Chapter 1: Introduction and review of literature
1. Interferons
1.7. Bioprocess Monitoring and Control
1.7.2. Biocalorimetry
Biocalorimetry facilitates a quantitative interpretation of metabolic heat generated from living systems as a useful „process signal‟ for monitoring purposes and „Biocalorimetry‟
had become popular among scientists and academics for monitor and control of biochemical reactions [133, 134]. Recently, biocalorimetry is gaining interest for its application in real-time bioprocess monitoring due to its non-invasive and instantaneous mode of operation [135]. Indeed, measured metabolic heat signals during a bioprocess provide a global insight into metabolic activity of living cells [136, 137]. Further studies illustrated that heat-flow biocalorimeters are high performing (bio) reactors suitable for all kind of bioprocess applications viz., cultivation of different cell lines, quantitative studies including process monitoring and control [138] and biothermodynamic studies [139]. For instance, authors first reported the existence of endothermic microbial growth by cultivation of acetotrophic methanogen, Methanosarcina barkeri in the BioRC1 Biocalorimeter [140]. In the case of a pure aerobic respiratory metabolism where the substrate is entirely converted into biomass, water, carbon dioxide and heat, the signal measured by a biocalorimeter (BioRC1) can be related to the actual biomass concentration as well as to specific growth rate. Product quality, as well as the productivity of a process is influenced by the specific growth rate and its fluctuation.
Controlling this CPP is an important step towards quality by design as encouraged by the FDA initiative (FDA 2004) [112]. The development of a robust biomass and specific growth rate estimator based on heat-measurements has its importance in the framework of the FDA‟s PAT initiative, since it gives a real-time insight into the on-going industrial processes [134]. Authors showed biocalorimetry as robust in-line monitoring and control of aerobic fed-batch cultures of crabtree-negative yeast cells with the help of reliable biomass and specific growth rate estimator based on heat flow measurements [134].
Also, the potential of the biocalorimetry to control the specific growth rate on real-time with the use of proportional integral (PI) feedback control was illustrated. DS and Biocalorimetry have proved to be potential and robust PAT tools to monitor biomass on real-time and also control of specific growth rate (CPP) for high yield recombinant protein production. Integration of a heat flow biocalorimetery and DS into a PAT platform would provide the end-user an insight into metabolic changes encountered in an on-going bioprocess and ensure a robust process control leading to enhanced product yield and quality. Studies addressing the feed-back control of CPP‟s viz., specific growth rate using different PAT tools during the production process of various recombinant proteins of therapeutic importance in P. pastoris are highlighted in Table 1.5.
Table 1.5 Feedback control strategies with different PAT tools implemented for production of different recombinant proteins of therapeutic importance in P. pastoris Strain Substrate Type of feedback
control
Product Reference
P. pastoris GS115 Methanol Mid-IR based control
Avidin [130]
P. pastoris GS115 Methanol Methanol sensor based control
Botulinum neurotoxin Α-galactosidase
[141]
P. pastoris GS115 Methanol Methanol sensor based control
ScFv A33- monoclonol antibody
[142]
P. pastoris X33 Methanol Methanol sensor based control
Rhizopusoryzae lipase
[143]
P. pastoris- PPC43AZ
Glycerol Capacitance based control
- [144]
P. pastoris X33 Glycerol DO based control single chain antibody
fragment (scFv)
[145]
Glycoengineered P. pastoris YGLY4140
Glycerol Methanol
NIR based control monoclonal antibody
[146]
P. pastoris Glycerol Calorimetry based control
HSA [147]
Definition of the Problem
The literature survey substantiates that recombinant huIFNα2b has been expressed on various platforms, which includes bacteria, yeast, insect cell lines, mammalian cell lines and plants. Expression of recombinant huIFNα2b in E. coli yielded high huIFNα2b titer but the expressed protein forms inclusion bodies and loses the activity during renaturation process. Also, the expressed protein lacks post-translational modifications and exhibited very less plasma life. The plasma life of huIFNα2b expressed by E. coli increased to certain extent through pegylation but could affect the patient‟s quality life on long-term use. To achieve the post translational modifications of the recombinant huIFNα2b insect cell lines and mammalian cell lines were considered as viable platform.
But, the expressed huIFNα2b yield is low and the protein is partially glycosylated.
Mammalian cell lines were used to express rightly processed glycosylated huIFNα2b but it is limited by low yield, high cost, challenges of scale-up, and the problems associated with purification. Other systems like milk of transgenic mice, hen egg and carrot plants were also attempted for the expression of recombinant huIFNα2b but suffer from bottlenecks viz., yield, activity and post translational modifications. Few literature reports are available for the expression of recombinant huIFNα2b in P. pastoris but failed to report the extent of post translational modifications of the expressed protein and the maximum protein yield reported was 600mg/L. No literature report available on real- time monitoring of the recombinant huIFNα2b production employing PAT tools addressing scope of CPPs and their influence on product (huIFNα2b) quality. The huIFNα2b drugs currently approved by FDA and therapeutically in use are expressed by E. coli system.
The present thesis work is aimed to address the gaps pertaining to recombinant huIFNα2b expression on various systems. The primary focus the present thesis is i) Design and development of high yield P. pastoris expression platform recombinant huIFNα2b, ii) development of effective process strategy by integrating PAT tools for real-time monitoring and control of P. pastoris cultivation for huIFNα2b production.
Glycoengineered P. pastoris strain is employed in this thesis work for successful expression of glycosylated huIFNα2b. Medium optimization studies employing the Design of experiments (DoE) and statistical optimization methods are performed to enhance the yield of recombinant huIFNα2b production in glycoengineered P. pastoris.
The reported suggests that DS and Biocalorimetry as potential PAT tools for successful monitoring of the biomass concentration and specific growth rate in different bioprocess systems. The CPPs associated with the glycoengineered P. pastoris fermentation for the production of glycosylated huIFNα2b could be monitored on real-time by DS, calorimetric and respirometric measurements. An effective process strategy ensuring high huIFNα2b production and its quality (desired glycosylation profile) could be developed by elucidation and control of CPPs by the application of PAT tools.
Objectives