Continuous Online Titer Monitoring in CHO Cell Culture Supernatant Using a Herringbone Nanofluidic Filter Array.

Online monitoring of monoclonal antibody product titers throughout biologics process development and production enables rapid bioprocess decision-making and process optimization. Conventional analytical methods, including high-performance liquid chromatography and turbidimetry, typically require interfacing with an automated sampling system capable of online sampling and fractionation, which suffers from increased cost, a higher risk of failure, and a higher mechanical complexity of the system. In this study, a novel nanofluidic system for continuous direct (no sample preparation) IgG titer measurements was investigated. Tumor necrosis factor α (TNF-α), conjugated with fluorophores, was utilized as a selective binder for adalimumab in the unprocessed cell culture supernatant. The nanofluidic device can separate the bound complex from unbound TNF-α and selectively concentrate the bound complex for high-sensitivity detection. Based on the fluorescence intensity from the concentrated bound complex, a fluorescence intensity versus titer curve can be generated, which was used to determine the titer of samples from filtered, unpurified Chinese hamster ovary cell cultures continuously. The system performed direct monitoring of IgG titers with nanomolar resolution and showed a good correlation with the biolayer interferometry assays. Furthermore, by variation of the concentration of the indicator (TNF-α), the dynamic range of the system can be tuned and further expanded.

Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation.

BACKGROUND: The production of chemicals via bio-based routes is held back by limited easy-to-use stabilisation systems. A wide range of plasmid stabilisation mechanisms can be found in the literature, however, how these mechanisms effect genetic stability and how host strains still revert to non-productive variants is poorly understood at the single-cell level. This phenomenon can generate difficulties in production-scale bioreactors as different populations of productive and non-productive cells can arise. To understand how to prevent non-productive strains from arising, it is vital to understand strain behaviour at a single-cell level. The persistence of genes located on plasmid vectors is dependent on numerous factors but can be broadly separated into structural stability and segregational stability. While structural stability refers to the capability of a cell to resist genetic mutations that bring about a loss of gene function in a production pathway, segregational stability refers to the capability of a cell to correctly distribute plasmids into daughter cells to maintain copy number. A lack of segregational stability can rapidly generate plasmid-free variants during replication, which compromises productivity. RESULTS: Citramalate synthase expression was linked in an operon to the expression of a fluorescent reporter to enable rapid screening of the retention of a model chemical synthesis pathway in a continuous fermentation of E. coli. Cells without additional plasmid stabilisation started to lose productivity immediately after entering the continuous phase. Inclusion of a multimer resolution site, cer, enabled a steady-state production period of 58 h before a drop in productivity was detected. Single-cell fluorescence measurements showed that plasmid-free variants arose rapidly without cer stabilisation and that this was likely due to unequal distribution of plasmid into daughter cells during cell division. The addition of cer increased total chemical yield by more than 50%. CONCLUSIONS: This study shows the potential remains high for plasmids to be used as pathway vectors in industrial bio-based chemicals production, providing they are correctly stabilised. We demonstrate the need for accessible bacterial 'toolkits' to enable rapid production of known, stabilised bacterial production strains to enable continuous fermentation at scale for the chemicals industry.

Deployment of metabolic heat rate based soft sensor for estimation and control of specific growth rate in glycoengineered Pichia pastoris for human interferon alpha 2b production.

Lack of appropriate process models, reliable online sensors, and process variability in bioprocess systems are poising challenges in real-time monitoring and control of critical process parameters (CPPs). This present investigation deals with the development of a non-invasive soft sensor by utilizing metabolic heat rate as input signal for online estimation of specific growth rate (µest) during the induction phase of glycoengineered Pichia pastoris for human interferon-alpha 2b (huIFNα2b) production. Feedforward strategy employing a predetermined exponential feeding of methanol during the induction phase was dealt at defined setpoint values (µSP). Standard PID controller with predetermined gain values regulated methanol feeding in accordance with the deviation from the µSP value. An adaptive PID (gain scheduling) significantly minimized the deviation of µ from its µSP value, reduced the amplitude of oscillation and achieved long-term controller stability. Robust control of methanol feeding by adaptive PID resulted in a 1.5 and 2.2-fold increase in productivity of huIFNα2b compared to standard PID and feedforward controls respectively. Moreover, adaptive PID control facilitated narrow range control of µ for longer durations (> 20 h) with a low average tracking error (< 6%) enumerating its scope of application in therapeutic protein production in near future.

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.

Batch and fed-batch fermentation of optically pure D (-) lactic acid from Kodo millet (Paspalum scrobiculatum) bran residue hydrolysate: growth and inhibition kinetic modeling.

A low-cost Kodo millet bran residue was utilized as feedstock for the production of D (-) lactic acid (DLA) using Lactobacillus delbrueckii NBRC3202 under anaerobic condition. Data culled from a series of batch fermentation processes with different initial Kodo millet bran residue hydrolysate (KMBRH) and DLA concentrations were used for kinetic model development. Both simulated and experimental data were in good agreement for cell growth, KMBRH utilization, and DLA formation. The values of kinetic constants specific growth rate, (µm = 0.17 h-1); growth (αP = 0.96 g.g-1) and non-growth (ßP = 1.19 g.g-1.h-1) associated constant for DLA production and the maximum specific KMBRH utilization rate, (qG, max = 1.18 g.g-1.h-1) were in good agreement with the literature reports. Kinetic analysis elucidated that L. delbrueckii growth was predominantly influenced by KMBRH limitation and highly sensitive to DLA inhibition. Fed-batch fermentation studies demonstrated the existence of substrate and product inhibition paving the scope for process intensification.

Optimization and effect of dairy industrial waste as media components in the production of hyaluronic acid by Streptococcus thermophilus.

Hyaluronic acid (HA) production using a dairy industrial waste is a more cost-efficient strategy than using an expensive synthetic medium. In this study, we investigated the production of HA using Streptococcus thermophilus under shake flask conditions using dairy industrial waste as nutritional supplements, namely whey permeate (WP) and whey protein hydrolysate (WPH). Preliminary screening using Plackett-Burman design exhibited WP, WPH, initial pH, and inoculum size as significant factors influencing HA titer. Response surface methodology design of four factors was formulated at three levels for enhanced production of HA. Shake flask HA fermentation by S. thermophilus was performed under global optimized process conditions and the optimal HA titer (342.93 mg L(-1)) corroborates with Box-Behnken design prediction. The molecular weight of HA was elucidated as 9.22-9.46 kDa. The ultralow-molecular weight HA reported in this study has a potential role in drug and gene delivery applications.