Protein purification, upstream and downstream processes
Resume:
Dr. Bejjani, Satyanarayana
Seoul, South Korea
*****.*******@***.***
Academic Qualifications
Ph.D. Biochemistry National Institute of Nutrition 2011 M.Sc. Plant science University of Hyderabad 2004
B.Sc. Biotechnology Kakatiya University 2002
Employment
International Vaccine Institute May 2018 - Present Associate Research Scientist - Seoul, South Korea
Project 1. TB Subunit Antigens (Ag 85B & Partial Triplet 85B) Project aims to develop, T Cell mediated antigen subunits (His tag) from Ecoli expressed in origami 2 DE3 Strains as inclusion body. The protein expression was tested using IPTG induced expression. Expressed proteins are purified using IMAC Chromatography under denaturing conditions using urea as a Chaotropic agent. Protein refolding was performed using step down dialysis for purity and functional analysis. Purified proteins were tested for purity using SDS-PAGE and western blot(turbo) for immunoreactivity. Impurities Endotoxin, NA Specifications were confirmed using PTS cartridges endosafe & Nano drop. Purified protein was analyzed for aggregation using SEC and Zeta sizer to confirm the homogeneity of the Drug substance for In vivo studies.
Project 2. TB Multiple Antigens (Fusion proteins MPT51, 64, 83) Project aims to develop upstream (shake flask) Screened and optimized transformation using DH5 Alpha cell, expression, growth, and induction conditions for protein expression (Shuffle T7 Express). Downstream purifications methods for process development of 3 multiple antigen (fusion proteins). After initial set of screening experiments, the sequence of steps used for purification of all the three proteins; were enabled by their iso-electric points (pI) which were between 4.55 and 4.95. Briefly, post-lysis, pH of the lysate supernatant was adjusted to ~3.8 with acetic acid; the precipitate formed is removed by centrifugation, and the clear supernatant is then loaded onto Cation-Exchange resin; the target proteins were eluted using pH-gradient between 3.8 and 7.5 using Tris buffer. The elutes containing the target protein were then pooled, their pH adjusted to 7.5, loaded onto Anion-Exchange resin and eluted using a salt gradient. Eshmuno CPX was used as the CEX resin and the Fractogel-DEAE was used as the AEX resin (both from Merck Millipore).
Project 3. Membrane proteins (Adjuvant TLR2 agonist) TB Specific adjuvant, Lipidated Rhizavidin construct is expressed to consist of a lipidation motif (derived from an E. coli heat shock protein HslJ) to which E. coli will add lipid on during translation of the protein; it can be heterogeneous in terms of the lipid composition (diacyl or triacyl). This pET24bplasmid was then transformed into BL21 CodonPlus (DE3) RIPL and other expression cells such as Lemo 21, C41 DE3, C43DE3 as part of overcoming leaky expression and recover more functional protein. Optimized purification using Affinity (Iminobiotin), Hydrophobic and Ion exchange resins for purification of membrane protein.
Project 4. Antigen Purification (O139, Cairo48 & 50) Developed chromatography free purification methods for multiple antigens from inactivated cholera cultures for Drug substance as potential vaccine candidates for tech transfer activity and part of Standards for collaborators. Methods involved Phenol chloroform extraction, Ethanol precipitation, Ultracentrifugation, filtration methods for Antigen developed. Project 5. Polysaccharide based antigens αglucan and LAM from TB cultures Supernatant was developed for assessing the immunogenicity of polysaccharide-based conjugates to Non-TB proteins. In this process flow using different molecular weight cut off filters were used to separate polysaccharides using TFF, followed by DEAE fractogel in Flow through mode to separate glucans which are neutral polysaccharide. Incase of LAM a lipid moiety of arabinomanan was extracted using detergent solubilization using Triton X114 and detergent rich layer was separated and analyzed further on SEC chromatography for purification.
