Medical Biotechnology Production of Pharmaceutical Proteins PDF

Production of Recombinant Proteins for Treatment of Human Diseases Medical Biotechnology Lv4 Lecture #2 Sami Mohamed Nasr, PhD Associate Professor BUC, School of Biotechnology  Purification of Recombinant Proteins Sample Preparation:...

Production of Recombinant Proteins for Treatment of Human Diseases Medical Biotechnology Lv4 Lecture #2 Sami Mohamed Nasr, PhD Associate Professor BUC, School of Biotechnology  Purification of Recombinant Proteins Sample Preparation:  Steps to Purify Recombinant Proteins Expressed as Inclusion Bodies Cell Harvest In E. coli, recombinant proteins accumulate Cell Lysis and IB Isolation in the cytoplasm in the form of Inclusion Centrifugation and IB Washing bodies (IB’s) IB Solubilization IB’s are insoluble aggregates of Purification misfolded protein that is lacking biological Refolding activity Further Purification  Chromatography Techniques for Recombinant Proteins Purification Gel Filtration Chromatography Ion-Exchange Chromatography High Pressure Liquid Chromatography (HPLC) Affinity Chromatography  Gel Filtration Chromatography A mixture of proteins in a small volume is applied to a column filled with porous beads. Because large proteins cannot enter the internal volume of the beads, they emerge sooner than do small ones  Ion-Exchange Chromatography This technique separates proteins mainly according to their net charge High Pressure Liquid Chromatography (HPLC) HPLC is an enhanced version of column techniques Separates proteins with great resolving power  Affinity Chromatography Affinity Purification in Recombinant Proteins using Fusion Tags Native Conditions Denaturing Conditions Cell Lysis Binding Buffer Binding Buffer including 8M Urea or 6M Guanidine HCl Binding to affinity column Wash Elute Pure Tagged Pure Denatured Protein Refolding Tagged Protein General purification steps of tagged proteins by affinity chromatography  Hydrophobic chromatography (HIC) Most hydrophobic amino acid residues are found inside proteins Bind with hydrophobic column low cost and biological activity Protein retention in the column, in the brine solution in low salt or proteins in aqueous solution  Protein Structure (Folding) Proteins fold into one or more specific spatial conformations to be able to perform their biological function  Methods For Protein Refolding Dilution Step Dialysis Pressure treatment 150–200 MPascal On-column refolding  Protein Biological Activity The biological activity of a recombinant protein is routinely measured using a bioassay depending on the protein function. E.g.; Enzyme assay Cell proliferation assay Functional ELISA  Bioreactors Large-scale recombinant biopharmaceutical production takes place in stainless steel, sterilizable bioreactors  Bioreactors Pilot-Scale Industrial-Scale  Scaling-Up the Cell Culture Bioreactors range in size from liters to cubic meters  Conclusions  Molecular cloning technologies offer powerful tools for the development of safer recombinant protein therapeutics  Care must be taken in designing constructs to avoid any unnecessary amino acid residues, immunogenicity, or other unwanted side effects  Mammalian cells, E. coli and yeasts are the most commonly used production hosts  The adoption of a new expression host is slow due to regulatory constraints  Recombinant protein therapeutics will have an expanding role in medicine for years to come