Ultra-Fast Middle-Up Reversed Phase Liquid Chromatography Analysis of Complex Bispecific Antibodies Obtained in Less Than One Minute.

This work illustrates the benefits and limitations of using ultra-short reversed phase liquid chromatography (RPLC) columns for the characterization of various complex bispecific antibodies after prolonged thermal stress at the middle-up level of analysis. First, we have demonstrated that alternative organic modifiers, such as isopropanol, can be used in RPLC mode without generating excessive pressure, thanks to the prototype 10 × 2.1 mm, 2.7 µm particle column. However, compared to acetonitrile, the selectivity was not improved, at least for the selected biopharmaceutical products. Importantly, very fast separations (sub-1 min) of high quality were systematically obtained for the different samples when using a spectroscopic detector, but a severe loss of performance was observed with mass spectrometry (MS) detection due to dispersion effects. Based on these results, there is a clear need to improve the interfacing between LC and MS (shorter/thinner tubing) to mitigate band broadening.

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products.

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic-inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.

Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins.

The purpose of this work was to study the potential of recently developed ultra-short column hardware for ion exchange chromatography (IEX). Various prototype and commercial columns having lengths of 5, 10, 15, 20 and 50 mm and packed with non-porous 3 µm particles were systematically compared. Both pH and salt gradient modes of elution were evaluated. Similarly, what has been previously reported for reversed phase liquid chromatography (RPLC) mode, an "on-off" retention mechanism was observed in IEX for therapeutic proteins and their fragments (25-150 kDa range). Because of the non-porous nature of the IEX packing material, the column porosity was relatively low (ε = 0.42) and therefore the volumes of ultra-short columns were very small. Based on this observation, it was important to reduce as much as possible all the sources of extra-column volumes (i.e. injection volume, extra-bed volume, detector cell volume and connector tubing volume), to limit peak broadening. With a fully optimized UHPLC system, very fast separations of intact and IdeS digested mAb products were successfully performed in about 1 min using an IEX column with dimensions of 15 × 2.1 mm. This column was selected for high-throughput separations, since it probably offers the best compromise between efficiency and analysis time. For such ultra-fast separations, PEEK tubing was applied to bypass the column oven (column directly connected) to the optical detector via a zero dead volume connection.

The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry.

The goal of the study was to evaluate the possibilities offered by a new generation of metal-free SEC column to perform direct SEC-MS of protein biopharmaceuticals using ammonium acetate as the main mobile phase additive. The prototype metal-free SEC column hardware used in this work was a polyether ether ketone (PEEK) infused stainless steel tube including PEEK frits. This PEEK-lined column provides a fully bioinert and metal-free fluidic path, while maintaining the stability of the metal hardware, and could be a good solution to limit possible undesired interactions between proteins and column wall/frits. This prototype metal-free SEC column was systematically compared with a conventional stainless-steel SEC column hardware packed with the same stationary phase material. Four different mAb products, namely trastuzumab, palivizumab, bevacizumab and NISTmAb, and one antibody drug conjugate (ADC), trastuzumab emtansine, were selected as test samples. It appears that peak symmetry, separation of low molecular weight species (LMWS), and the recovery of high molecular weight species (HMWS) were significantly improved for the different biopharmaceutical products on the metal-free SEC column. It has also been demonstrated that the largest differences between standard and metal-free SEC columns were observed for the most basic mAbs (high pI), which confirms that electrostatic interactions between the mAb and the metallic parts of the column (frits and inlet tube) could be responsible for the issues observed when performing SEC analysis with volatile mobile phase. Finally, it was feasible to perform SEC-MS analysis for a wide range of biopharmaceutical products using volatile mobile phase. Our results also highlight that an inappropriate column could bias the quantification of size variants when using MS-compatible mobile phases. Therefore, metal-free column, such as the PEEK-lined column, should be preferentially selected for SEC-MS analysis.

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies.

This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.

Empirical correction of non-linear pH gradients and a tool for application to protein ion exchange chromatography.

