High recovery of intracellular recombinant HBsAg from Pichia pastoris via continuous pulsed laser cell disruption system optimized by response surface methodology

Abstract

In our previous study, we demonstrated that continuous power laser could be a clean, rapid, and convenient alternative to the other conventional disruption techniques for the release of recombinant hepatitis B surface antigen (rHBsAg) from Pichia pastoris. In the current work, we examined the effect of pulsed laser in the continuous laboratory-scale process on cell disruption. Design-of-experiments (DOE) methodology was used for optimization of cell disruption process to obtain the highest protein concentration in the disruption buffer. Our investigations for the pulsed laser at wavelength of 1,064 nm demonstrated that for disrupting P. pastoris cell and releasing rHBsAg, the laser power was the most influential factor, and laser pulse duration and cycle number were in the second and third places. According to the results, the effect of laser power and pulse duration (time) had a direct relationship with protein concentration. For the number of cycles, however, increasing the value from the lowest point at first led to the enhancement and then reduction of protein concentration. The maximum cell disruption and rHBsAg release were recorded for the laser system in the energy input of 284 mW and the pulse duration of 100 mSec after four complete rounds of circulation. C 2018

HBsAg Production in Methanol Controlled <i>P. pastoris</i> GS115 Mut^S Bioreactor Process

“…The final estimated purity of HBsAg VLPs is ~90 %. To achieve purities up to 99 %, CsCl method can be used, however it is expensive and time-consuming [23]. According to Gurramkonda et al [20], a purity increase from 90 % to 99 % leads to a 3-fold protein loss.…”

Numerical and Experimental Biomass Separation from Fermentation Process by Minihydrocyclones

“…The process was followed by the separation of biomass from the supernatant and then biomass was disrupted in a bead mill apparatus. It is not necessary to concern the cell viability in separation process [23][24][25][26][27][28][29][30]. Samples were taken from the fermenter and entered the hydrocyclone.…”

Accurate and cost‐effective prediction of HBsAg titer in industrial scale fermentation process of recombinant Pichia pastoris by using neural network based soft sensor

“…Wet cell weight value was measured after separation of biomass (cell pellets) by centrifugation (3,000 g /20 Min). The DCW was determined after drying biomass at 80 °C for 48 H . The HBsAg concentration was measured by sandwich ELISA, where sheep polyclonal antibodies against HBsAg were coated on the plate and conjugated with horseradish peroxidase .…”

“…In this way, potential challenges in the following process could be predicted and resolved . The common specific analytical technique for quantifying the recombinant protein, in this case, HBsAg, is the enzyme‐linked immunosorbent assay (ELISA) test . However, this off‐line method is slow, time‐consuming, and relatively expensive, especially for intracellular recombinant proteins.…”