On-line coupling of size exclusion chromatography with mixed-mode liquid chromatography for comprehensive profiling of biopharmaceutical drug product

Abstract

A methodology based on on-line coupling of size exclusion chromatography (SEC) with mixed-mode liquid chromatography (LC) has been developed. The method allows for simultaneous measurement of a wide range of components in biopharmaceutical drug products. These components include the active pharmaceutical ingredient (protein) and various kinds of excipients such as cations, anions, nonionic hydrophobic surfactant and hydrophilic sugars. Dual short SEC columns are used to separate small molecule excipients from large protein molecules. The separated protein is quantified using a UV detector at 280 nm. The isolated excipients are switched, online, to the Trinity P1 mixed-mode column for separation, and detected by an evaporative light scattering detector (ELSD). Using a stationary phase with 1.7 μm particles in SEC allows for the use of volatile buffers for both SEC and mix-mode separation. This facilitates the detection of different excipients by ELSD and provides potential for online characterization of the protein with mass spectrometry (MS). The method has been applied to quantitate protein and excipients in different biopharmaceutical drug products including monoclonal antibodies (mAb), antibody drug conjugates (ADC) and vaccines.

Introduction

As progress in biotechnology continues, a large number of therapeutic proteins have been developed, including monoclonal antibodies (mAbs), antibody drug conjugates (ADC) and protein conjugated vaccines. Monoclonal antibodies (mAbs) represent the largest segment and the most rapidly growing class of biotherapeutics in development for many different disease indications including cancer, arthritis, etc. [1], [2], [3]. Novel mAb molecules are entering clinical studies at a rate of nearly 40 per year, and the research pipeline includes about 250 therapeutic mAbs in clinical studies. The potential for introducing new functionality onto a mAb by conjugating drugs to the antibody was realized decades ago [4]. Obstacles related to design, engineering, manufacturing and clinical evaluation of these products have gradually been overcome, and as of 2011, most major pharmaceutical companies and many biotechnology firms are developing antibody-drug conjugates (ADCs) [5], [6]. In addition to mAbs and ADCs, there has been rapid increase in the number of vaccines currently in development, with most of them exploring novel mechanisms, adjuvants and/or delivery systems not only for traditional prophylactic use, but also for therapeutic use [7], [8].

Biopharmaceutical drug products consist of an active ingredient (mAb, ADC or vaccine) and multiple inactive ingredients (excipients). Proteins can vary significantly in their biophysical and biochemical properties including molecular weight, isoelectric point, primary and secondary structure, etc. Various kinds of excipients are used in biopharmaceutical drug products to enhance stability, control pH and adjust tonicity [9]. The excipients are diversified, including cations, anions, zwitterions (e.g. amino acids), hydrophobic surfactants, hydrophilic sugars, etc. During the development of a biopharmaceutical drug product, a variety of analytical methods are needed to quantify all components in the dosage form to ensure the integrity of the formulated drug product.

The quantity of protein in biopharmaceutical drug product is an important metric to measure at different steps of bioprocess development. Numerous methods are available for protein quantification. Spectroscopic methods such as UV [10], Lowry [11], Bradford [12], and bicinchoninic acid (BCA) [13] assays have been widely used to determine the concentration of protein in different matrices. HPLC methods have also been evaluated for the quantitation of proteins [14], [15]. A number of different analytical techniques have been used to analyze various excipients in pharmaceutical formulation. The techniques include ion chromatography with conductivity detection (IC-CD) [16], [17], hydrophilic interaction chromatography with evaporative light-scattering detection or charged aerosol detection (HILIC-ELSD or CAD) [18], [19], mixed-mode liquid chromatography (LC) with CAD [20], [21], and micellar electrokinetic chromatography (MEKC) with UV detection [22]. IC-CD is the most commonly used method for ion analysis. IC-CD provides good separation selectivity and high detection sensitivity of a variety of cations and anions. However, this technology requires a cation exchange column to separate cations [16], and an anion exchange column to separate anions [17]. Risley et al. demonstrated the use of a HILIC-ELSD method for separation and detection of multiple excipients including sugars, anions and zwitterions [18]. The method was also validated for the quantitation of the API Gemcitabine and the excipient mannitol in the dosage form. Zhang and co-workers evaluated the mixed-mode LC-CAD as a powerful generic method for simultaneous determination of over twenty positive and negative pharmaceutical counterions. The performance of the method was demonstrated in the analysis of APIs and their counterions in several small molecule drug products [21]. The use of capillary electrophoresis for excipients analysis has been explored by Altria et al. They used MEKC to separate wide a range of pharmaceuticals and excipients [22]. MEKC provided fast separation of API and excipients usually within 10 min. However, the MEKC method was limited by relatively poor reproducibility and sensitivity as compared to the HPLC method. Many laboratories have reported on the quantitation of API, counter ions and/or excipients in small molecule drug product [18], [21]. Surprisingly, we have found no work in the literature on simultaneous quantitation of API and excipients in large molecule drug products. The few reports that have been published on excipients in large molecule drug products, were focused on nonionic surfactant such as PS80 and PS20 [23], [24], [25], [26]. Tani et al. described a method for quantitation of PS80 by size exclusion chromatography (SEC) [23]. PS80 was separated from other excipients, but proteins (MW > 50,000 Da) such as mAbs, caused interference. Hewitt et al. developed a mixed-mode LC method for quantitation of PS20 in a mAb drug product. PS20 was well separated from other excipients and the mAb. However, the mAb and other excipients eluted as one peak in the void volume [26].

In this work, online coupling of SEC-UV with mixed-mode LC-ELSD was developed as an integrated and generic approach to the separation and detection of all or most components (including protein, anionic, cationic, zwitterionic, and nonionic excipients) in a biopharmaceutical drug product. Comprehensive two-dimensional liquid chromatography (2-D LC) has been widely used in proteomic and non-proteomic applications [27], [28], [29], [30], [31]. The commonly used configuration for 2DLC include ion exchange LC with reverse phase LC (IE × RP) for proteomic applications, and normal phase LC with reverse phase LC (NP × RP) for non proteomic applications. In an ideal comprehensive 2D-LC setup, all of the effluent from the primary column in the first dimension should be sampled into the column in the second dimension. Heart-cutting 2-D LC has also been explored by a number of groups, and used to separate and quantitate the targeted analyte in the complex sample matrix [32], [33]. In a practical heat-cutting 2-D LC system, only the eluent that contains the peaks of interest monitored during the first dimension of separation is redirected to the second dimension of separation. This work represents the first effort to explore the combination of four different separation mechanisms (size exclusion, anion exchange, cation exchange and reverse phase) in a heart-cutting 2-D LC setup. This allows for the analysis of a complex mixture of biopharmaceutical formulations. The general applicability of the combination was demonstrated in the simultaneous separation of the active protein ingredient and the inactive excipients in the formulations from different modalities of biopharmaceutical products including mAb, ADC and vaccine.