14 February / March 2017
Faster Analysis of Monoclonal Antibodies Using Silica Monoliths Designed for Bioanalysis
Tom Kupfer1 1
2 , Benjamin Peters1 , Egidijus Machtejevas2 , Gisela Jung1 , Peter Knoell1
Merck KGaA, Instrumental Analytics R&D, Darmstadt, Germany Merck KGaA, Advanced Analytical, Darmstadt, Germany
The chromatographic analysis of biomolecules, such as monoclonal antibodies (mAbs), requires new types of columns providing a good permeability, improved mass transfer and selectivity properties. Silica monoliths with larger mesopores are a powerful tool for the fast separation of biomolecules. In this article, the immobilisation of protein A on those widepore silica monoliths is described for the titre determination of monoclonal antibodies. The protein A modified monolith provided highly reproducible and fast, one minute, separation of mAbs. Furthermore, the high reproducibility of immobilisation method with three batches and the column stability over 5,000 runs are demonstrated.
Introduction
In the last two decades, the pharmaceutical market has changed dramatically from small molecules to protein-based drugs and antibodies, which have a higher potential for targeting extraneous substances [1]. This is supported by the fact that six of the top ten best-selling pharmaceuticals are based on monoclonal antibodies. In comparison to chemical entities, the analysis of mAbs is complex and requires several separation techniques, e.g. affinity chromatography, reversed phase chromatography, size exclusion chromatography and ion-exchange chromatography. Especially, the strong growth in development of biosimilars due to expiring patents of those blockbusters, led to increased requirements concerning column performance in HPLC. Recently, a survey of several HPLC column manufacturers offered the introduction of almost 20 new columns in 2016, addressing biomolecules in different separation modes [2].
The analysis of biomolecules using conventional HPLC columns is usually associated with limited accessibility of surface for larger molecules, slow diffusion leading to extremely broad peaks with severe tailing, and possibly conformational changes during elution. One of the reasons for those observations is the mesopore size ranging from 8-15 nm. The separation quality could be improved by using column materials with wider mesopores of ca. 30 nm or larger [3-5].
During development, process monitoring, and quality control testing of mAbs, fast and precise analytical methods are necessary. The most important technique for observation of mAb titer is affinity chromatography using protein A. Protein A, a cell wall protein from Staphylococcus aureus, was the first isolated protein with a high affinity to the Fc region of immunoglobulin G (IgG) [6]. Its high selectivity and high resistance against temperature, pH and tryptic cleavage make protein A a powerful tool in antibody purification [7]. Today, the use of protein A as a ligand on chromatographic media is one the most employed methods for antibody capture during the purification process. In comparison to process protein A media, only few vendors provide analytical protein A columns for process monitoring and quality control. The majority of those columns are packed with particles limited in column backpressure, chromatographic performance and application of ‘dirty’ samples, such as harvest cell culture fluids. Hence, sample preparation is more complex and the limited column backpressure result in long analysis time due to lower flow rates.
Figure 1: Separation of cetuximab (1 mg/mL) and BSA (1 mg/mL) by immobilised rSPA silica monolith. Chromatographic conditions: Stepwise gradient: 100 mM sodium phosphate pH 7.4/100 mM sodium phosphate pH 2.5; 0.25 min 100/0, 0.25-0.26 min 0/100, 1.00 min 0/100, 1.00-1.01 100/0, 1.01-2.50 min 100/0; flow rate: 2.0 mL/min; detection: 280 nm; injection: 10 µL; temperature: 25°C
Silica monoliths, which consist of one continuous piece of silica, are an alternative to fully porous or superficially porous particles, and could be used [8]. Silica monoliths are preferred for the application and fast separation of ‘dirty’ samples due to their bimodal pore structure consisting of large through pores (macropores) and smaller micro- or mesopores [9]. They are prepared according to a sol-gel process leading to a bimodal pore structure where both macropores and mesopores could be controlled individually [9, 10]. The design of larger macropores offers good flow
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