Optimizing Messenger RNA Analysis Using Ultra-Wide Pore Size Exclusion Chromatography Columns.

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl2) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.

Fast and efficient size exclusion chromatography of adeno associated viral vectors with 2.5 micrometer particle low adsorption columns.

More and more transformative gene therapies (GTx) are reaching commercialization stage and many of them use Adeno Associated Viruses (AAVs) as their vector. Being larger than therapeutic antibodies, their size variant analysis poses an analytical challenge that must be addressed to speed up the development processes. Size exclusion chromatography (SEC) can provide critical information on the quality and purity of the product, but its full potential is not yet utilized by currently applied columns that are (i) packed with relatively large particles, (ii) prepared exclusively in large formats and (iii) built using metal hardware that is prone to secondary interactions. In this paper, we investigate the use of state-of-the-art sub-3 µm particles to address existing limitations. A prototype 2.5 µm column was found to deliver superior kinetic efficiency, significant reduction in run times and increased resolution of separations. No evidence for shear or sample sieving effects were found during comparisons with conventional 5 µm columns. Moreover, use of low adsorption hardware enabled the application of a wide range of mobile phase conditions and a chance to apply a more robust platform method for several AAV serotypes. The resulting method was tested for its reproducibility as well as utility for critical quality attribute assays, including multiangle light scattering based (MALS) measurements of size and molar mass. Thus, a new tool for higher resolution, higher throughput size variant analysis of AAVs has been described.

Pressure-Enhanced Liquid Chromatography, a Proof of Concept: Tuning Selectivity with Pressure Changes and Gradients.

Many chromatographers have observed that the operating pressure can dramatically change the chromatographic retention of solutes. Small molecules show observables changes, yet even more sizable effects are encountered with large biomolecules. With this work, we have explored the use of pressure as a method development parameter to alter the reversed-phase selectivity of peptide and protein separations. An apparatus for the facile manipulation of column pressure was assembled through a two-pump system and postcolumn flow restriction. The primary pump provided an eluent flow through the column, while the secondary pump provided a pressure-modulating flow at a tee junction after the column but ahead of a flow restrictor. Using this setup, we were able to quickly program various constant pressure changes and even pressure gradients. It was reconfirmed that pressure changes impact the retention of large molecules to a much greater degree than small molecules, making it especially interesting to consider the use of pressure to selectively separate solutes of different sizes. The addition of pressure to bring the column operating pressure beyond 500 bar was enough to change the elution order of insulin (a peptide hormone) and cytochrome C (a small serum protein). Moreover, with the proposed setup, it was possible to combine eluent and pressure gradients in the same analytical run. This advanced technique was applied to improve the separation of insulin from one of its forced degradation impurities. We have referred to this method as pressure-enhanced liquid chromatography and believe that it can offer unseen selectivity, starting with peptide and protein reversed-phase separations.

Development of an innovative salt-mediated pH gradient cation exchange chromatography method for the characterization of therapeutic antibodies.

The successful application of monoclonal antibodies (mAb) in oncology and autoimmune diseases paved the way for the development of therapeutic antibodies with a wider range of structural and physico-chemical properties. A pH-gradient combining 2-(N-morpholino)ethanesulfonic (MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers and mediated with potassium chloride was developed to sufficiently retain acidic mAbs (pI < 7) in cation exchange chromatography (CEX), while keeping suitable separation performance for basic mAbs (pI > 7). Firstly, the MES and HEPES buffers were individually evaluated in their useful pH range by applying a salt gradient. The performance of a salt-mediated pH gradient combining the MES and HEPES buffers was then compared to a commercial pH gradient kit. The developed conditions were found superior to the salt-gradient approaches and provided a useful alternative to commercial pH gradient kits. In this study, the developed conditions were applied to separate a bispecific antibody (BsAb) from its two parental mAbs.

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.

A generic workflow for the characterization of therapeutic monoclonal antibodies-application to daratumumab.

