Polysorbates (PS) are added to biopharmaceutical formulations to stabilize proteins and monoclonal antibodies (mAbs) and to prevent aggregation, denaturation, and surface adsorption. Polysorbate analysis can be challenging because of their inherent complexity, polydispersity, and their presence in matrices containing high concentrations of proteins and other excipients such as sugars, amino acids, salts, and buffers. This instalment of "Biopharmaceutical Perspectives" provides an overview of different approaches for the qualitative and quantitative analysis of polysorbates, its impurities and degradants in raw material, intermediate dilutions, drug substance, and drug products. Various gas (GC) and liquid chromatography (LC) methods and accompanying detection techniques are briefly discussed and illustrated with results obtained at the authors' laboratory.

Analytical requirements in the biopharmaceutical, pharmaceutical, and food industries, among several others, are more demanding than ever. Chromatographic techniques are great tools to acquire detailed information on a vast number of molecules and sample types. The present challenge in research and development (R&D), as well as in quality control (QC) laboratories, is to collect as much sample information as possible. However, even with the current one-dimensional (1D) analytical portfolio, it is not possible to fully ensure that all the relevant information from a sample has been captured. This article illustrates the power of an online two-dimensional liquid chromatographic (2D-LC) setup to unravel the complexity of biopharmaceutical and pharmaceutical samples. This technology tremendously increases the resolving power in all areas where LC is applied and drastically reduces the risk of missing information about the sample.

Fully automated analysis of multiple structural attributes of monoclonal antibodies (mAbs) using three-dimensional liquid chromatography-mass spectrometry (3D-LC-MS) is described. The analyzer combines Protein A affinity chromatography in the first dimension (1D) with a multimethod option in the second dimension (2D) (choice between size exclusion (SEC), cation exchange (CEX), and hydrophobic interaction chromatography (HIC)) and desalting SEC-MS in the third dimension (3D). This innovative 3D-LC-MS setup allows simultaneous and sequential assessment of mAb titer, size/charge/hydrophobic variants, molecular weight (MW), amino acid (AA) sequence, and post-translational modifications (PTMs) directly from cell culture supernatants. The reported methodology that finds multiple uses throughout the biopharmaceutical development trajectory was successfully challenged by the analysis of different trastuzumab and tocilizumab samples originating from biosimilar development programs.

Comprehensive two-dimensional liquid chromatography (2D-LC) was used for detailed profiling of various nonionic ethoxylated surfactants applied in pharmaceutical formulations. Hydrophilic-interaction chromatography (HILIC) and reversed-phase liquid chromatography (LC) were used as the first and second dimensions, respectively. Detection was performed with evaporative light-scattering detection (ELSD) for general profiling and with single-quadrupole mass spectrometry (MS) for structure elucidation of individual peaks and for class-type confirmation of peak-groups.

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development.

In recent years, 2D-LC has been highly promising for the detailed characterization and comparability assessment of protein biopharmaceuticals such as monoclonal antibodies. This Application Note describes the use of the Agilent 1290 Infinity II 2D-LC solution in the comparison of an infliximab originator and candidate biosimilar. RPLC×RPLC and SCX×RPLC peptide mapping revealed important differences that could be attributed to a double mutation in the heavy chain of the candidate biosimilar using quadrupole time-of-flight mass spectrometry.

Two-dimensional liquid chromatography (2D-LC) has in recent years seen an enormous evolution, and with the introduction of commercial instrumentation, the technique is no longer considered a specialist tool. One of the fields where 2D-LC is being widely adopted is in the analysis of biopharmaceuticals, including monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). These molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry (MS) to its limits. Using practical examples from the authors' laboratory complemented with background literature, the possibilities of on-line 2D-LC for the characterization of mAbs and ADCs are presented and discussed.

Unexpected and unusual reactivity of 2-methylimidazolium salts toward aryl-N-sulfonylimines and aryl aldehydes is here reported. Upon reaction with aryl-N-sulfonylimines, the addition product, arylethyl-2-imidazolium-1-tosylamide (3), is formed with moderate to good yields, while upon reaction with aldehydes, the initial addition product (6) observed in NMR and HPLC-MS experimental analysis is postulated by us as an intermediate to the final conversion to carboxylic acids. Studies in the presence and absence of molecular oxygen allow us to conclude that the imidazolium salts is crucial for the oxidation. A detailed mechanistic study was carried out to provide insights regarding this unexpected reactivity.

A high-resolution UHPLC analysis of xanthones present in the extracts of the pericarp of mangosteen (Garcinia mangostana L.) was carried out on an Agilent 1290 Infinity II LC. Excellent resolution and peak capacity (300 calculated at half height) were obtained within a total analysis time, including column reconditioning, of 16.5 minutes. The main xanthone (α-mangostin) was quantified in two extracts. To ensure and evaluate the purity of target compound peaks, a significant increase in peak capacity is required. Therefore, the analysis was repeated using high-resolution sampling 2D-LC with the Agilent 1290 Infinity II 2D-LC solution. The original separation was coupled online to a second-dimension separation for more resolution on a selection of peaks eluting from the first dimension. Diode array detection (DAD) and mass spectrometry (MS) were used for detection and

Antibody-drug conjugates might be the magic bullets referred to by Paul Ehrlich over 100 years ago. Together with a huge therapeutic potential, these molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry to its limits. The use of multiple heart-cutting (mLC-LC) and comprehensive (LC×LC) multidimensional LC in combination with high resolution mass spectrometry for the characterization of the lysine conjugated antibody-drug conjugate ado-trastuzumab emtansine, commercialized as Kadcyla, is presented. By combining protein and peptide measurements, attributes such as drug loading, drug distribution and drug conjugation sites can be assessed in an elegant manner.

