Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept.

Due to the particular elution mechanism observed with large solutes (e.g., proteins) in liquid chromatography, column length has less impact in controlling their retention compared to small solutes. Moreover, long columns-in theory-just broaden the peaks of large solutes since a great part of the column only acts as void (extra) volume. Such a theory suggests that using very short columns should result in comparable separation quality versus using long columns and make it possible to perform faster (high-throughput) analyses. Therefore, the elution behavior of various therapeutic monoclonal antibodies and their fragments (25-150 kDa) has been investigated using modern instrumentation and column formats. The possibilities offered by narrow-bore columns packed with state-of-the-art 2.7 µm superficially porous particles with 5, 50, 100, and 150 mm lengths have been compared. In particular, the impact of gradient steepness and column length on separation efficiency was evaluated. Using 5 mm × 2.1 mm columns, it has become possible to separate antibody fragments and antibody-drug conjugate species in less than 30 s. Such fast methods can be very useful for high-throughput screening purposes in biopharmaceutical industries.

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.

Proof of Concept To Achieve Infinite Selectivity for the Chromatographic Separation of Therapeutic Proteins.

Reversed phase liquid chromatography (RPLC) is a widely used technique for the analytical characterization of proteins biopharmaceuticals, due to its inherent compatibility with mass spectrometry (MS). However, this chromatographic mode suffers from limited selectivity when analyzing large molecules. Due to the on/off mechanism observed with large solutes in RPLC (S values were higher than 100 for intact proteins), we have developed a new analytical strategy based on the use of multi-isocratic elution mode, to achieve arbitrary selectivity for protein variants. In this work, it has been demonstrated that the combination of multi-isocratic steps and very short steep gradient segments at solute elution allows one to set the selectivity as desired, while maintaining sharp peaks due to significant band compression effects. The strategy was successfully applied to the analysis of intact and subunits of monoclonal antibodies (mAbs) as well as antibody-drug conjugates (ADCs), illustrating the possibility to achieve a uniform peak distribution (equidistant band spacing) and much higher resolution than in the case of common linear, multilinear, or nonlinear gradients.

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.

Development of a LC-MS/MS method for the determination of isomeric glutamyl peptides in food ingredients.

A liquid chromatography with tandem mass spectrometry method was developed for the determination of 27 glutamyl di- and tripeptides in food ingredients. Such compounds are of importance for the food industry, as they can modulate the perception of basic tastes (sweet, salty, and umami). Due to their high polarity, the hydrophilic interaction chromatography mode was selected to have sufficient retention on the column and the best separation was obtained on an amide hybrid silica stationary phase packed with 1.7 µm particles. Thorough optimization of the mobile phase was performed as the start-composition had to be free of ammonium to avoid on-column cis-trans isomerization of the first eluting proline dipeptide. A baseline separation was achieved for all α and γ isomers whereas only a partial resolution was obtained for γ-Glu-Leu and γ-Glu-Ile, for which only the position of a methyl group differs. A fast sample preparation, based on successive dilutions, was performed before injection into the liquid chromatography with tandem mass spectrometry system. The developed method was then applied for the semi-quantification of glutamyl di- and tri-peptides in four different food ingredients. The methodology will further support the optimization of production processes to select the conditions for which the peptide concentrations would be the highest.

Adding a new separation dimension to MS and LC-MS: What is the utility of ion mobility spectrometry?

Ion mobility spectrometry is an analytical technique known for more than 100 years, which entails separating ions in the gas phase based on their size, shape, and charge. While ion mobility spectrometry alone can be useful for some applications (mostly security analysis for detecting certain classes of narcotics and explosives), it becomes even more powerful in combination with mass spectrometry and high-performance liquid chromatography. Indeed, the limited resolving power of ion mobility spectrometry alone can be tackled when combining this analytical strategy with mass spectrometry or liquid chromatography with mass spectrometry. Over the last few years, the hyphenation of ion mobility spectrometry to mass spectrometry or liquid chromatography with mass spectrometry has attracted more and more interest, with significant progresses in both technical advances and pioneering applications. This review describes the theoretical background, available technologies, and future capabilities of these techniques. It also highlights a wide range of applications, from small molecules (natural products, metabolites, glycans, lipids) to large biomolecules (proteins, protein complexes, biopharmaceuticals, oligonucleotides).