In the present analytical workflow, chromatographic methods have been developed and hyphenated to mass spectrometry (MS) for the characterization of protein size, charge, hydrophobic, and hydrophilic variants of daratumumab. Multiple critical quality attributes (CQAs) were characterized in forced degraded daratumumab sample, using size exclusion, ion exchange (IEX), and hydrophobic interaction (HIC) chromatography coupled to fluorescence detection for relative quantification and fractionation. Mass assignment was performed by using a fast, non-denaturing and universal size exclusion chromatography (SEC) method prior to native MS analysis of the collected fractions (off-line approach). This allowed the identification of N-terminal lysine clipping, and the extent of glycation and oxidation at intact protein level. Finally, middle-up analysis of daratumumab was performed using reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography coupled to MS to obtain a comprehensive overview of all PTMs after the forced stressed conditions and a fine characterization of the glycosylation profile. Conveniently, the presented workflow maintains the established golden standard non-denaturing chromatography techniques and additionally introduces a straightforward and automated desalting procedure prior to MS analysis. Therefore, it is expected that the off-line coupling of SEC, IEX, and HIC to SEC-MS has great potential to be implemented in routine characterization of mAbs. Graphical abstract ᅟ.

Is hydrophobic interaction chromatography the most suitable technique to characterize site-specific antibody-drug conjugates?

Antibody drug conjugates (ADCs) belong to the fastest growing class of therapeutic agents for cancer therapy. In preclinical and clinical studies, there is a significant number of site-specific ADCs (also known as third generation ADCs), which are more homogeneous than their previous generations. These new ADC formats, in which the inter-chain disulphide bridges (hinge cysteines) are not reduced, also need to be deeply characterized. In particular, various quality attributes (QAs) have to be determined, such as free antibody level, average drug to antibody ratio (DAR) and drug distribution. In this contribution, a non-commercial site-specific conjugated ADC has been analyzed by RPLC. Our results demonstrated that RPLC has a huge potential to determine QAs and can replace the historically used HIC methods as RPLC provides better separation quality for such type of ADCs. Site-specific ADCs can be analyzed in RPLC at intact level without the need for sample preparation. A further advantage of RPLC is that it enables the direct coupling to MS and thus allows the fine identification of all eluting species.

Computer-assisted UHPLC-MS method development and optimization for the determination of 24 antineoplastic drugs used in hospital pharmacy.

This study reports the use of retention modeling software for the successful method development of 24 injectable antineoplastic agents. Firstly, a generic screening of several stationary and mobile phases (using various organic modifiers and pH) was achieved. Then, an optimization procedure of mobile phase temperature, gradient profile and mobile phase binary composition was conducted through only 28 real experiments using retention modeling software for data treatment. Finally, the optimized separation was achieved with a mobile phase consisting in 10 mM acetic acid at pH 5.1 (A) and acetonitrile (B). A Waters CORTECS® T3 column (100 × 2.1 mm, 1.6 µm) operated at 25 °C with a gradient time of 17.5 min (0-51%B) at a flow rate of 0.4 mL/min was used. The prediction offered by the retention model was found to be highly reliable, with an average error lower than 1%. A robustness testing step was also assessed from a virtual experimental design. Success rate and regression coefficient were evaluated without the need to perform any real experiment. The developed LC-MS method was successfully applied to the analysis of pharmaceutical formulations and wiping samples from working environment.

Apparent efficiency of serially coupled columns in isocratic and gradient elution modes.

The goal of this work was to understand the variation of apparent efficiency when serially coupling columns with identical stationary phase chemistries, but with differences in their kinetic performance. For this purpose, a mathematical treatment was developed both for isocratic and gradient modes to assess the change in plate numbers and peak widths when coupling arbitrary several columns. To validate the theory, experiments were also carried out using various mixtures of compounds, on columns packed with different particle sizes, to mimic highly efficient (new, not used) and poorly efficient columns (used one with many injections). Excellent agreement was found between measured and calculated peak widths. The average error in prediction was about 5% (which may be explained by the additional volume of the coupling tubes). In isocratic mode, the plate numbers are not additive when the coupled columns possess different efficiencies, and a limiting plate count value can be calculated depending on the efficiency and length of the individual columns. Theoretical efficiency limit can also be determined assuming one column in the row with infinite efficiency. In gradient elution mode, the columns' order has a role (non-symmetrical system). When the last column has high enough efficiency, the gradient band compression effect may outperform the competing band broadening caused by dispersive and diffusive processes (peak sharpening). Therefore, in gradient mode, the columns should generally be sequentially placed according to their increasing efficiency.

Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry.

