Capillary Electrophoresis in a QC environment
by Dr. Vikas Dhingra, Biochemistry Group Leader
Over the past couple of years, Capillary Electrophoresis (CE) has shown great promise in replacing traditional analytical techniques, especially conventional electrophoresis and chromatography. The CE technology is designed to separate analytes (Protein, Nucleic acid) based on their size-to-charge ratio in a 30-75 cm long 50-100µM (i.d) capillary filled with an electrolyte. Field strengths of up to 1kV/cm and currents of 10-20mA are used. The narrow capillary enables the application of high electric fields, and thus faster run times, without overheating problems associated with the high voltages used. CE separations typically take 20-45 minutes with a low intake of chemicals and samples. There are many detection methods possible: UV/VIS, PDA, Fluorescence, etc. The results obtained are further processed by chromatography based software. The other advantages of capillary electrophoresis methodology include automation, increased accuracy, and improved sensitivity, efficiency, and resolution of protein separation. This article describes the advantages of several of these CE applications used in a biopharmaceutical QC environment.
Capillary Gel Electrophoresis (CGE)
CGE with UV and LIF detection is used to detect the size, purity and heterogeneity of a protein product. Gel filled capillary provides a robust replacement to traditional gel electrophoresis and has the advantage of automation, quantitation and sensitivity. In order to achieve a size based separation though the gel matrix, the proteins are denatured in the presence of SDS. This also allows the analytes having similar chargeto- mass ratio to be resolved by size. Separation is usually performed on a bare fused silica capillary with -15 kV separation voltage applied over 35 minutes.
Capillary Isoelectric Focusing (CIEF)
CIEF is used to determine the charge heterogeneity of protein products. Protein samples are introduced into a capillary with a mixture of ampholyte buffers which are a polymerized mixture of monomers that contain weakly acidic and weakly basic groups. By using an acidic solution on the anode side of the capillary and a basic solution on the cathode side and applying electric field, the ampholyte molecules orient themselves with respect to their pKa values, generating a pH gradient within the capillary. Proteins migrate through the ampholyte mixture and focus at a pH where they become electrically neutral (pI). After focusing, the zones are mobilized to the detection window by applying pressure, gravity or a chemical method together with focusing potential. At Lancaster Laboratories, separation is usually performed on a neutral capillary filled with Ampholyte/ sample mixture with a 15 kV focusing voltage applied over 6 minutes. The sample is then mobilized to the detection window at 21kV using 350mM Acetic acid. The Proteins are detected at 280 nm.
Capillary Zone Electrophoresis – Glycan Analysis (CZE-LIF)
CZE-LIF is useful in analyzing the complex glycans and characterizing glycoproteins in protein products. The coupling of CE with on column Laser Induced Fluorescence (LIF) detection has the advantage of highly sensitive and short analysis time, ability to separate and quantify isomeric species. The CZE-LIF assay used for profiling protein glycosylation involves three major steps: 1) Releasing Nlinked oligosaccharides with the Nglycosidase PNGase-F; 2) Labeling with a charged fluorophore (8-aminopyrene-1,3,6- trisulfonate (APTS), 8-aminonapthalene- 1,3,6-trisulfonate (ANTS) or 2- aminopyridine (2-AP) or 2-aminobenzamide (2-AB); and 3) Sample analysis using CZELIF. At Lancaster Laboratories, we adopt an APTS derivatization strategy and a separation methodology to allow routine separation of the G1 isomers on a Beckman Coulter PA800 plus CE system.
Capillary Ion Analysis (CIA)
CIA is used to determine the residual ions (Bromide, Sulfate, Acetate, Ammonium, Sodium, Chloride, etc.) in a protein product. Anions and Cations are charged polar species and UV transparent, hence can be analyzed on a CE format with reverse polarity using a dynamically coated bare-fused silica capillary and indirect mode of detection. The CE method and the separation conditions are robust and offer a huge advantage against the traditional gravimetric analysis and titration methods. The detection range of the anions and cations using the CE methodology ranges from 1-100ppm.