Alexandre Goyon

Evaluation of size exclusion chromatography columns packed with sub-3 μm particles for the analysis of biopharmaceutical proteins

The aim of this study was to evaluate the practical possibilities and limitations of several recently introduced size exclusion chromatographic (SEC) columns of 150×4.6mm, sub-3μm (Agilent AdvanceBioSEC 2.7μm, Tosoh TSKgel UP-SW3000 2.0μm, Phenomenex Yarra SEC X-150 1.8μm and Waters Acquity BEH200 1.7μm) for the separation of biopharmaceutical proteins. For this purpose, some model proteins were tested, as well as several commercial therapeutic monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs). Calibration curves were drawn to highlight the applicability of these new SEC columns for the separation of mAbs, ADCs and their aggregates, despite some differences in their nominal pore diameter (vary from 150 to 300Å). The kinetic performance (van Deemter curves and kinetic pots) was evaluated. Columns packed with 1.7-2.0μm particles improved the plate count by a factor of 1.5-2 compared to 2.7μm particles, which is in agreement with theoretical expectations. Finally, possible secondary hydrophobic and/or electrostatic interactions between the SEC stationary phases and biopharmaceutical proteins were systematically studied. Significant differences in nonspecific interactions were observed, with hydrophobic interactions generally exerting more influence than electrostatic interactions. The use of a novel bond chemistry with the AdvanceBioSEC column was found highly effective to limit non-specific interactions and pave the way to further improvements for column provider. At the end, the average resolutions achieved on the four sub-3μm SEC columns between monomer and dimer structures were comparable for ten approved mAbs products.

Comprehensive study on the effects of sodium and potassium additives in size exclusion chromatographic separations of protein biopharmaceuticals

HIGHLIGHTSThe impact of mobile phase salt additives on SEC measurements was evaluated.Mobile phase with low salts molarity (<0.2 M) may induce a bias in SEC analysis.Lower ionic interactions obtained with potassium salts compared to sodium salts.Tryptophan fluorescence can discriminate the propensity of mAbs to interact. ABSTRACT To separate proteins solely based on their difference in hydrodynamic volume in size exclusion chromatography (SEC), the ionic strength of the mobile phase has to be increased in order to avoid secondary ionic interactions between proteins and the stationary phase. However, adding salts to the mobile phase can have a serious effect on protein aggregation and can lead to artifacts. In the present study, several monoclonal antibodies (mAbs) and the antibody‐drug conjugate (ADC), trastuzumab emtansine were selected to study the effect of mobile phase salt additive on aggregation measurements. In a first instance, the same aggregation ratios between the dimeric and monomeric forms of ten mAbs approved by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) were obtained with three UHP‐SEC columns. However, SEC analysis using various amounts of NaCl provided surprising results for rituximab, e.g. presence of 0.8% aggregates with a mobile phase containing 0.2 M NaCl, while no aggregates were observed without NaCl in the mobile phase. Despite the absence of monomeric protein adsorption at the surface of the SEC resin, the comparison of sodium‐ and potassium‐based salts demonstrated the superiority of potassium‐based salts to reduce possible secondary electrostatic interactions, mainly between protein dimers and the SEC support as well as to lower protein‐salts interaction. To investigate the effect of mobile phase salt additives on SEC measurements, fluorescence spectroscopy provided insights related to the possible contribution of protein tertiary structure. Indeed, biopharmaceuticals could be classified depending on the exposure of their tryptophan residues to the solvent in order to understand their propensity to interact with the stationary phase or/and to undergo self‐association.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. I – Non-denaturing chromatographic techniques

Size-, charge- and hydrophobicity-related variants of a biopharmaceutical product have to be deeply characterized for batch consistency and for the assessment of immunogenicity and safety effects. Size exclusion chromatography (SEC) and ion exchange chromatography (IEX) are considered as the gold standard for the analysis of high molecular weight species (HMWS) and charge-related variants, respectively. Hydrophobic interaction chromatography (HIC) has drawn renewed attention to monitor the small drug payload distribution in the cysteine-linked antibody-drug conjugates (ADC). These three chromatographic techniques, namely SEC, HIC and IEX, are historical, non-denaturing and robust approaches widely used for the characterization of biopharmaceutical proteins. Despite the broad spectrum of monoclonal antibodies (mAbs) structures, isoelectric points (pIs) and hydrophobicities, generic protocols can be applied to separate their size-, charge- and hydrophobicity-related variants, using the last generation of chromatographic columns and appropriate mobile phase conditions. Straightforward protocols are described in this manuscript with representative chromatograms of ten distinct Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved therapeutic mAb products to illustrate the performance of the SEC, IEX and HIC methods.

Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies

Despite the popularity of therapeutic monoclonal antibodies (mAbs), data relative to their ionic physico-chemical properties are very scarce in the literature. In this work, isoelectric points (pIs) of 23 Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAbs were determined by imaged capillary isoelectric focusing (icIEF), and ranged from 6.1 to 9.4. The obtained values were in good agreement with those calculated by both Vector NTI and MassLynx softwares. icIEF can therefore be considered as a reference technique for such a determination. The relative percentages of acidic and basic variants determined by cation exchange chromatography (CEX) using both salt- and pH-gradients were comprised between 15% and 30% for most mAbs and were in good agreement with each other, whereas generic icIEF seems to overestimate the amount of acidic charge variants in mAb products. To our knowledge, this is the first study focusing on the ionic properties of a wide range of FDA and EMA approved reference mAbs, using both generic chromatographic and electrophoretic methodologies. To illustrate the interest of the study for mAb developability purposes, ionic properties of a clinical mAb candidate (dalotuzumab) were also investigated.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. II – Enzymatic and chemical sample preparation

The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Structural elucidation and quality control (QC) analysis of biopharmaceutics are usually performed at intact, subunit and peptide levels. In this paper, general sample preparation approaches used to attain peptide, subunit and glycan level analysis are overviewed. Protocols are described to perform tryptic proteolysis, IdeS and papain digestion, reduction as well as deglycosylation by PNGase F and EndoS2 enzymes. Both historical and modern sample preparation methods were compared and evaluated using rituximab and trastuzumab, two reference therapeutic mAb products approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA). The described protocols may help analysts to develop sample preparation methods in the field of therapeutic protein analysis.

An Online Four-Dimensional HIC×SEC-IM×MS Methodology for Proof-of-Concept Characterization of Antibody Drug Conjugates

There are currently two main techniques allowing the analytical characterization of interchain cysteine-linked antibody drug conjugates (ADCs) under native conditions, namely, hydrophobic interaction chromatography (HIC) and native mass spectrometry (MS). HIC is a chromatographic technique allowing the evaluation of drug load profile and calculation of average drug-to-antibody ratio (DAR) in quality control laboratories. Native MS offers structural insights into multiple ADC critical quality attributes, thanks to accurate mass measurement. However, both techniques can lead to misinterpretations or incomplete characterization when used as standalone methods. Online coupling of both techniques can thus potentially be of great interest, but the presence of large amounts of nonvolatile salts in HIC mobile phases makes it not easily directly compatible with native MS. Here, we present an innovative multidimensional analytical approach combining comprehensive online two-dimensional (2D)-chromatography that consists of HIC and size-exclusion chromatography (SEC), to ion mobility and mass spectrometry (IM-MS) for performing analytical characterization of ADCs under nondenaturing conditions. This setup enabled comprehensive and streamlined characterization of both native and forced degraded ADC samples. The proposed 4D methodology might be more generally adapted for online all-in-one HIC×SEC-IM×MS analysis of single proteins or analysis of protein complexes in nondenaturing conditions.

The importance of system band broadening in modern size exclusion chromatography

&NA; In the last few years, highly efficient UHP‐SEC columns packed with sub–3 &mgr;m particles were commercialized by several providers. Besides the particle size reduction, the dimensions of modern SEC stationary phases (150 × 4.6 mm) was also modified compared to regular SEC columns (300 × 6 or 300 × 8 mm). Because the analytes are excluded from the pores in SEC, the retention factors are very low, ranging from −1 < k < 0, resulting in very small column band variance. Therefore, the contribution of the system itself to peak variance can become significant under UHP‐SEC conditions. The goal of this study was to evaluate the loss of efficiency observed with three different instruments (regular HPLC, non‐optimized UHPLC and fully optimized UHPLC) offering different system variances. It appears that the new 150 × 4.6 mm, sub–3 &mgr;m SEC columns cannot be employed on a regular HPLC instrument, since the efficiency loss was equal to 60–85%, when analyzing mAb sample. Optimized UHPLC systems having very low extra‐column volumes (typically Vec < 10 &mgr;L) have therefore to be used to properly operate these columns. Due to the instrument contribution to band broadening, the apparent efficiency of SEC columns packed with sub–2 &mgr;m particles can indeed be hampered when using inappropriate system. Considering the extra‐column band broadening contribution of current UHPLC instruments, a further decrease of SEC column dimension is therefore not desired. Highlights150 × 4.6 mm SEC columns cannot be operated on conventional systems.With mAbs, only 15–40% of the column potential is utilized on regular HPLC.HPLC systems also have a huge impact on the apparent elution time.A further decrease of SEC column dimension is not desired.

