Bioseparation I Centrifugation

What is Bioseparation?  Purification or separation of a specific material of interest from contaminants in a manner that provides a useful end product - protein of interest in lab, drug product in pharmaceutical company  All purification strategies require compromises - high product purity is usually obtained at high cost but with reduced yield. - protein of interest in lab, drug product in pharmaceutical company Thus different types of separation strategies must be adopted.

Designing a product purification strategy 1. prioritize the goals of the purification process 2. select a source for the product 3. develop an assay for the product of interest 4. determine the optimal methods to:  - release the product from its source in a soluble form  - reduce the volume of the product  - separate the product from its impurities  - purify the product of interest 5. Verify the identity and purity of the product

Stages of a Bioseparation Strategy 1. Solubilization of the product of interest 2. Separation of product from contaminating solid material (centrifugation, filtration) 3. Separation of product from divergent impurities (eg. salt fractionation or organic extraction) 4. Purification of product from similar impurities (PAGE, HPLC) 5. Product polishing, concentration and preparation for end use

Methods for Bioseparation  1. Fractionation and clarification methods  2. Low resolution purification methods  3. High resolution methods - Electrophoresis (agarose or polyacrylamide) - Chromatography (TLC, column chromatography, HPLC and Gel permeation chromatography(GPC)/size exclusion chromatography

Analysis of Product and Yield  After each purification step you must  - measure the amount of desired protein present  - measure the total amount of protein present  This allows SPECIFIC ACTIVITY to be determined: =the amount or units of the protein of interest total amount of protein in the sample

Biologically active molecules  Enzyme assays involve the measurement of either the appearance or disappearance of substrate or the product.  Enzyme activity is expressed as UNITS  International Unit (IU) of enzyme activity : amount of enzyme necessary to catalyze transformation of 1 umole of substrate o product per minute under optimal conditions.  SI unit of enzyme activity: amount of enzyme necessary to catalyze transformation of 1 mole of substrate to product per second under optimal conditions.

Common problems with Bioseparation and Purification Strategies. Most commonly, loss of the product at a specific purification step.  Possible causes:  - discarding the fraction that contains the product of interest  - performing separation using poor quality reagents  - poor product stability  - unexpected precipitation of the product  NEVER DISCARD ANYTHING UNTIL AFTER FINAL ANALYSIS!

Principles of Filtration - based on the principle that particles smaller than the filter pore size will pass through the filter. Larger particles will be trapped by the filter. 4 components.  1. Filter itself  2. Support for the filter  3. vessel to receive the filtrate  4. Driving force (such as gravity or a vacuum) to push the movement of fluids and particles through the filter.

Types of Filtration and Filters 1. Macrofilters or depth filters 2. Microfilter eg. HEPA filters (High Efficiency Particulate Filters) in biological safety hoods to protect from contamination or animal cages to protect from infection. 3. Ultrafilters - Fractionation - Concentration - Desalting

Dialysis and Reverse Osmosis Use membranes similar to those used for ultrafiltration.  DIALYSIS – based on the difference in concentration of solutes between one side of the membrane and the other eg desalting of buffer is done this way.  REVERSE OSMOSIS – used to remove very low molecular weight materials, including salts, from a liquid eg. Water.

Principles of Centrifugation  1. SEDIMENTATION  - over time you would expect particles to sediment (move through the liquid and settle out at the bottom). Largest particles would be closest to the bottom and a series of layers would form.  A centrifuge accelerates the rate of sedimentation by rapidly spinning the samples. This creates a centrifugal force many times that of gravity.

 2. CENTRIFUGAL FORCE - the speed of rotation is revolutions per minute (RPM) - the distance from the center is called radius of rotation (r) Relative Centrifugal Force (RCF) = r (RPM/1,000) 2 (units are xg) Where, r: radius of rotation in centimeters RPM: speed of rotation in rotations per minute

Factors influencing the rate of sedimentation  1. Relative centrifugal field (  RCF then  sedimentation rate)  2. Viscosity of the medium  3. Size of the particle 4. Difference in density between the particle and the medium

Modes of Centrifugation 1. DIFFERENTIAL CENTRIFUGATION  - separates a sample into two phases; a pellet and a supernatant 2. DENSITY CENTRIFUGATION  - a density gradient is a column of fluid that increases in density from the top of the centrifuge tube to the bottom…..particles separate into discrete bands.  - Step gradient  - Continuous gradient 3. CONTINUOUS CENTRIFUGATION  - Mode of centrifugation for large samples, such as separating cells from fermentation broth.