies applied. Therefore, it is crucial to identify the physiological responses of the cells triggered by induction and, in addition, to incorporate this issue on the development of cultivation strategies. Besides, the manufacturing of more accessible and high quality drugs relies on the simultaneous optimization of upstream and downstream processes, including as well economic viability analysis of the integrated protein production and purification steps. The main objective of this research project is to intensify, integrate and optimize the processes for pneumococcal surface protein A production and purification, which can be used to prevent infections caused by Streptococcus pneumoniae in the form of a conjugated vaccine or as a subunit protein vaccine. From bench-scale bioreactor (stirred tank or overpressure operated airlift) batch cultures, using different media (complex or defined), inducers (IPTG or lactose) and induction temperatures, ordinary and detoxified E. coli BL21(DE3) biomasses containing the target protein PspA4Pro will be generated under distinct operation conditions. These biomasses will be further processed at Butantan Institute, where different purification protocols will be evaluated. The purification protocols that lead to satisfactory performance in terms of product purity and quality, together with the respect cultivation-induction conditions, will be integrated and evaluated in terms of economic viability by the "Recombinant Protein Virtual Biorefinery", which will be implemented using open software EMSO (Environment for Modelling, Simulation and Optimization). The results generated by all combinations of production /purification processes studied will be evaluated in terms of recombinant protein yield, purity and cost. The most promising cultivation/purification strategies regarding product quality and economic viability will be identified and further scaled up as high cell density cultivation conducted in fed-batch mode. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

CAMPANI, GILSON; RIBEIRO, MARCELO P. A.; ZANGIROLAMI, TERESA C.; LIMA, V, FERNANDO. A hierarchical state estimation and control framework for monitoring and dissolved oxygen regulation in bioprocesses. Bioprocess and Biosystems Engineering, v. 42, n. 9, p. 1467-1481, SEP 2019. Web of Science Citations: 0.

CAMPANI, GILSON; DA SILVA, GABRIEL GONCALVES; ZANGIROLAMI, TERESA CRISTINA; PERENCIN DE ARRUDA RIBEIRO, MARCELO. Recombinant Escherichia coli cultivation in a pressurized airlift bioreactor: assessment of the influence of temperature on oxygen transfer and uptake rates. Bioprocess and Biosystems Engineering, v. 40, n. 11, p. 1621-1633, NOV 2017. Web of Science Citations: 2.