Brentuximab vedotin (Adcetris) is a cysteine-linked antibody-drug conjugate (ADC) used in the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). In this study, the drug payload and glycan modifications of this ADC were simultaneously characterized using a unique LC-MS middle-up analysis, involving hydrophilic interaction chromatography (HILIC). This work demonstrates that HILIC is an effective and complementary analytical technique to reversed phase liquid chromatography (RPLC) for subunit-level characterization of immuno-conjugates.

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates.

Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics. ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DARav), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach.

Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

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.

Development of a fast workflow to screen the charge variants of therapeutic antibodies.

Chemical or enzymatic modifications of therapeutic monoclonal antibodies (mAbs) having high risk towards safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic mAbs process development, a variety of analytical techniques have to be used for the thorough characterization and quantitative monitoring of CQAs. This paper describes the development of a rapid analytical platform to assess and rank charge variants of mAbs. The workflow is first based on a cation exchange chromatography (CEX) comparative analysis of intact IgGs versus F(ab)'2 and Fc sub-domains generated by IdeS digestion. This analytical procedure was validated with FDA and EMA approved mAbs. Then, functional assays and peptide mapping can be performed in a second instance. This approach can be used during the early stage of drug research and development to screen lead molecules and select optimized candidates (best clone, best formulation) which could be "easily" developed (OptimAbs).

Separation of antibody drug conjugate species by RPLC: A generic method development approach.

This study reports the use of modelling software for the successful method development of IgG1 cysteine conjugated antibody drug conjugate (ADC) in RPLC. The goal of such a method is to be able to calculate the average drug to antibody ratio (DAR) of and ADC product. A generic method development strategy was proposed including the optimization of mobile phase temperature, gradient profile and mobile phase ternary composition. For the first time, a 3D retention modelling was presented for large therapeutic protein. Based on a limited number of preliminary experiments, a fast and efficient separation of the DAR species of a commercial ADC sample, namely brentuximab vedotin, was achieved. The prediction offered by the retention model was found to be highly reliable, with an average error of retention time prediction always lower than 0.5% using a 2D or 3D retention models. For routine purpose, four to six initial experiments were required to build the 2D retention models, while 12 experiments were recommended to create the 3D model. At the end, RPLC can therefore be considered as a good method for estimating the average DAR of an ADC, based on the observed peak area ratios of RPLC chromatogram of the reduced ADC sample.

Optimization of non-linear gradient in hydrophobic interaction chromatography for the analytical characterization of antibody-drug conjugates.

The goal of this work was to evaluate the potential of non-linear gradients in hydrophobic interaction chromatography (HIC), to improve the separation between the different homologous species (drug-to-antibody, DAR) of commercial antibody-drug conjugates (ADC). The selectivities between Brentuximab Vedotin species were measured using three different gradient profiles, namely linear, power function based and logarithmic ones. The logarithmic gradient provides the most equidistant retention distribution for the DAR species and offers the best overall separation of cysteine linked ADC in HIC. Another important advantage of the logarithmic gradient, is its peak focusing effect for the DAR0 species, which is particularly useful to improve the quantitation limit of DAR0. Finally, the logarithmic behavior of DAR species of ADC in HIC was modelled using two different approaches, based on i) the linear solvent strength theory (LSS) and two scouting linear gradients and ii) a new derived equation and two logarithmic scouting gradients. In both cases, the retention predictions were excellent and systematically below 3% compared to the experimental values.

Impact of organic modifier and temperature on protein denaturation in hydrophobic interaction chromatography.