Clonz Biotech July 2015 – April 2018
Scientist - Hyderabad, India
Project 1. Process development for monoclonal antibody (Rituixmab & Transzumab) Developed a process from the clarified harvest of CHO cultures using TFF, followed by platform purification of monoclonal antibody using Capture, Intermediate and Polishing steps using Protein A (Mab select Sure Lx), CEX (SP Sepharose) and AEX (Q Sepharose) chromatography, respectively. Separation steps at each step of chromatography involves in selective binding of the feed properties such as pH and conductivity to resin buffers and elution of the monoclonal antibody at low pH for affinity purification and hold time at low pH for viral inactivation. IEX chromatography were performed either in flow through mode or bind and elute mode for removal of impurities and recovery of Monoclonal antibody. Process flow at each step of chromatography was tested using SDS-PAGE, A280nm & ELISA for protein quantification, western blot & ELISA for immunoreactivity for QC analysis of monoclonal antibody for step recovery, yield and identity.
Intermediate impurity analysis was checked using HIC (Phenyl sepharose) and SEC chromatography for dimers and aggregate separation. The final DS obtained after purification were concentrated using ultrafiltration using 30kDa molecular weight membranes for concentration. Project 2. Process development for monoclonal antibody fragment (Ranibizumab) Ranibizumab is the Fab moiety of a recombinant humanized monoclonal antibody with Anti-VEGF functions for Macular degeneration. Process development of Fab using Migula strain, was developed which accumulates in the periplasmic space. The cell suspension was clarified from cell debris using physicochemical methods of homogenization & sonication to lysis the cells for solubility. Heat hold step to precipitation of impurities and flocculation of insoluble cell debris. Process improvement was monitored by checking in line parameters A280nm, SDS-PAGE, western blot and ELISA to monitor the consistency and validation of chromatography runs. The process involved Capto L affinity chromatography capture step followed by intermediate purification using Capto Q & Cation (SP Sepharose) for separation and remove cell derived impurities, product related aggregates and variants formed during different stages of purification. Optimized Scale down models for purification of monoclonal antibody and scale up activity.
Synteny Life Sciences 2014 - 2015
Project Manager - Hyderabad, India
CRO: Regular duties included monitoring
Protein Purification and Expression in bacteria, optimizing the production using shake flasks cultures. Testing the purity and analysis using SDS-PAGE, HPLC Protein estimation methods, related to bio-therapeutics (biochemical assays (Bradford, Modified Lowry, BCA). Perform membrane filtration methods such as TFF, Ultrafiltration. Antibody development and titer determination by Elisa and Western blots University of Alberta 2011 - 2014
Post Doc Fellow - Edmonton Alberta
Project 1. Bioavailability of Antihypertensive peptides from (Ovo transferrin) using Caco-2 cell line
This project aims to identify Antihypertensive peptides (Ovo transferrin) derived tripeptides bioactivity using Transcellular transport and pharmacokinetics using Caco-2 cells. Bioactivity testing for various peptides quantification using transport mechanisms using trans well cell culture plates and analyzing the transported peptides by using RP-HPLC & UPLC methods. Bioassays for cell proliferation and cytotoxicity activities using mammalian Cell Culture methods like Caco-2 cells, EAhy926 were studied as part of their culture conditions and functional activity. Steered efforts in identifying egg white peptides (anti- hypertensive activity) bioavailability which are low in molecular weight using Caco-2 monolayers and their transport mechanisms which implicates their bioavailability. Project 2. Bioavailability of collagen peptides using Caco-2 cells This project aims to identify collagen hydrolysate and peptides absorption using different mimetic peptides of proline -hydroxyproline variants as stimulators of collagen constituent. Different amino acids analogs were studied to understand the absorption rates of peptides using UPLC. National Institute of Nutrition 2005 - 2011
PhD Biochemistry
Project: Ferritin purification and bioavailability Hypothesis is to understand the nonheme nature of ferritin iron and its bioavailability. To understand Non heme nature of iron ferritin, purified homogenous ferritin and developed a very sensitivity Elisa method for first time for ferritin quantitation. On-heme nature of ferritin iron and its interactions with dietary factors was tested for the first time in Caco-2 cell culture. The stability of the ferritins to gastric and intestinal stimulations was done at invitro and in vivo conditions to test non-heme nature using chromatography (gel filtration) and spectroscopy CD & fluorescence to understand the stability of ferritin under simulated gastric and intestinal conditions. Awards & Honors
Awarded summer fellowship from (Jawaharlal Nehru Centre for Advanced Scientific Research) JNCAR Bangalore 2002-2003
Awarded Junior Research fellow by Indian council of Medical Research (ICMR) in the year 2004.
