BioOutsource

Protein Characterisation

High Performance Liquid Chromatography (HPLC) Testing

Throughout the production of bio pharmaceuticals, characterisation of the raw material and the product is needed.

HPLC is an analytical technique used to separate, identify and quantify molecules. The separation technique used varies with the molecule and can include size-exclusion and ion exchange. There are a range of detectors available for use within HPLC and these include UV/ VIS absorbance.

Our Agilent 1200 Series HPLC system can offer effective separation and identification, with the method customised to the analyte of interest. This technique allows for the separation of very similar products, enabling the detection and quantification of related impurities.

Gradient or isocratic methods are used to analyse either raw materials or products for a wide range of molecules including:

  • Small molecules
  • Peptides
  • Proteins

Analyses include:

  • Purity
  • Identification
  • Content
  • Impurities

High performance liquid chromatography (HPLC) is a chromatographic technique where the sample is injected onto a column (stationary phase) and a solvent or mix of solvents (mobile phase) is pumped through the column under high pressure. The time at which constituents of the sample elute from the column depends on the molecules being analysed, the interactions between the stationary phase and the solvent(s) used.

In normal phase (NP-HPLC), the mobile phase is non-polar and the stationary phase is polar. In reverse-phase (RP-HPLC), the polarity of the stationary and mobile phases is reversed, allowing only hydrophobic interactions with the analytes. Polar analytes elute first followed by non-polar. RP-HPLC is more widely used, as it is a sensitive and flexible technique.

Size exclusion chromatography (SEC) technique separates particles based on their size, or hydrodynamic volume. Particles of different sizes elute at different rates through the column, the larger eluting first as they are too large to penetrate the pores in the stationary phase. SEC is widely used for the analysis for large molecules, such as proteins and polymers. Since SEC measures hydrodynamic volume, rather than molecular weight, it may be used to assess the tertiary structure of a protein. SEC will differentiate between proteins with the same sequence/molecular weight, but with different molecular size due to variations in folding. This may be used as a comparative technique in batch release. It is a recommended technique in the ICH Q6B guidelines for assessment of molecular weight or size, and also for liquid chromatographic patterns.

Ion Exchange Chromatography (IEX / IEC) - in this technique proteins including antibodies, small nucleotides and amino acids may be separated depending on their net charge. Either cation or anion exchange chromatography may be employed, retaining positively-charged or negatively charged analyte ions, respectively. Since proteins have numerous functional groups containing positive and/or negative charges, ion exchange chromatography is a useful method for purification and separation of proteins. It is one of the recommended techniques for liquid chromatographic patterns in the ICH Q6B guidelines.

Benefits of BioOutsource analytical testing include:

  • GMP compliant services
  • BioWeb – online publishing of data and results allowing the vendor audit of the data
  • High speed turnaround of studies – Sample receipt to final certificate of analysis in one week

Results

All reports are available online following completion of the analysis. The final report can be custom designed to the sponsor's requirement.

SDS-PAGE (reducing/non-reducing/native)

Protein separation by SDS-PAGE (polyacrylamide gel electrophoresis) can be used to estimate relative molecular mass (molecular weight) and determine the relative abundance of major proteins in a sample and to determine the distribution of proteins among fractions. 

The test protein is prepared in solution with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. The SDS binds to and linearises the protein giving an approximately uniform mass to charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein.  Besides the addition of SDS, proteins may also be heated to near boiling in the presence of a reducing agent (such as DTT), which further denatures the proteins by reducing disulfide linkages, breaking up quaternary protein structure.  This is known as reducing SDS-PAGE, and is most commonly used. Non-reducing SDS-PAGE (no boiling and no reducing agent) may be used in situations when native structure is important in further analysis.

Isoelectric focusing (IEF)

Isoelectric focusing is a technique for separating different molecules by their charge differences.  It is a type of zone electrophoresis, usually performed in a polyacrylamide gel, that takes advantage of the fact that a molecule's charge changes with the pH of its surroundings.

Molecules to be focused are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes).  An electric current is passed through the medium, creating a "positive" anode and "negative" cathode end. Negatively charged molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end.  When the applied proteins reach the gel fraction that has a pH that is the same as their isoelectric point (pI), their charge is neutralised and migration ceases.  The pH gradient is initially established before adding the particles of interest by first subjecting a solution of small molecules such as polyampholytes with varying pl values to electrophoresis.

Gels with large pores are used in this process to eliminate any "sieving" effects, or artefacts in the pI caused by differing migration rates for proteins of differing sizes. Isoelectric focusing can be used as an identity test when the migration pattern on the gel is compared to a suitable standard preparation and IEF calibration proteins.  It can also be used as a limit test when the density of a band is compared subjectively with the density of bands appearing in a standard preparation, or it can be used as a quantitative test when the density is measured using a densitometer to determine the relative concentration of protein in the bands.  IEF can resolve proteins that differ in pl value by as little as 0.01.

Electrophoretic (Western) blotting

An analytical technique in which prior to protein immobilization on the PVDF or nitrocellulose membranes, sample proteins are separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) before detection using monoclonal or polyclonal antibodies.  This technique can provide information about molecular weight and the potential existence of different isoforms of the proteins under study.

pH, appearance and osmolality

pH, appearance and osmolality are general tests required for final product testing.

The assessment of particles in solution is determined by visual inspection. The pH (potential of hydrogen) is determined using a validated Mettler Toledo SevenEasy™ S20K pH meter and InLab® Expert Pro combination electrode, checked for accuracy using standards. Osmolality is measured using the Osmomat® 030, which is calibrated by performing measurements with distilled water and two calibration standards.

Osmolality (osmoles per kg solution) is a measure of the total concentration of dissolved particles in a solution, without regard for homogeneity of the molecular species or the molecular weights, size or density. Any substance dissolved in a solvent affects four colligative properties of the solvent-solute mixture: decrease in vapour pressure, decrease in freezing point, increase in boiling point and change in osmotic concentration. All of these properties are interrelated and the accurate determination of any one of these properties allows the estimation of the other three and is a measure of the osmotic concentration of the solution.

Freezing point depression is one of the easiest methods for determination of the osmotic concentration of biological fluids. The sample is placed in a cooling chamber maintained below the freezing point of the solution, and then supercooled which initiates crystallisation. Crystal formation results in the release of heat, causing the sample to warm to the point at which ice and solution exist in equilibrium.

Osmotic concentration (osmolality) is expressed in units of milliOsmoles (mOsm) per kg of water where one Osm is equivalent to one mM of dissolved solute particles. A solution containing one Osmole (1000 mOsm) of dissolved solute per kg of water lowers the freezing point of water by 1.858oC. Therefore, the freezing point depression of the sample can be converted to units of osmolality by dividing by 1.858. This calculation is performed automatically by the osmometer.