Computer assisted liquid chromatographic method development for the separation of therapeutic proteins.

This review summarizes the use of computer assisted liquid chromatographic method development for the analytical characterization of protein biopharmaceuticals. Several modes of chromatography including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and some perspectives are discussed. For all these chromatographic modes, the most important variables for tuning retention and selectivity are exposed. Then, the retention models that were applied in the literature in RPLC, IEX and HIC are described and critically discussed. Finally, some representative examples of separation of therapeutic proteins and mAbs are shown, to illustrate the possibilities offered by the retention modeling approach. At the end, the reliability of the models was excellent, whatever the chromatographic mode, and the retention time prediction errors were systematically below 2%. In addition, a significant amount of time can be saved during method development and robustness testing.

Evaluation of thermally pretreated silica stationary phases under hydrophilic interaction chromatography conditions.

Three novel hydrophilic interaction chromatography columns packed with bare silica 2.6 µm superficially porous particles were evaluated. These stationary phases undergo a different pretreatment temperature (400, 525, and 900°C) that might influence their kinetic performance and thermodynamic properties. In the first instance, we demonstrated that the performance of these columns was inferior to the commercial ones in the low plate count range (10 000 plates), but was more favorable for N values beyond 40 000 plates. Thanks to its high permeability and reasonable flow resistance (φ = 695), together with a minimum reduced heights equivalent to a theoretical plate value of only 2.4, the stationary phase pretreated at 400°C was particularly attractive for N > 70 000 plates with a remarkably low impedance value (E = 2488). In a second step, the impact of pretreatment temperature was evaluated using two mixtures of polar substances, namely nucleobases and derivatives, as well as nicotine and derivatives. Retentions and selectivities achieved on the tested stationary phases were appropriate, but selectivity differences were minor when modifying pretreatment temperature from 400 to 525°C. When we increased the pretreatment temperature up to 900°C, the surface chemistry was more seriously modified. Finally, the columns presented a good stability even at high temperature (70°C), especially for the phases pretreated at 400 and 525°C.

Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies.

Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ~250 Å and macropores of ~ 1.1 µm, a column packed with 3.6 µm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 µm particles having pore size of ~300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene-divinyl benzene) organic monolithic column, with a macropore size of approximately 1 µm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 µm and fully porous 1.7 µm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 µm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 µm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.

Comparison of various silica-based monoliths for the analysis of large biomolecules.

In the present study, three types of silica-based monoliths, i.e. the first and second generations of commercial silica monolithic columns and a wide-pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first-generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa) but very close with smaller ones (1.3-5.8 kDa). In contrast, the wide-pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide-pore prototype material, which led to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica-based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa).

Evaluation of a sheathless nanospray interface based on a porous tip sprayer for CE-ESI-MS coupling.

The hyphenation of capillary electrophoresis (CE) with mass spectrometry (MS) is a powerful method to obtain high efficient, sensitive, and selective analyses. The successful coupling with electrospray ionization (ESI) source requires closed electric circuits for both the CE separation and the ESI processes. A wide range of interfaces has been proposed to satisfy this requirement. Among them, the new high sensitivity porous sprayer based on a porous tip achieves the electric connection by inserting the capillary outlet made of a porous material into an ESI needle filled with a conductive liquid and independently grounded. This device is compatible with the minute flow rates exhibited in CE and therefore makes possible the use of a nano-electrospray behavior. In this work, this interface was evaluated for hyphenating a CE with a single quadrupole MS instrument for low molecular weight analytes. Investigations aimed at highlighting the most influent parameters thanks to a design of experiments, reaching the best performance in terms of sensitivity and stability. MS signal intensities of various pharmaceutical compounds (e.g. amphetamines, ß-blockers) emphasized high sensitivity and efficiency, while repeatability, expressed as relative standard deviation of corrected heights and areas, was suitable for quantitative purposes (<5%).

Characterization of drug-protein interactions by capillary electrophoresis hyphenated to mass spectrometry.