This concept article reports a practical solution to improve the linearity of effluent pH response as observed in pH gradient cation exchange chromatography (CEX). When performing pH gradient CEX, it is not easy to develop buffer systems that will universally provide pH response proportional with the mobile phase (buffer) composition. It is an especially challenging pursuit when exploring MS compatible buffers (e.g. ammonium-acetate, ammonium-carbonate). In addition to "non-proportional" behavior from the mobile phase composition, the chromatographic column itself will sometimes impose an unpredictable impact on the effluent pH. Here, we propose a simple approach based on the on-line measurement of effluent pH response, conversion of pH to mobile phase volume fraction (φ) and then generation of the inverse response function in the time domain. In the end, when setting the inverse function as the gradient program instead of a linear gradient, an improved - ideally linear - pH response can be produced. A simple Excel tool was developed to assist analysts with this correction procedure, and it has been made available by download for public use.

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems.

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.

Aptamer-based immunoaffinity LC-MS using an ultra-short column for rapid attomole level quantitation of intact mAbs.

Quantification of proteins in biofluids has largely involved either traditional ligand binding assays or "bottom-up" mass spectrometry. Recently, top-down mass spectrometry using reversed-phase liquid chromatography (RPLC) paired with high-resolution mass spectrometry (HRMS) has emerged as a promising technique, due to the potential of better identification of post-translational modifications (PTMs), lack of downstream interferences, and less time-consuming sample preparation and analysis times. However, it can be difficult with this approach to robustly obtain high-fidelity MS data, especially when pushing for low limits of detection. To address these issues, we developed a chromatographic device with an optimized form factor and stationary phase to improve protein recovery, while reducing run times. We have observed that by using this device, it is possible to achieve attomole quantitation of mAbs without the addition of carrier proteins and with over three-fold higher throughput than columns employed in previous studies. Moreover, we have devised a novel affinity capture method, based on repurposing a unique aptamer ligand that can give 93% recovery of mAb using only a 2 h incubation. When hyphenated together, these two technologies greatly improve the ability to analyze proteins in complex matrices.

New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies.

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH). These three phases were compared with two commercial RPLC phases. In terms of protein recovery, the new ES-LH stationary phase clearly outperforms the other phases for any type of biopharmaceutical sample, and can already be successfully used at a temperature of only 60°C. In terms of retention, the new ES-LH and C1 materials were the less retentive ones, requiring lower organic solvent in the mobile phase. However, it is important to mention that the stability of C1 phase was critical under acidic, high temperature conditions. Finally, some differences were observed in terms of selectivity, particularly for the ES-LH column. Besides the chemical nature of the stationary phase, it was found that the nature of organic modifier also plays a key role in selectivity.

Negative gradient slope methods to improve the separation of closely eluting proteins.

In the present work, we describe the fundamental and practical advantages of a new strategy to improve the resolution of very closely eluting peaks within therapeutic protein samples. This approach involves the use of multiple isocratic steps, together with the addition of a steep negative gradient segment (with a decrease in mobile phase strength) to "park" a slightly more retained peak somewhere along the column (at a given migration distance), while a slightly less retained compound can be eluted. First, some model calculations were performed to highlight the potential of this innovative approach. For this purpose, the retention parameters (logk0 and S) for two case studies were considered, namely the analysis of a mixture of two therapeutic mAbs (simple to resolve sample) and separation of a therapeutic mAb from its main variant (challenging to resolve sample). The results confirm that the insertion of a negative segment into a multi-isocratic elution program can be a good tool to improve selectivity between critical peak pairs. However, it is also important to keep in mind that this approach only works with large solutes, which more or less follow an "on-off" type elution behavior. Two real applications were successfully developed to illustrate the practical advantage of this new approach, including the separation of a therapeutic mAb from its main variant possessing very close elution behavior, and the separation of a carrier protein from an intact mAb as might be encountered in a quantitative bioanalysis assay. These two examples demonstrate that improved selectivity can be achieved for protein RPLC through the inclusion of a negative gradient slope that selectively bifurcates the elution of two or more peaks of interest.

