Characterisation and separation of infectious bursal disease virus-like particles using aqueous two-phase systems

Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination programs.Virus-like particles(VLPs)are recognised as a safe and potent recombinant vaccine platform.This research work explores the characterisation and separation of infectious bursal disease virus-like particles(IBD-VLPs)from crude feedstock.Various characteristics were studied with highperformance size-exclusion chromatography(HP-SEC),sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE)and transmission electron microscopy(TEM)analyses.Subsequently,the separation of IBD-VLPs using polyethylene glycol(PEG)/sodium citrate-based aqueous two-phase systems(ATPSs)was conducted and optimised.Moreover,a scale-up study of the best ATPS constituted of 15%PEG 6000,11%sodium citrate and 10%crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted.The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBDVLPs of>97%in the interphase between the PEG-rich top and salt-rich bottom phases.These optimised systems also showed high removal efficiencies of impurities of>95%.The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.

UV/Vis-based process analytical technology to improve monoclonal antibody and host cell protein separation

Process analytical technology(PAT) is gaining more interest in the biomanufacturing industry because of its potential to improve operational control and compliance through real-time quality assurance.Currently, biopharmaceutical producers mainly monitor chromatographic processes with ultraviolet/visible(UV/Vis) absorbance. However, this measurement has a very limited correlation with purity and quantity. The current study aims to determine the concentration of monoclonal antibody(mAb) and host cell proteins(HCPs) using a build-in UV/Vis monitoring during Protein A affinity chromatography and to optimize the separation conditions for high purity of mAb and minimizing the HCPs content. The eluate was analyzed through in-line UV/Vis at 280 and 410 nm, representing mAb and HCPs concentration,respectively. Each 0.1 column volume(CV) fraction of UV/Vis chromatogram peak area were calculated,and different separation conditions were then compared. The optimum conditions of mAb separation were found as 12 CV loading, elution at pH 3.5, and starting the collection at 0.5 CV point, resulting in high m Ab recovery of 95.92% and additional removal of 49.98% of HCP comparing with whole elution pool. This study concluded that UV/Vis-based in-line monitoring at 280 and 410 nm showed a high potential to optimize and real-time control Protein A affinity chromatography for mAb purification from HCPs.

Raman spectroscopy as process analytical tool in downstream processing of biotechnology

Downstream processing or product recovery plays a vital role in the development of bioprocesses.To improve the bioprocess efficiency,some unconventional methods are much required.The continuous manufacturing in downstream processing makes the Process Analytical Technologies(PATs)as an important tool.Monitoring and controlling bioprocess are an essential factor for the principles of PAT and quality by design.Spectroscopic methods can apply to monitor multiple analytes in real-time with less sample processing with significant advancements.Raman spectroscopy is an extensively used technique as an analytical and research tool owing to its modest process form,non-destructive,non-invasive optical molecular spectroscopic imaging with computer-based analysis.Generally,its application is essential for the analysis and characterization of biological samples,and it is easy to operate with minimal sample.The innovation on various types of enhanced Raman spectroscopy was designed to enhance the Raman analytical technique.Raman spectroscopy could couple with chemometrics to provide reliable alternative analysis method of downstream process analysis.Thus,this review aims to provide useful insight on the application of Raman spectroscopy for PAT in downstream processing of biotechnology and Raman data analysis in biological fields.

Cryo-copolymerization preparation of dextran-hyaluronate based supermacroporous cryogel scaffolds for tissue engineering applications

Dextran-hyaluronate （Dex-HA） based supermacroporous cryogel scaffolds for soft tissue engineering were prepared by free radical cryo-copolymerization of aqueous solutions containing the dextran methacrylate （Dex-MA） and hyaluronate methacrylate （HA-MA） at various macromonomer concentrations under the freezing condition. It was observed that the suitable total concen- tration of macromonomers for the preparation of Dex-HA cryogel scaffold with satisfied properties was 5% （w/w） at the HA-MA concentration of 1% （w/v）, which was then used to produce the test scaffold. The obtained cryogel scaffold with 5% （w/w） macromonomer solution had high water permeability （5.1 × 10 ^-2m2） and high porosity （92.4%）. The pore diameter examined by scanning electron microscopy （SEM） was in a broad range of 50-135 um with the mean pore diameter of 91 um. Furthermore, the cryogel scaffold also had good elastic nature with the elastic modulus of 17.47±1.44 kPa. The culture of 3T3-L1 preadipocyte within the scaffold was investigated and observed by SEM. Cells clustered on the pore walls and grew inside the scaffold indicating the Dex-HA cryogel scaffold could be a promising porous biomaterial for applications in tissue engineering.