The demand for analytical techniques to evaluate and measure drug-plasma protein interactions continues to increase. The binding of drugs to plasma proteins is an important parameter to determine during the drug development process because it impacts both pharmacokinetics and pharmacodynamics. Among the numerous methods that have been proposed to perform such studies, CE in frontal analysis mode (CE/FA) is attractive because it consumes a relatively low amount of samples, is fast, and enables analyses under near-physiological conditions. Most CE/FA applications have been performed with UV detection and often lack sensitivity. In this study, CE was hyphenated to MS to enhance the sensitivity of the method and to evaluate strong drug-plasma protein interactions. To adapt the previously developed CE/FA-UV method to CE/FA-MS, different parameters were considered, such as the buffer composition, the rinsing step, and the ESI and MS parameters. The most critical aspect involved obtaining stable MS signals. Good results were achieved due to careful optimization of the ESI and MS parameters, among which the sheath liquid composition appeared to be the most significant. Interactions between six drugs and α(1) -acid glycoprotein and three drugs and BSA, including basic, neutral, and acidic drugs, were measured with the optimized CE/FA-MS method. The obtained affinity constants ranged from 1·10(-4) M(-1) to 2·10(-5) M(-1) and were in good agreement with the results that were obtained by CE/FA-UV and equilibrium dialysis.

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.

Impact of mobile phase temperature on recovery and stability of monoclonal antibodies using recent reversed-phase stationary phases.

Recent reversed-phase wide-pore stationary phases were evaluated for the separation of intact monoclonal antibodies and their fragments. Two types of stationary phases were tested: Phenomenex Aeris Widepore, with 3.6 µm core-shell particles and Waters Acquity BEH300 with 1.7 µm fully porous particles. A systematic investigation was carried out using model IgG1 and IgG2 antibodies, namely rituximab, panitumumab, and bevacizumab. It appeared that adsorption of these antibodies on the stationary phase was significantly higher compared to proteins of equivalent size. The adsorption was particularly important for the intact antibodies of 150 kDa and for the largest fragments of 50 to 100 kDa (i.e., heavy chain, -fraction of antigene-binding). The present study demonstrated an obvious relationship between adsorption phenomenon and the unwanted strong secondary interactions (ionic and hydrogen bond) of the stationary phase. Thus, adsorption was more pronounced on the Aeris column because of the stronger ion exchange contribution of this stationary phase. Using C4 phase instead of C18 at 50-70°C, there is a slight reduction (5-20%) in adsorption. Two solutions were proposed to decrease the strength of secondary interactions and thus resolve (or at least diminish) adsorption issue. First, increasing mobile phase temperature up to 80-90°C appeared as a promising solution. However, temperature should be used with caution as it can partially damage large biomolecules. A compromise between residence time and temperature should be found. Second, it is recommended to add a small amount of an ancillary solvent, such as n-butanol to the mobile phase. Indeed, the hydroxyl group of n-butanol probably interacts with water adsorbed on the residual silanol groups "to shield" silanols.

New insights in pharmaceutical analysis.

The research unit of pharmaceutical analytical chemistry (PAC) has been active in the field of separation sciences for many years. Liquid chromatography (LC) and its latest improvements such as ultra-high performance chromatography (UHPLC) and supercritical fluid chromatography (SFC) are deeply and thoroughly studied, from a fundamental viewpoint to its various application capabilities. Electro-driven separations such as capillary electrophoresis (CE) are also a major field of interest, especially for macromolecules, and low cost. All these techniques are investigated with various detection modes including mass spectrometry (MS) for various applications where high sensitivity and selectivity is needed. Extracting the relevant information from the overwhelming amount of data generated by modern analytical platforms has become an important issue for knowledge discovery in various research fields. The appropriate treatment of such data is therefore of crucial importance to provide valuable information. Numerous works in our research group have demonstrated the usefulness of statistical and mathematical methodologies to improve quality of the results. Therefore, well-established chemometric approaches (e.g. design of experiments, multivariate data analysis, etc.) are implemented to optimize the analytical process from method development to data analysis.

Application of prediction intervals to the interpretation of the robustness study of a UHPLC method for the separation of cannabinoids.

