Real-time metabolic heat-based specific growth rate soft sensor for monitoring and control of high molecular weight hyaluronic acid production by Streptococcus zooepidemicus.

This present investigation addressing the metabolic bottleneck in synthesis of high MW HA by Streptococcus zooepidemicus and illustrates the application of calorimetric fed-batch control of µ at a narrower range. Feedforward (FF) and feedback (FB) control was devised to improve the molecular weight (MW) of HA production by S. zooepidemicus. Metabolic heat measurements (Fermentation calorimetry) were modeled to decipher real-time specific growth rate, [Formula: see text] was looped into the PID circuit, envisaged to control [Formula: see text] to their desired setpoint values 0.05 [Formula: see text], 0.1 [Formula: see text], and 0.15 [Formula: see text] respectively. Similarly, a predetermined exponential feed rate irrespective of real-time µ was carried out in FF strategy. The developed FB strategy established a robust control capable of maintaining the specific growth rate (µ) close to the [Formula: see text] value with a minimal tracking error. Exponential feed rate carried out with a lowest [Formula: see text] of 0.05 [Formula: see text] showed an improved MW of HA to 2.98 MDa and 2.94 MDa for the FF and FB-based control strategies respectively. An optimal HA titer of 4.73 g/L was achieved in FF control strategy at [Formula: see text]. Superior control of µ at low [Formula: see text] value was observed to influence HA polymerization positively by yielding an improved MW and desired polydispersity index (PDI) of HA. PID control offers advantage over conventional fed-batch method to synthesize HA at an improved MW. Calorimetric signal-based µ control by PID negates adverse effects due to the secretion of other end products albeit maintaining regular metabolic activities. KEY POINTS: First report to compare HA productivities by feedforward and feedback control strategy. Inherent merits of regulating µ at narrower range were entailed. Relationship between operating µ and HA molecular weight was discussed.

Deciphering the role of dissolved oxygen and N-acetyl glucosamine in governing higher molecular weight hyaluronic acid synthesis in Streptococcus zooepidemicus cell factory.

The present study is focused on systematic process and kinetic investigation of hyaluronic acid (HA) production strategy unraveling the role of dissolved oxygen (DO) and N-acetyl glucosamine (GlcNAc) towards the enhancement of HA titer and its molecular weight. Maintaining excess DO levels (10-40% DO) through DO-stat control and the substitution of GlcNAc at a range (5-20 g/L) with glucose (Glc) critically influenced HA production. DO-stat control strategy yielded a promising HA titer (2.4 g/L) at 40% DO concentration. Controlling DO level at 20% (DO-stat) was observed to be optimum resulting in a significant HA production (2.1 g/L) and its molecular weight ranging 0.98-1.45 MDa with a consistent polydispersity index (PDI) (1.57-1.69). Substitution of GlcNAc with Glc at different proportions explicitly addressed the metabolic trade-off between HA titer and its molecular weight. GlcNAc substitution positively influenced the molecular weight of HA. The highest HA molecular weight (2.53 MDa) of two-fold increase compared with glucose as sole carbon substrate and narrower PDI (1.35 ± 0.18) was achieved for the 10:20 (Glc:GlcNAc) proportion. A novice attempt on modeling the uptake of dual substrates (Glc and GlcNAc) by Streptococcus zooepidemicus for HA production was successfully accomplished using double Andrew's growth model and the kinetic parameters were estimated reliably.

Biocalorimetric monitoring of glycoengineered P. pastoris cultivation for the production of recombinant huIFNα2b: A quantitative study based on mixed feeding strategies.

Real-time monitoring of glycoengineered Pichia pastoris by employing process analytical technology (PAT) tools is vital for gaining deeper insights into the therapeutic protein production process. The present study focuses on influence of mixed feed carbon substrates during the induction phases of glycoengineered P. pastoris cultivation, for recombinant human interferon α2b (huIFNα2b) production by employing calorimetric (biological heat rate, q B ) and respirometric (oxygen uptake rate and carbon dioxide evolution rate) measurements. Mixed feed stream of carbon substrates (methanol + glycerol, methanol + sorbitol) at a predetermined "C-molar ratios" were added during the induction phases. Methanol- and sorbitol-based mixed feeding approach resulted in an improved huIFNα2b titer of 288 mg/L by channeling of methanol predominantly towards an optimal functioning of AOX expression system. A stand-off between biomass yield YXSand biomass heat yieldYQX coefficient, degree of reduction of methanol and its cosubstrate (glycerol and sorbitol) determines the fraction of carbon energy channeled toward biomass and protein production, under strict aerobic conditions. Calorespirometric monitoring and assessment of thermal yields enables a reliable prediction of process variables, leading to futuristic efficient PAT-based feed rate control.