Chromatography

Chromatography is the separation of components within a mixture, based on a specific physical molecular attribute. BioPharmaSpec is proficient in a variety of chromatographic techniques and can apply these to your biopharmaceutical product in order to provide quality physicochemical characterization data.

Chromatographic Techniques

There are several orthogonal chromatographic techniques available for the detailed characterization of biopharmaceuticals, offering a high selectivity for separating variants based on hydrophobicity, charge and size.

Reversed Phase-HPLC (RP-HPLC), where components are separated based on their hydrophilicity/hydrophobicity, benefits from its high resolving power and its ability to be linked directly to mass spectrometry. The use of Ultra High Pressure Liquid Chromatography (UPLC) systems has also become more routine.

Ion Exchange chromatography (IEX), is a widely used technique where separation is achieved through charge differences. Among different IEX techniques available, cation exchange chromatography (CEX) is most used for protein characterization.

Size Exclusion Chromatography (SEC) separates biomolecules according to their hydrodynamic radius, i.e. based on the size of the components. A main advantage is the mild mobile phase conditions which cause minimal impact on the conformational structure of the protein. Various detectors may be utilized in SEC, including refractive Index (RI), ultraviolet (UV) and multi-angle laser-light scattering (MALS).

Hydrophilic interaction chromatography (HILIC) provides separation of analytes based on their polarity utilizing a hydrophilic stationary phase with reverse-phase type eluents. Hydrophobic Interaction Chromatography (HIC) separates proteins according to reversible hydrophobic interactions between immobilized hydrophobic ligands on the column and non-polar regions of the protein.

Applications of Chromatography

RP-HPLC is particularly useful for separating deamidated and oxidized peptides from their native counterparts and is often used in peptide mapping experiments to assess these Post Translational Modifications (PTMs). Using a limited proteolysis approach, heterogeneity of specific regions of monoclonal antibodies can also be investigated. However, it is the robust coupling of RP-HPLC to mass spectrometry (LC-MS) which has revolutionized primary protein structure and impurity/degradation product characterization.

IEX can be viewed as reference technique for the qualitative and quantitative evaluation of charge heterogenicity. It can also be used to purify isoforms within a biopharmaceutical product for further structural analysis and ultimate identification (e.g. deamidated forms and for monoclonal antibodies with heavy chain C-terminal Lysine).

SEC can be used in conjunction with MS detection to separate components based on their size. As it determines hydrodynamic volume, not molecular weight, it can assess folding in protein tertiary structure. UV, RI and Multi Angle Laser-Light Scattering (MALS) can also be used alongside SEC to provide an assessment of protein aggregation.

HILIC is particularly useful in glycosylation and carbohydrate analysis separating fluorescently labeled glycans, such as 2-AB, to assess the glycan population (glycoforms).

HIC is commonly used for purification of proteins, but it has application in the determination of the drug antibody ratio (DAR) of Antibody -Drug Conjugate products (ADCs)

Electrophoresis

Electrophoresis is a general term that describes the migration and separation of charged particles (ions) under the influence of an electric field. It is a classical technique used to separate biopharmaceutical samples in order to provide a physicochemical assessment based on size (e.g. to assess identity and/ or purity) or charge (e.g. to detect sialic acid variants or deamidation). There are many types of electrophoresis available. BioPharmaSpec provide the below methods for your electrophoresis requirements.

Electrophoretic Techniques

Separation by Size

Most separations based on size are performed using traditional gel based (polyacrylamide) electrophoresis (SDS-PAGE) or the newer technique of capillary gel electrophoresis (CGE or CE-SDS). In these techniques, molecular weights of components are defined via comparison to data obtained from analysis of a range of protein markers with known molecular weights. CE-SDS uses UV at 280nm to detect separated protein components and thus provides more reliable quantitative information relative to that obtained from densitometric analysis of gels.

Separation by Charge

Separations based on charge are performed using traditional IsoElectric Focusing (IEF) on immobilized pH gradient gels (IPG) or the newer technique of imaging capillary isoelectric focusing (icIEF). The isoelectric points (pIs) of components within a biopharmaceutical product are defined via comparison to data obtained from analysis of a range of protein or small molecule markers with known pIs. As with CE-SDS, icIEF uses UV at 280nm to detect separated protein components and in the same way provides a more reliable quantitative information relative to that obtained from densitometric analysis of gels.

Although BioPharmaSpec scientists would recommend icIEF for the best resolution, other techniques such as Ion Exchange Chromatography (IEX) are also available.