Therapeutic Fc-fusion proteins: Current analytical strategies.

Fc-Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc-Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps. Due to their highly diverse structures, the analytical characterization of Fc-Fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. This can be explained, for example, by the presence of numerous sialic acids, leading to high diversity in terms of isoelectric points and complex glycosylation profiles including multiple N- and O-linked glycosylation sites. In this review, we highlight the wide range of analytical strategies used to fully characterize Fc-fusion proteins. We also present case studies on the structural assessment of all commercially available Fc-fusion proteins, based on the features and critical quality attributes of their ligand-binding domains.

Use of Ultra-short Columns for Therapeutic Protein Separations, Part 2: Designing the Optimal Column Dimension for Reversed-Phase Liquid Chromatography.

In the first part of the series, it was demonstrated that very fast (<30 s) separations of therapeutic protein species are feasible using ultra-short (5 × 2.1 mm) columns. In the second part, our purpose was to find the appropriate column length; therefore, a systematic study was performed using various custom-made prototype reversed-phase liquid chromatography (RPLC) columns ranging from 2 to 50 mm lengths. It was found that on a low dispersion ultrahigh-pressure liquid chromatography instrument, columns between 10 and 20 mm were most effective when made with 2.1 mm i.d. tubing. However, with the same LC instrument, 3 mm i.d. columns as short as ∼5 to 10 mm could be effectively used. In both cases, it has been found to be best to keep injection volumes below 0.6 µL, which presents a potential limit to further decreasing column length, given the current capabilities of autosampler instrumentation. The additional volume of the column hardware outside of the packed bed (extra-bed volume) of very small columns is also a limiting factor to decrease the column length. For columns shorter than 10 mm, columns' extra-bed volume was seen to make considerable contributions to band broadening. However, the use of ultra-short columns seemed to be a very useful approach for RPLC of large proteins (>25 kDa) and could also work well for ∼12 kDa as the lowest limit of molecular mass. In summary, a renewed interest in the use of ultra-short columns is warranted, and additional method development will be to the benefit of the biopharmaceutical industry as there is an ever-increasing demand for faster, yet accurate assays (e.g., high-throughput screening) of proteins.

Determination of size variants by CE-SDS for approved therapeutic antibodies: Key implications of subclasses and light chain specificities.

In the present work, a generic non-reducing capillary electrophoresis sodium dodecyl sulphate (nrCE-SDS) method was tested for a wide range of 26 FDA and EMA approved monoclonal antibodies (mAbs) and 2 antibody drug conjugates (ADCs) as well as for the NISTmab, in a QC environment (e.g. testing quality requirements for batch manufacturing or batch release). This method allows obtaining rapidly and accurately the amount of size variants in drug products within about 40 min and may be used for batch release and consistency as well as for stability and shelf-life. First, the method repeatability was found to be excellent in terms of relative migration times and relative proportions of fragments (average RSD values of 0.3 and 0.2 %, on relative migration times and relative percentages of fragments, respectively), thanks to the addition of an internal standard. A panel of chimeric, humanized and human mAbs were tested, belonging to different subclasses (heavy chain gamma 1, 2, 2/4 and 4) and light chain types (κ or λ) and produced in different cell lines (CHO, NS0 and SP2/0). For all these biopharmaceutical products, the amount of H2L2 species was comprised between 90.9 % and 97.7 %, except for the two mAbs belonging to the IgG1λ subclass, namely avelumab and belimumab, which were prone to partial reduction during the sample preparation at 70 °C. Based on the CE-SDS results obtained for a diverse panel of therapeutic antibodies investigated in this study, and covering a wide range of structural and physico-chemical properties, a specification on the intact antibody content (H2L2) greater than 90 % can be achieved.

Practical considerations on the particle size and permeability of ion-exchange columns applied to biopharmaceutical separations.

