PREPARATION OF RECOMBINANT PROTEINS Bi7430c Molecular Biotechnology Practicals 1 2 3 4 5 6 7 8 9 10 11 LIGATION OF TARGET GENE INTO PLASMID TRANSFORMATION AND SELECTION CULTIVATION AND EXPRESSION PURIFICATION SCALE-UP •shake flasks – up to 1L (uncontrolled conditions) •laboratory fermentor – up to 10L (sterilized whole with medium, tubes, filters...) •pilot scale – hundreds of L •industrial bioreactor – thousands of L (sterilized with steam through pipes) • MANUFACTURE •a cell line is established from a single clone and is used to make-up the master cell bank (MCB) •MCB must be characterized and tested for contaminants (bacteria, fungi, mycoplasmas, viruses) •cells from MCB are expanded to form the working cell bank (WCB), which is characterized for cell viability prior to use in the manufacturing process CONDITIONS TO CONTROL •factors influencing the growth of microorganisms and production of metabolites • •temperature •concentration of oxygen •pH •pressure •extracellular concentration of substances and water •agitation • •production medium • TEMPERATURE •psychrophiles •mezophiles – E. coli (5 to 40°C) •thermophiles temperature 1 2 3 •↑t = ↑growth and synthesis of metabolites •above 45°C proteins losing 3D structure •above 60°C half of proteins denatured • 1.solidification of lipids in membranes 2.growth is proportional to temperature 3.denaturation of proteins TEMPERATURE •t measurement •thermometer • •t maintenance •thermal jacket •water in/out • •across all operating processes, temperature variations of as little as 1°C can limit production OXYGEN •aerobic – supply of oxygen necessary •agitation and aeration •shape of cultivation flask •increased pressure •additives • •microaerophile •hard to maintain standard conditions • •anaerobic •facultative (S. cerevisiae +O2 = growth and production of acids, -O2 = ethanol) •obligatory – complete elimination of O2 during cultivation is necessary; N2 and CO2 atmosphere OXYGEN •fostering the optimal growth environment within a bioreactor requires adding the right amounts of reactant gasses to a vessel to support a cell culture • •the amounts of gasses (oxygen) vary depending on the size and growth stage of a culture pH •H+ interact with proteins and lipids in the cell membrane •if the difference between extracellular and intracellular pH increases, energetic demand on cells grow to maintain the intracellular pH • •filamentous fungi – can withstand even lower pH •yeasts – pH usually 4 to 7 •bacteria – pH usually 4 to 9 •mammalian cells – very sensitive to pH changes • •some microorganisms have different pH optimum for growth and production of metabolites (A. niger growth 5-7, citric acid production 2-3, gluconic acid 5-6) pH •pH measurement •electrode • •pH maintenance •acid (phosphoric acid) •base (ammonium) • •metabolic changes in organisms and improperly calibrated sensors can result in ineffective pH control and negatively impact an operation PRESSURE •obligatory basophiles •they can not withstand atmospheric pressure •marine microorganisms found in oceans are not suitable for laboratory/industrial use • •lots of microorganisms can form spores that are resistant to high pressures and temperatures – problems with sterilization PRESSURE •precise control of the pressure is necessary for ensuring adequate delivery of oxygen from a gas stream to cell culture • •even slight variations in local pressures can stunt cellular growth and limit product formation EXTRACELLULAR CONCENTRATION OF SUBSTANCES •halophiles – tollerant up to 25% of NaCl •osmophiles – especially fungi (growth even on solid substrates like fermented soya, using only atmospheric moisture) • •very high concentration of substrates in extracellular environment •dehydration of cells •inhibition of important enzymes in cells •sweet wine – osmotolerant yeasts AGITATION •agitation and rocking processes help transfer nutrients and oxygen to a cell culture within a bioreactor • •while these mixing processes are critical to achieving optimal productivity, imprecise and ineffective machinery can lead to excessive spinning or shaking – and destroy a cell culture (filamentous fungi) CALIBRATION! •for life sciences manufacturers, calibration issues can result in deviations and quarantined batches and requires increased maintenance • •lack of traceability can result in potential compliance issues MEDIUM •nutrition sources •sources of proteins, vitamins, mineral and carbohydrates •peptone (hydrolysed protein), tryptone (digested casein), yeast extract •energy sources •sources of carbohydrates •glucose, glycerole •essential minerals •sources of micro and macro minerals •present in peptone and meat extract •buffering agents •maintain optimum pH •specific amino acids, phosphates, citrates and zwitterions •selective agents •allow the growth of only specific bacteria •antibiotics, tellurites, azides, bile salts •solidifying agents •agar plates, gelatin •specific substances such as growth factors, enzymes may be incorporated into the medium for specific bacteria •commercial or DIY •LB (Miller/Lennox/low salt) •minimal salt/mineral (M9) •Terrific Broth TB •SOB •SOC • •commercial •EnPresso™ B Growth System (5x more protein compared to traditional culture media) OPERATION MODES Characteristics Batch Fed-batch Continuous Cultivation system Closed-type Semi-closed type Open type Addition of fresh nutrition No Yes Yes Volume of culture Constant Increases Constant Removal of wastes No No Yes Chance of contamination Minimum Intermediate Maximum Growth phase Lag, log, stationary, decline Lag, log, stationary, decline Lag and log Log phase Shorter Longer Longest, continuous Density of bacteria Changes with time Changes with time Remains same Product yield Low Medium High PROCESSING •