Awarded Distinguished poster in International congress of Nutrition, BANGKOK, Thailand-2009
PepsiCo travel grant for ICN (International congress of Nutrition) Technical & Specialized Skills
Protein methods:
Transformation, expression and purification of inclusion body characterization and refolding.
Affinity purification using Protein A and Capto L, IMAC, Ion exchange, Gel filtration chromatography, Determination of protein structure using CD, fluorescence spectroscopy, Gel Electrophoresis, SDS- PAGE.
Filtration methods:
Clarification using (Hallow fiber cartridge) TFF, Ultrafiltration and concentration using stirred cell, Endotoxin removal, LAL test.
Analytical methods:
Characterization with analytical methods such a s R P -HPLC, Zeta Sizer FPLC, UPLC, UV s pectroscopy for proteins, small molecules, and metabolites. Model peptides design, Stability studies, Pharmacokinetics of ACE 1 peptides invitro using cell cultures. Cell culture:
Caco-2 cell culture, Endothelial cell line Eahy926, HepG2 and fibroblasts. Cryopreservation of cells and reviving, Analyzing the absorption of protein and peptides through invitro methods Caco-2 monolayers.
Insilico Design:
Model mimetic peptides design, stability testing invitro and in Exvivo models. Immunological methods:
Fluorescence Microscopy, Agarose gel electrophoresis, Flow cytometer, ELISA, western blotting, Immunoprecipitation, Production and Purification of antibody and raising polyclonal antisera in rabbits, ELISA titer determination, HRP conjugation of Antibody. Computer proficiency:
MS word, Excel, PowerPoint, Empower, Unicorn, SoftMax pro Safety training:
WHMIS, MSDS, Labels
Publications
1. Bejjani Satyanarayana, Raghu Pullakhandam, Punjal Ravinder and Nair K. M. Gastric digestion of pea ferritin and modulation of its iron bioavailability by ascorbic acid and phytic acid in Caco-2 cells. World journal of gastroenterology 2007 Apr 14; 13 (14): 2083-8.
2. Bejjani Satyanarayana and Jianping Wu Transcellular transport of IRW a ovotransferrin derived antihypertensive peptide transport in Caco-2 cells by RP- HPLCMS/MS(J Agric Food Chem. 2013 61 (7)1487-1492. Conferences
1. ICN 2009 Bangkok, Thailand 4th – 9th October Iron content and bioavailability in six rice genotypes using Caco-2 cell model Awarded distinguished poster. Bejjani Satyanarayana, Raghu Pullakhandam and Nair K.M.
2. Harvest Plus workshop on Caco-2 cell based bioavailability-screening methods held at department of Human nutrition, Ohio state university, Columbus, USA during 12 and 13th September 2006. Raghu Pullakhandam Bejjani Satyanarayana and Nair K.M. Modulation of pea ferritin iron bioavailability by ascorbic acid and phytic acid: Studies using coupled in vitro digestion/caco2 cell model. 3. 75th Annual meeting of Society of Biological Chemists, India (SBC, INDIA) JNU, New Delhi December 8th -11th 2006. Bejjani Satyanarayana, Raghu Pullakhandam, Punjal Ravinder and Nair K. M. Ascorbic acid and phytic acid modulate pea ferritin iron bioavailability in Caco-2 Cell Model. Poster no.47 4. XXXVII Annual Conference of Nutrition Society of India. National Institute of Nutrition, Hyderabad. November 18th -19th 2005. Bejjani Satyanarayana, Raghu, P, Vasuprada Iyengar and Nair, KM. Novel method for assessing iron density in staple foods: development of dot blot for phytoferritin. Abstracts, poster no21.
Contact this candidate