The goal of this study was to better understand the chromatographic conditions in which monoclonal antibodies (mAbs) of broad hydrophobicity scale and a cysteine conjugated antibody-drug conjugate (ADCs), namely brentuximab-vedotin, could denaturate. For this purpose, some experiments were carried out in HIC conditions using various organic modifier in natures and proportions, different mobile phase temperatures and also different pHs. Indeed, improper analytical conditions in hydrophobic interaction chromatography (HIC) may create reversed-phase (RP) like harsh conditions and therefore protein denaturation. In terms of organic solvents, acetonitrile (ACN) and isopropanol (IPA) were tested with proportions ranging from 0 to 40%. It appeared that IPA was a less denaturating solvent than ACN, but should be used in a reasonable range (10-15%). Temperature should also be kept reasonable (below 40°C), to limit denaturation under HIC conditions. However, the combined increase of temperature and organic content induced denaturation of protein biopharmaceuticals in all cases. Indeed, above 30-40°C and 10-15% organic modifier in mobile phase B, heavy chain (HC) and light chain (LC) fragments dissociated. Mobile phase pH was also particularly critical and denaturation was significant even under moderately acidic conditions (pH of 5.4). Today, HIC is widely used for measuring drug-to-antibody ratio (DAR) of ADCs, which is a critical quality attribute of such samples. Here, we demonstrated that the estimation of average DAR can be dependent on the amount of organic modifier in the mobile phase under HIC conditions, due to the better recovery of the most hydrophobic proteins in presence of organic solvent (IPA). So, special care should be taken when measuring the average DAR of ADCs in HIC.

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. II- Identification of sub-units for the characterization of even and odd load drug species.

This paper is the second part of a two-part series dedicated to the development of an on-line comprehensive HICxRPLC-UV/MS method for the characterization of a commercial inter-chain cysteine-linked ADC (brentuximab vedotin, Adcetris(®)). The first part focused on the optimization of the chromatographic conditions. In the second part of this series of papers, the structural characterization of the Brentuximab Vedotin was extensively discussed. With the combination of HIC and RPLC-MS data, the average DAR was easily measured in HIC and, at the same time, the predominant positional isomers were identified in RPLC-MS in one single injection. It was also demonstrated that the retention data obtained in the first and second dimensions was particularly useful to assist ADC characterization through the identification of sub-units. Using this methodology, the presence of odd DARs (1, 3 and 5) and their relative abundance was assessed by a systematic evaluation of HIC x RPLC-UV/MS data for both commercial and stressed ADC samples. Finally, once the exhaustive characterization of ADC was completed, MS could be conveniently replaced by UV detection to quickly assess the conformity of different ADCs batches.

Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatoraphy, part 2: Optimization of the phase system.

The goal of this second part was (i) to evaluate the performance of commercially available HIC columns and (ii) to develop a fast and automated "phase system" (i.e. stationary phase and salt type) optimization procedure for the analytical characterization of protein biopharmaceuticals. For this purpose, various therapeutic mAbs (denosumab, palivizumab, pertuzumab, rituximab and bevacizumab) and a cysteine linked ADC (brentuximab-vedotin) were selected as model substances. Several HIC column chemistries (butyl, ether and alkylamide) from different providers were evaluated in four different buffer systems (sodium acetate, sodium chloride, ammonium acetate and ammonium sulfate). As stationary phases, the historical TSK gel Butyl NPR phase and the brand new Thermo MAbPac HIC-10 were found to be the most versatile ones in terms of hydrophobicity, peak capacity and achievable selectivity. As salt types, ammonium sulfate and sodium acetate were found to be particularly well adapted for the analytical characterization of mAbs and ADCs, but it is important to keep in mind that a concentration 2 to 3-times higher of sodium acetate versus ammonium sulfate is required to achieve a similar retention in HIC. After selection of the most appropriate phase systems, the optimization of the separation can be carried out by computer assisted retention modeling in a high throughput manner.

Practical method development for the separation of monoclonal antibodies and antibody-drug-conjugate species in hydrophobic interaction chromatography, part 1: optimization of the mobile phase.

The goal of this work is to provide some recommendations for method development in HIC using monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) as model drug candidates. The effects of gradient steepness, mobile phase pH, salt concentration and type, as well as organic modifier were evaluated for tuning selectivity and retention in HIC. Except the nature of the stationary phase, which was not discussed in this study, the most important parameter for modifying selectivity was the gradient steepness. The addition of organic solvent (up to 15% isopropanol) in the mobile phase was also found to be useful for mAbs analysis, since it could provide some changes in elution order, in some cases. On the contrary, isopropanol was not beneficial with ADCs, since the most hydrophobic DAR species (DAR6 and DAR8) cannot be eluted from the stationary phase under these conditions. This study also illustrates the possibility to perform HIC method development using optimization software, such as Drylab. The optimum conditions suggested by the software were tested using therapeutic mAbs and commercial cysteine linked ADC (brentuximab-vedotin) and the average retention time errors between predicted and experimental retention times were ∼ 1%.