Separation of monoclonal antibody charge state variants by open tubular capillary electrochromatography with immobilised protein as stationary phase
Introduction
Monoclonal antibodies (mAbs) are one of the most promising classes of therapeutic protein biopharmaceuticals, and are used in cancer therapy owing to their characteristics including improved tolerance, good efficacy, high specificity, and limited side effects [1], [2]. Most of the currently approved mAbs belong to the immunoglobulin G (IgG) class. These mAbs (appropriately 150 kDa) are tetrameric glycoproteins including two identical heavy chains and two identical light chains, which are connected through several inter- and intra-chain disulfide bonds at their hinge region [3]. During mAb production, a wide range of post-translational modifications, including formation of disulfide bonds, glycosylation, fragmentation, deamidation, methionine oxidation, N-terminal cyclization, C-terminal processing and sequence alteration can occur [4], [5], [6], [7]. These modifications alter the biochemical and biophysical properties of the proteins [8]. Charge state variants, possibly originated from the formation of disulfide bonds, glycosylation, fragmentation, deamidation, C-terminal processing [5], are one of the most commonly observed and extensively characterized types of heterogeneous proteins, which are usually classified as acidic or basic variants depending on their isoelectric points (pI) relative to the main form.
The complexity of mAbs will make the heterogeneity characterization of mAbs very challenging. Therefore, many methods have been developed to detect the charge, size, and molecular heterogeneities of mAbs, including ion exchange chromatography (IEX) [9], [10], [11], reversed-phase liquid chromatography (RPLC) [12], [13], hydrophobic interaction chromatography (HIC) [14], size exclusion chromatography (SEC) [15], sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary isoelectric focusing (cIEF) [16], capillary zone electrophoresis (CZE) [17], [18], and mass spectrometry (MS) [19]. Among these methods, IEX is the most widely used for the characterization of charge state variants of therapeutic proteins [20]. Separation of mAb charge state variants, that are often present as minor peaks in an IEX separation, is critical to the understanding of product quality and stability. So the emphasis in the development of separation methods is to resolve minor forms and distinguish them from the major form of the mAb. Among various modes of capillary electrophoresis (CE), capillary electrochromatography (CEC) is a promising hybrid technique, where the separation results from a combination of electrophoretic migration and chromatographic retention. It combines high selectivity of high-performance liquid chromatography and high efficiency of CE, and has shown great potential to achieve high efficiency for the analysis of complex biomolecules. As the core of CEC, various forms of columns including packed, monolithic, and open tubular (OT) columns are used [21], [22]. Among these columns, OT columns have advantages such as no bubble formation, simple column preparation, and simple instrumental handling [23]. Many approaches have been utilized for preparation of OT columns, including chemically bonded ligands, sol-gel derived phases, molecularly imprinted polymers, porous layers, physically attached or adsorbed phases, and nanoparticle coatings [24]. Among these approaches, layer-by-layer self-assembly method has been proven to be a powerful method to design function-specific films at the nanoscale level due to its advantages including simplicity, stability and low cost [25].
Poly(diallydimethylammonium chloride) (PDDA) is a cationic water soluble polyelectrolyte, with highly hydrophilic and positively charged quaternary ammonium groups. Recently, it has been used to fabricate coating in OT-CEC by the layer-by-layer self-assembly [21], [26], [27], [28], [29], [30]. Some nanomaterials have been applied in CEC as novel stationary phases via the layer-by-layer self-assembly, such as silica nanoparticles [26], graphene oxide and reduced graphene oxide [27], [28], cyclodextrin-modified gold nanoparticles [29], carboxyl modified magnetic nanoparticles [30].
Proteins have been utilized as stationary phases in CEC for the separation of enantiomers [31], [32], [33]. The basic protein lysozyme (Lys) was directly adsorbed on the capillary wall and used as chiral stationary phase for separation of enantiomers of four amino acids and mephenytoin [31]. Polydopamine-graphene oxide nanocomposite was used as a medium to immobilise bovine serum albumin (BSA) in the poly(dimethylsiloxane) microchannel and BSA was employed as a stationary phase for chiral separation of tryptophan, threonine, and dipeptide [32]. Avidin was covalently immobilised onto the inner surface of aldehyde group modified silica capillary through a Schiff base formation reaction between aldehyde group and amino groups in avidin [33]. And avidin was used as a stationary phase for chiral separation of abscisic acid and arylpropionic acids [33]. Since proteins are amphoteric and possess many functional moities, if they were used as stationary phases in OT-CEC, they could potentially yield good selectivity for separation of mAbs charge state variants.
The aim of this work was to develop an OT-CEC method to detect the charge heterogeneity of mAbs (rituximab, trastuzumab, cetuximab). BSA was selected as a stationary phase for preparation of the BSA coated PDDA OT column. A simple and reliable procedure based on layer-by-layer self-assembly method was utilized to immobilise BSA on the inner surface of a positively charged PDDA coated capillary. Negatively charged BSA was adsorbed to the surface of PDDA coated OT column through electrostatic interaction. The performance of the BSA coated PDDA OT column was evaluated by separation of three basic proteins and three mAbs. Furthermore, the separation conditions and performance for basic proteins and mAbs charge state variants were investigated. The stability and repeatability of the OT column were also investigated.
Section snippets
Materials and chemicals
All chemicals and reagents were analytical grade or better. Fused-silica capillaries (50 μm i.d. × 365 μm o.d.) were purchased from Hebei Yongnian Ruipu Chromatogram Equipment Company (Handan, China). Phosphoric acid (H3PO4) and N,N-dimethylformamide (DMF) were purchased from Tianjin Damao chemical reagent factory (Tianjin, China). PDDA solution, 20% in water, with an average molecular weight (Mw) between 400 and 500 kDa, was acquired from Sigma-Aldrich (Saint Louis, MO, USA).
Characterization of BSA coated PDDA OT column
The inner surface morphologies of a bare fused-silica capillary, PDDA and BSA coated PDDA OT columns were investigated by SEM, as shown in Fig. 2. The inner surface of a bare fused-silica capillary was very smooth (Fig. 2A). After modification of PDDA, a series of small hills were observed on the inner surface of capillary and the inner surface became rough possibly due to the random coil of PDDA chain under high salt concentration [36], as shown in Fig. 2B. The SEM of the cross-section of PDDA
Conclusion
In this work, an electrostatic self-assembly approach was developed for preparation of BSA coated PDDA OT column. The column was successfully utilized for separation of basic proteins and mAbs charge state variants in CEC. The BSA coating significantly improved the separation of proteins and effectively reduced the adsorption of proteins to the capillary wall. Especially, the BSA coating showed the special separation ability for the charge state variants of mAbs. In addition, the column
Acknowledgements
We are grateful to the financial support of Natural Science Foundation of Guangdong Province, China (2015A030311013) and National Natural Science Foundation of China (21175048, 21675056).
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The first two authors contributed equally to this work.