Design of Experiments (DoE) is a well-established tool used for analytical methods robustness studies, because of its ability to assess the effect of a great number of factors in a minimal number of experiments. However, when assessing the robustness of an analytical method the analysis of the individual effect of each factor is not sufficient on its own. Some factors may not influence the robustness of the method, but their effect combined with the effects of other factors may have a significant contribution on the robustness of the method, which is not given by conventional analysis of DoE results. The aim of this work is to propose, in addition to the analysis of the individual effects of the factors, to estimate the joint effect of the factors by means of the matrix experimental results prediction interval. This prediction interval is the interval in which, with a given probability, should fall the next results, therefore it is an interesting tool to estimate the variation limits of the method results during routine use. We also propose the use of two other prediction intervals which can help to analyze the DoE results and give a conclusion on the method robustness. The first one is based on the DoE experimental error information, and it gives an estimation of the experimental error component impact on the factors joint effect. The second one is based on the factors non-significance limits, and it provides the information regarding the factors impact on the responses in the case where the conditions are, by definition, robust. We applied these proposals to the robustness study of a UHPLC method for the separation of phytocannabinoids and we could demonstrate that, in addition to the calculated effects values and robustness information, the use of the prediction intervals information provided additional information that allowed a better interpretation of the method performance parameters.

Sub/supercritical fluid chromatography versus liquid chromatography for peptide analysis.

The aim of this study was to evaluate the potential of ultra-high performance supercritical fluid chromatography (UHPSFC) for peptide analysis by comparing its analytical performance to several chromatographic approaches based on reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and mixed-mode liquid chromatography. First, the retention behavior of synthetic peptides with 3 to 30 amino acids and different isoelectric points (acid, neutral, and basic) was evaluated. For all the tested conditions (13 peptides in 8 conditions), only 4 results were not exploitable (not retained or not eluted), confirming that all the tested chromatographic conditions can be successfully applied when analyzing a wide range of diverse peptides. Average tailing factor were quite comparable across all chromatographic modes, while the best peak capacity values were obtained under mixed-mode LC conditions. Selectivity for each chromatographic mode was also evaluated for six closely related peptides having minor modifications on their structures. The LC-based chromatographic modes confirmed their superior selectivity over UHPSFC. By contrast, when analyzing short peptides (di- or tripetides), UHPSFC was the only technique allowing to simultaneously separate highly polar and less polar peptides within the same run confirming its unique versatility. In addition, the sensitivity of each chromatographic approach was accessed by for two representative peptides by both UV and MS detection. With UV detection, limit of detection (LOD) values were comparable among the different chromatographic modes, ranging from 0.5 to 2 µg mL-1. However, major differences were found when employing MS detection (LOD values ranged from 0.05 to 5 µg mL-1). The best results were obtained under HILIC conditions, followed by SFC, and finally mixed-mode LC and RPLC modes.

Decellularized Spinach Biomaterials Support Physiologically Relevant Mechanical Cyclic Strain and Prompt a Stretch-Induced Cellular Response.

Recently, decellularized plant biomaterials have been explored for their use as tissue engineered substitutes. Herein, we expanded upon the investigation of the mechanical properties of these materials to explore their elasticity as many anatomical areas of the body require biomechanical dynamism. We first constructed a device to secure the scaffold and induce a strain within the physiological range of the normal human adult lung during breathing (12-20 movements/min; 10-20% elongation). Results showed that decellularized spinach leaves can support cyclic strain for 24 h and displayed heterogeneous local strain values (7.76-15.88%) as well as a Poisson's ratio (0.12) similar to that of mammalian lungs (10.67-19.67%; 0.01), as opposed to an incompressible homogeneous standard polymer (such as PDMS (10.85-12.71%; 0.4)). Imaging and mechanical testing showed that the vegetal scaffold exhibited strain hardening but maintained its structural architecture and water retention capacity, suggesting an unaltered porosity. Interestingly, we also showed that cells seeded on the scaffold can also sense the mechanical strain as demonstrated by a nuclear reorientation perpendicular to strain direction (63.3° compared to 41.2° for nonstretched cells), a nuclear location of YAP and increased expression of YAP target genes, a high cytoplasmic calcium level, and an elevated expression level of collagen genes (COL1A1, COL3A1, COL4A1, and COL6A) with an increased collagen secretion at the protein level. Taken together, these data demonstrated that decellularized plant leaf tissues have an inherent elastic property similar to that found in the mammalian system to which cells can sense and respond.