Characterization of 30 therapeutic antibodies and related products by size exclusion chromatography: Feasibility assessment for future mass spectrometry hyphenation

Despite the popularity of targeted and immune therapies, the number of studies dealing with the quantitation of aggregates for Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAb and related products are still very scarce in literature. In this work, 30 therapeutic proteins including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), Fc-fusion proteins and a bi-specific antibody (bsAb) were investigated using size exclusion chromatography (SEC). Their levels of high molecular weight species (HMWS) were experimentally estimated between 0.1% and 13.1%. Except for blinatumomab, etanercept and pembrolizumab, the HMWS amount for the other antibodies was well below the limit of 5% usually set a specification for therapeutic mAbs in the biopharmaceutical industry. The main chromatographic peak shape of 24 therapeutic antibodies and the NIST mAb [1] was found suitable (0.8<As<1.5) with a generic SEC method involving potassium-based salts mobile phase. Conversely, only acidic therapeutic proteins (pI<7) could be successfully analyzed with a mass spectrometry (MS) compatible mobile phase containing 100mM ammonium acetate. This study aimed to provide HMWS data for 30 therapeutic proteins covering a wide range of physico-chemical properties with molecular weights between 54 and 153kDa, pI values comprised between 6.1 and 9.4 and hydrophobic interaction chromatography (HIC) retention factors ranging from 1.2 to 6.0 for the mAbs.

Extending the limits of size exclusion chromatography: Simultaneous separation of free payloads and related species from antibody drug conjugates and their aggregates

Size exclusion chromatography (SEC) is commonly performed in isocratic conditions to separate partially excluded molecules from the pores of the stationary phase, based on their difference in hydrodynamic volume. In this work, a baseline resolution was obtained between the monomeric antibody drug conjugate (ADC) and high molecular weight species (HMWS). Besides HMWS, small free payloads, linkers and linker-payloads of ADCs, which would not be discriminated solely based on their size (MW < 1.5 kDa), were also separated on the same SEC column by applying sequentially an acetonitrile gradient after the elution of the largest species. Such an approach allowed a simultaneous i) measurement of the HMWS amount under native conditions, and ii) quantitation of the free payloads, within one generic SEC run. For this purpose, a state-of-the-art 150 × 4.6 mm SEC column packed with 2.0 μm particles and 250 Å pore size, was selected to achieve fast separations of the species within 10 min. A second dimension (RPLC) was also developed to further extend the possibility offered by this experimental setup. The SECxRPLC multiple heart cutting mode was operated by using a modern 2D-LC instrument containing twelve 120 μL sampling loops. Repeatabilities (0.01% < RSD < 3.68%) and recoveries (between 82% and 107%) were found to be suitable with both approaches (SEC and SECxRPLC), whereas the LOQs remain similar. Finally, the SEC method was applied for the screening of ADC crude reaction mixtures, whereas the SEC x RPLC method facilitated separating some additional impurities. The streamlined methodology will further support the development and characterization of ADC products.

High-resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

The determination of mAb critical quality attributes (CQA) is crucial for their successful application in health diseases. A generic CZE method was developed for the high‐resolution separation of various mAb charge variants, which are often recognized as important CQA. A dynamic coating of the capillary was obtained with polyethylene oxide (PEO), whereas Bis‐Tris allowed the analysis of mAbs under native conditions at pH 7.0. The effect of PEO and Bis‐Tris concentrations, as well as the nature of the acidic counter ion on the method performance was systematically studied. The %RSD on migration times was below 5% on three different CE instruments using the optimized method. Additional charge variants (in particular acidic variants) were resolved for 10 out of 17 mAbs compared to a reference CZE approach involving the use of ε‐amino‐caproic acid (EACA), triethylenetetramine (TETA), and hydroxypropylmethyl cellulose (HPMC). The amount of basic and acidic charge variants of 17 Food and Drug Administration (FDA) approved mAbs covering a wide range of physico‐chemical properties, e.g., pI between 8.0 and 9.4 and different hydrophobicity, were mainly comprised between 5–15% and 15–30%, respectively. It is noteworthy that applications for the quality control in hospitals as well as for the combination of the immune checkpoint inhibitors nivolumab and ipilimumab were presented.