Capillary electrophoresis of intact basic proteins using noncovalently triple‐layer coated capillaries

Abstract

The usefulness of a noncovalent, positively charged capillary coating for the efficient analysis of intact basic proteins with CE was studied. Capillaries were coated by subsequent flushing with solutions of 10% w/v Polybrene (PB), 3% w/v dextran sulfate (DS), and again 10% w/v PB. Coating characterization studies showed that stable coatings could be produced which exhibited a pH-independent and highly reproducible EOF. The PB-DS-PB coating was evaluated with Tris phosphate BGEs of various pH using the four basic model proteins: alpha-chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme. Typical migration time RSDs for the proteins were less than 0.85%, and apparent plate numbers were above 125,000 using a capillary length of 40 cm. The high separation efficiency allowed detection of several minor impurities in the model proteins. Using a BGE of medium pH, the CE system with triple-layer coating appeared to be useful for the repeatable profiling of recombinant humanized mouse monoclonal immunoglobulin G(1) showing a characteristic pattern of glycoforms. The CE system was also applied to the characterization of two llama antibodies, which were produced in Saccharomyces cerevisiae, revealing the presence of a side product in one of the antibodies. The high migration time stability allowed the reliable determination of antibody-antigen binding by monitoring migration time shifts. Finally, the feasibility of using the PB-DS-PB coated capillaries for CE with mass spectrometric detection was shown by the characterization of the impure llama antibody sample.

Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Setup and detector performance

“…In order to avoid adsorption of the basic lysozyme onto the fused-silica capillary wall -and thus allow efficient CE -the capillary was coated with multiple layers of charged polymers (PB-DS-PB) resulting in a positively charged surface. The production and performance of this coating has been described previously [29][30][31]. As determined by comparing protein fluorescence signals obtained with bare and coated fused-silica capillaries, presence of the coating did not cause any background fluorescence.…”

“…The capillaries had an id of 75 mm, an od of 375 mm and total/effective lengths of 73/47 cm. New bare fused-silica capillaries were rinsed with 1 M sodium hydroxide for 30 min at 20 psi, and deionised water for 15 min at 20 psi, before they were coated with a triple-layer coating of PB-DS-PB according a procedure described by Haselberg et al [29]. The capillary coating was daily regenerated by subsequently rinsing for 15 min at 10 psi with 10 mM phosphoric acid, 3% w/v PB in water, deionised water and BGE.…”

A novel cationic triblock copolymer as noncovalent coating for the separation of proteins by CE

“…When the pH was above 5.0, the electrophoretic velocity of lysozyme became faster than that of cytochrome c. However, when the pH was below 5.0, the electrophoretic velocity of lysozyme was slower than that of cytochrome c. This can be explained by the net positive charge in cytochrome c increasing much more than lysozyme at low pH, as was calculated by Protein Calculator v 3.3 (http:// www.scripps.edu/$cdputnam/protcalc.html) [45].…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…We covalently coated the bare fused silica capillary with cPEI in contrast to dynamic polymeric coatings such as polybrene or hydroxypropylmethylcellulose, which require frequent recoating and can potentially contaminate the MS source [248][249][250][251]. Although the coating is positively charged and rhIFN-β1 has up to four sialic acids, the net charge of the various proteoforms is positive at pH 2.5, reducing the possibility of protein interaction with the coating.…”

“…N-glycans produces in CHO cells consist of the biantennary G2F core polysaccharide (Figure 2-4) [252], to which are attached, among others, repeating N-acetyllactosamine (LacNAc) extensions and up to four LacNAc antennae, many of which are terminated with sialic acid (NeuAc) with very small amounts of (NeuGc) [253][254][255]. The mass spectrometric analysis of native glycans cannot distinguish between hexoses (Hex) as well as N-acetylhexosamines (HexNAc).…”