The goal of this study was to better understand the possibilities and limitations of modern cation exchange chromatography (CEX) columns for the separation of protein biopharmaceuticals (typically mAbs and related products). Several commercial and research columns consisting of a non-porous polymeric core particle with a thin hydrophilic coating and grafted ion-exchanger sulfonate groups, were compared. The impact of particle size, porosity and packing pressure on the separation of therapeutic proteins was evaluated in a systematic way. First, it was shown that the porosity of modern CEX columns depends on the applied conditions, and lower apparent porosity as well as increased column pressures were observed when using low ionic strength mobile phase (less than 0.01 M NaCl), due to swelling. Column pressure seemed to be dependent on the 1/dp3 to 1/dp5 relationships with particle size, depending on whether 0.3 M NaCl or pure water was used as mobile phase, respectively. Using 5 cm long columns packed with 2 or 2.5 µm particles could easily result in higher than 1000 bar pressure drops when the mobile phase ionic strength is low. Therefore, it is recommended that particle size not be decreased to below 2.5 µm so that technologies can remain compatible with the current state of ultra-high pressure (UHPLC) instrumentation. This recommendation is underscored by the fact that a decrease in particle size does not produce improved separations, since the particles are non-porous (no intra-particle diffusion nor resistance to mass transfer) and that large solutes follow an on-off (bind and elute) type retention mechanism. The only advantage of CEX columns packed with small particles is that they can provide more specific surface area per unit length of column, and thus facilitate higher throughput methods. In conclusion, it appears that there is no need to further decrease the particle size in CEX since decreasing their particle size may result in more drawbacks than benefits.

Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 2: Evaluation of recent stationary phases.

In this second part of the series, recently commercialized cation exchanger stationary phases were systematically investigated for their capabilities to separate therapeutic monoclonal antibodies. It was demonstrated that the different combinations of stationary and mobile phases result in diverse retention, selectivity and efficiency. Hence, the whole phase system (combination of stationary and mobile phase) should be considered when developing a method. In addition, retention behavior is mAb dependent and should be individually optimized. Another interesting observation was that in cation exchange chromatographic separations of large proteins, the particle size of the columns probably impacts retention rather than efficiency, due to the non-porous particle structure - and therefore the higher specific surface area of smaller particles -. Particle size influences the specific surface area and total porosity. Therefore, columns packed with larger particles showed lower retention (when the ion exchanger group was the same e.g. strong exchanger sulfonic group) while no link was observed between efficiency and particle size. The retention, efficiency and selectivity of the studied columns were quite different and strongly dependent on the elution mode (i.e. salt gradient, pH gradient or combined salt/pH gradient mode). The columns can be considered to be complementary, suggesting that it is useful to have more than one type of column on hand while developing new charge variant assays. Moreover, this work shows that it is especially attractive to make use of short, narrow bore ion exchange columns that offer the possibility to perform 4-6 min long separations of both intact and partially digested antibodies.

Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation.

Cation exchange chromatography (CEX) of therapeutic monoclonal antibodies is generally performed with either salt gradient (MES buffer + NaCl) or using commercial pH gradient buffer. The goal of this study was to find out some alternative buffer systems for CEX separation of mAbs, which may offer alternative selectivity, while maintaining similar peak shapes. Among the new buffers that were tested, (N-morpholino)ethanesulfonic acid (MES) / 1,3-diamino-2-propanol (DAP), and citric acid / 2-(cyclohexylamino)ethanesulfonic acid (CHES) systems were particularly promising, especially when combining them with a moderate salt gradient of NaCl. This two buffer system provides an equivalent or slightly better separation than the standard, mobile phases for therapeutic mAbs. It was also demonstrated that working with salt-mediated pH gradients, allows to extend the possibilities in method development, since the concentration of salt in the mobile phase has a significant impact on selectivity. Using HPLC modeling software (Drylab), it was possible to successfully develop CEX methods for authentic mAb samples within only 6 h, by optimizing the gradient steepness and salt concentration in the B eluent.