Computational fluid dynamic modeling of alternating tangential flow filtration for perfusion cell culture.

Alternating tangential flow (ATF) filtration has been successfully adopted as a low shear cell separation device in many perfusion-based processes. The reverse flow per cycle is used to minimize fouling compared with tangential flow filtration. Currently, modeling of the ATF system is based on empirically derived formulas, leading to oversimplification of model parameters. In this study, an experimentally validated porous computational fluid dynamic (CFD) model was used to predict localized fluid behavior and pressure profiles in the ATF membrane for both water and supernatant solutions. The results provided numerical evidence of Starling flow phenomena that has been theorized but not previously proven for the current operating parameters. Additionally, feed cross flow velocity was shown to significantly impact the localized flux distribution; higher feed cross flow rates lead to an increased localized permeate flux as well as irreversible and reversible fouling resistance. Further, the small average permeate flux values of 2 L·m-2 ·h-1 traditionally used in perfusion bioreactor membranes lead to approximately 50% of the membrane length utilized for permeate flow during each pressure and exhaust phase, leading to a full membrane utilization during one ATF cycle. Our preliminary CFD results demonstrate that local flux and resistance distribution further elucidate the dynamics of ATF membrane fouling in a perfusion-based system.

Design of a novel continuous flow reactor for low pH viral inactivation.

Insufficient mixing in laminar flow reactors due to diffusion-dominated flow limits their use in applications where narrow residence time distribution (RTD) is required. The aim of this study was to design and characterize a laminar flow (Re 187.7-375.5) tubular reactor for low pH viral inactivation with enhanced radial mixing via the incorporation of curvature and flow inversions. Toward this aim, the reactor described here, Jig in a Box (JIB), was designed with a flow path consisting of alternating 270° turns. The design was optimized by considering the strength of secondary flows characterized by the Dean No., the corresponding secondary flow development length, and the reactor turn lengths. Comprehensive CFD analysis of the reactor centerline velocity profile, cross-sectional velocity, and secondary flow streamlines confirmed enhanced radial mixing due to secondary flows and changes in flow direction. For initial CFD and experimental studies the reactor was limited to a 16.43 m length. Pulse tracer studies for the reactor were computationally simulated and experimentally generated to determine the RTD, RTD variance, and minimum residence time for the tracer fluid elements leaving the reactor, as well as to validate the computational model. The reactor was scaled length wise to increase incubation time and it was observed that as the reactor length increases the RTD variance increases linearly and the dimensionless RTD profile becomes more symmetrical and tighter about the mean residence time.

Optimization of norovirus virus-like particle production in Pichia pastoris using a real-time near-infrared bioprocess monitor.

The production of norovirus virus-like particles (NoV VLPs) displaying NY-ESO-1 cancer testis antigen in Pichia pastoris BG11 Mut(+) has been enhanced through feed-strategy optimization using a near-infrared bioprocess monitor (RTBio(®) Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real-time. The production of NoV VLPs displaying NY-ESO-1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two-fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP-NY-ESO-1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L(-1) during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1-NY-ESO-1 yield of 0.85 g L(-1) . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518-526, 2016.

Secreted production of assembled Norovirus virus-like particles from Pichia pastoris.

BACKGROUND: Norovirus virus-like particles (NoV VLPs) have recently been explored as potential vaccine platforms due to their ability to produce an effective immune response. Expression of the main structural protein, VP1, leads to formation of self-assembled particles with similar characteristics to the original virus. These NoV VLPs have been expressed in Escherichia coli, yeast and insect cells. Expression in E. coli and insect cells share downstream processing issues due to the presence of inclusion bodies or the need for numerous purification steps. NoV VLPs have also been produced in the yeast P. pastoris; however the protein was only expressed intracellularly. RESULTS: We have successfully expressed and secreted the VP1 protein in the novel P. pastoris strain, Bg11, using the methanol inducible pJ912 expression vector, containing the cDNA of NoV VP1. Expression of the VP1 protein in Bg11 was carried out in a 1.5 L bioreactor resulting in a total yield of NoV VLPs greater than 0.6 g/L. NoV VLPs obtained from the culture supernatant were purified via ion-exchange chromatography, resulting in particles with a purity over 90%. The average size of the particles after purification was 40 nm. Transmission electron microscopy was used to visualize the morphology of the particles and saliva-binding assay confirmed that the NoV VLPs bind to Histo-Blood Group Antigens (HBGA). CONCLUSIONS: In this study we describe the expression and characterization of fully assembled Norovirus virus-like particles obtained from P. pastoris. The particles are similar in size, morphology and binding capacity, as previously described, for the original NoV. Our results detail the successful expression and secretion of VLPs in P. pastoris, improving their candidacy as a vaccine platform.

Process development and production of cGMP grade Melan-A for cancer vaccine clinical trials.

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body's immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%.

Methods for chromatographic removal of endotoxin.

Endotoxin removal is critical when producing therapeutic proteins in bacterial systems. This hydrophobic compound can be removed through chromatography or filtration, but presents unique challenges dependent upon protein composition as well as production scale. Here we present a robust method for endotoxin removal at the pilot production scale using fast protein liquid chromatography and buffers specifically engineered for endotoxin removal.

Expression and purification of cGMP grade NY-ESO-1 for clinical trials.

NY-ESO-1 is a cancer testis antigen expressed in numerous cancers. Initial tests have shown its efficacy as a cancer vaccine, stimulating the body's own immune response against the invading tumor. To produce enough material for phase I clinical trials, a process using current good manufacturing practices to produce clinical grade material was developed and executed. His-tagged NY-ESO-1 was expressed in C41DE3 Escherichia coli under control of the T-7 promoter. NY-ESO-1 was produced in a 20 L fed-batch fermentation utilizing a pH-stat control scheme. The protein was then purified from inclusion bodies using a three-column process that achieved a yield of over 3.4 g and endotoxin below the detection limit of 0.005 EU/µg protein.