On-line two-dimensional liquid chromatography (2D-LC) embracing mainly comprehensive LC (LCxLC) and multiple heart-cutting LC (mLC-LC) offers new opportunities for in-depth characterization of pharmaceuticals. Reversed-phase LC x reversed-phase LC using different column chemistries and mobile phases provides good orthogonality for a wide range of applications related to small molecule drugs. Moreover, hardware configurations and software are now commercially available to perform LCxLC and mLC-LC measurements in a reproducible manner.

Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both R&D and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cation-exchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and (2)D retention time opening interesting perspectives for use in QA/QC testing.

Trastuzumab (marketed as Herceptin), is a monoclonal antibody used in the treatment of HER2 positive metastatic breast cancer. After tryptic digestion, over 100 peptides with varying physicochemical properties present in a wide dynamic concentration range are produced. High peak capacity is required for a detailed peptide mapping of such complex samples. Comprehensive two‐dimensional liquid chromatography (LC×LC) is a very powerful tool to do this. This Application Note demonstrates the use of the Agilent 1290 Infinity 2D‐LC Solution to perform a combination of hydrophilic interaction liquid chromatography and reversed‐phase liquid chromatography. The system is coupled on‐line with an Agilent 6530 Accurate‐Mass Q‐TOF LC/MS System. Analysis of Monoclonal Antibody Digests with the Agilent 1290 Infinity 2D‐LC Solution Part 2: HILIC × RPLC‐MS

Comprehensive two-dimensional liquid chromatography (LC×LC) using the Agilent 1290 Infi nity 2D-LC Solution with DAD and MS detection is applied for the analysis of citrus oils. The developed method uses normal phase liquid chromatography in the fi rst dimension and reversed-phase liquid chromatography in the second dimension. This combination provides good orthogonality and is valuable for profi ling various citrus oils. The method also enables the detection of (potentially) carcinogenic furocoumarins in citrus oil samples. Profi ling of Citrus Oils and Determination of Furocoumarins in Citrus Oils Using the Agilent 1290 Infi nity 2D-LC Solution

The determination of genotoxic impurities (GIs) in drug substances and pharmaceutical products is an emerging topic in pharmaceutical quality control. GIs are intermediates or reactants in the synthetic pathway of a drug substance and should be monitored at ppm (mu g/g drug substance) or even ppb (ng/g) levels. This is several orders of magnitude lower than in classical impurity analysis (0.05% or 500 ppm level) or in residual solvent analysis. Analytical methods for the determination of GIs include gas chromatography (GC) and liquid chromatography (LC), both often combined with mass spectrometry (MS) detection. Some typical examples of GIs trace analysis using GC and LC are presented. The potential of on-line reaction monitoring is also discussed.

Two primary classes of perfluorinated acids are perfluorinated sulfonates: perfluorooctane sulfonate (PFOS) and perfluorinated carboxylic acids: perfluorooctanoate (PFOA, C8 acid). They are generally the most prominent perfluorinated contaminant in biological samples from around the world. In this study reversed phase and chiral columns were evaluated for the separation of isomers of branched perfluoroalkyl compounds using SFC-MS-TOF. On Cellulose tris (4methylbenzoate) stationary phase using MeOH/H2O (5 %)/ NH4HCO2 (10 mM)/CO2 mobile phase fast baseline separation of the isomers of Perfluoro-3-methylheptane sulfonate (P3FOS), Perfluoro-3-methylheptanoic acid (P3FOA), Perfluoro-4-methylheptane sulfonate (P4FOS), Perfluoro-4-methylheptanoic acid (P4FOA), Perfluoro-5-methylheptane sulfonate (P5FOS), and Perfluoro-5-methylheptanoic acid (P5FOA) in isocratic mode was developed with potential for related isomers.

Supercritical fl uid chromatography (SFC) in combination with mass spectrometric detection (MS) is a valuable technique for the determination of glycerol and mono-, di-, and triglyceride impurities at trace levels in biodiesel. The diluted biodiesel sample is analyzed without derivatization on a cyanopropyl silica column. Using MS detection in fast polarity switching positive and negative ion detection mode, detection limits of 0.02% (w/w) were reached for glycerol and all glycerides (mono-, di-, and tri-). Good repeatability was obtained, which allowed the system to be used for qualitative as well as for quantitative analysis, with an analysis time of less than 6 minutes. Authors Maria Rambla Alegre, Melissa N. Dunkle, Gerd Vanhoenacker, Frank David, and Pat Sandra Research Institute for Chromatography Kennedypark 26 B-8500 Kortrijk, Belgium Martin Vollmer Agilent Technologies Inc. Waldbronn, Germany