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Messenger RNA (mRNA) can be sequenced via indirect approaches such as Sanger sequencing and next generation sequencing (NGS), or direct approaches like bottom-up mass spectrometry (MS). Direct sequencing allows the confirmation of RNA modifications. However, the conventional bottom-up MS approach involves time-consuming in-solution digestions that require a large amount of sample, and can lead to the RNase contamination of the LC-MS system and column. Here, we describe a platform that enables online nucleotide mapping of mRNAs via the use of immobilized RNase cartridges and 2D-LC-MS instrumentation. The online approach was compared to conventional offline digestion protocols adapted from two published studies. For this purpose, five model mRNAs of varying lengths (996-4521 nucleotides) and chemistries (unmodified uridine vs 5-methoxyuridine (5moU)) were analyzed. The profiles and sequence coverages obtained after RNase T1 digestion were discussed. The online nucleotide mapping achieved comparable or slightly greater sequence coverage for the 5 mRNAs (5.8-51.5%) in comparison to offline approaches (3.7-50.4%). The sequence coverage was increased to 65.6-85.6 and 69.7-85.0% when accounting for the presence of nonunique digestion products generated by the RNase T1 and A, respectively. The online nucleotide mapping significantly reduced the digestion time (from 15 to <5 min), increased the signal intensity by more than 10-fold in comparison to offline approaches.
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ARN Mensajero , ARN Mensajero/análisis , ARN Mensajero/genética , Mapeo Nucleótido/métodos , Espectrometría de Masas , Cromatografía Liquida , Uridina/análogos & derivados , Uridina/química , Humanos , Ribonucleasa T1/metabolismoRESUMEN
Agile analytical approaches are needed for fast and comprehensive characterization of peptide drug candidates. In this study, a unified and versatile multiplex platform was developed to expedite method development and enable the routine determination of multiple quality attributes simultaneously. The platform integrates the automation of size exclusion chromatography (SEC), reversed phase liquid chromatography coupled to reversed phase liquid chromatography (RPLC-RPLC), and hydrophilic interaction liquid chromatography hyphenated to charged aerosol detection (HILIC-CAD). Various therapeutic peptide constructs, including macrocyclic peptides and disulfide constrained peptides, across different lots were studied. The effect of the mobile phase acetonitrile content on the impurity profiles was systematically studied using two SEC columns. A prototype MaxPeak Premier SEC 125 Å column packed with BEH PEO particles achieved the separation of impurities (>2.0% area), whereas no impurities could be observed with an ACQUITY UPLC Protein BEH SEC 125 Å column packed with BEH diol particles. Comprehensive impurity profiling and expedited method development was performed utilizing RPLC-RPLC. Each peptide was analyzed by a combination of 12 conditions in the second dimension, using four columns with octadecyl, phenyl-hexyl, and cyano bonded phases, and three mobile phases with various solvents, modifiers, and pH compositions. Additionally, a HILIC-CAD method was developed for the quantification of TFA, commonly present in peptide products.
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Plasmid DNA (pDNA) is an essential tool in genetic engineering that has gained prevalence in cell and gene therapies. Plasmids exist as supercoiled (SC), open circular (OC), and linear forms. Plasmid multimerization can also occur during the manufacturing process. Even though the SC forms are thought to provide optimal knock-in (KI) efficiency, there is no strong consensus on the effect of the topological forms and multimers on the functional activity. In addition, the results obtained for conventional pDNAs (>5 kbp) do not necessarily translate to smaller pDNAs (â¼3 kbp). In this study, a workflow was developed for the analytical and functional characterization of pDNA topological forms and multimers. An anion exchange chromatography (AEC) method was first developed to quantify the topological forms and multimers. Four AEC columns were initially compared, one of which was found to provide superior chromatographic performance. The effect of mobile phase pH, various salts, column temperature, and acetonitrile content on the separation performance was systematically studied. The method performance, including precision and accuracy, was evaluated. The final AEC method was compared to capillary gel electrophoresis (CGE) by analyzing several pDNA sequences and lots. A forced degradation study revealed unexpectedly high degradation of the SC forms. Finally, the KI efficiency was compared for the SC and OC forms, and the multimers.
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Plásmidos , Plásmidos/genética , Cromatografía por Intercambio Iónico , Electroforesis Capilar , ADN/química , Concentración de Iones de HidrógenoRESUMEN
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.
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Cromatografía en Gel , ARN Mensajero , ARN Mensajero/genética , ARN Mensajero/química , Cromatografía en Gel/métodos , Humanos , Porosidad , Peso Molecular , Cloruro de Magnesio/químicaRESUMEN
Health authorities have highlighted the need to determine oligonucleotide aggregates. However, existing technologies have limitations that have prevented the reliable analysis of size variants for large nucleic acids and lipid nanoparticles (LNPs). In this work, nucleic acid and LNP aggregation was examined using prototype, low adsorption ultrawide pore size exclusion chromatography (SEC) columns. A preliminary study was conducted to determine the column's physicochemical properties. A large difference in aggregate content (17.8 vs 59.7 %) was found for a model messenger RNA (mRNA) produced by different manufacturers. We further investigated the nature of the aggregates via a heat treatment. Interestingly, thermal stress irreversibly decreased the amount of aggregates from 59.7 to 4.1% and increased the main peak area 3.3-fold. To the best of our knowledge, for the first time, plasmid DNA topological forms and multimers were separated by analytical SEC. The degradation trends were compared to the data obtained with an anion exchange chromatography method. Finally, unconjugated and fragment antigen-binding (Fab)-guided LNPs were analyzed and their elution times were plotted against their sizes as measured by DLS. Multi-angle light scattering (MALS) was coupled to SEC in order to gain further insights on large species eluting before the LNPs, which were later identified as self-associating LNPs. This study demonstrated the utility of ultrawide pore SEC columns in characterizing the size variants of large nucleic acid therapeutics and LNPs.
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The recent clinical and commercial success of lipid nanoparticles (LNPs) for nucleic acid delivery has incentivized the development of new technologies to manufacture LNPs. As new technologies emerge, researchers must determine which technologies to assess and how to perform comparative evaluations. In this article, we use a quality-by-design approach to systematically investigate how the mixer technology used to form LNPs influences LNPstructure. Specifically, a coaxial turbulent jet mixer and a staggered herringbone microfluidic mixer were systematically compared via matched formulation and process conditions. A full-factorial design-of-experiments study with three factors and three levels was executed for each mixer to compare process robustness in the production of antisense oligonucleotide (ASO) LNPs. ASO-LNPs generated with the coaxial turbulent jet mixer were consistently smaller, had a narrower particle size distribution, and had a higher ASO encapsulation as compared to the microfluidic mixer, but had a greater variation in internal structure with less ordered cores. A subset of the study was replicated for mRNA-LNPs with comparable trends in particle size and encapsulation, but more frequent bleb features for LNPs produced by the coaxial turbulent jet mixer. The study design used here provides a road map for how researchers may compare different mixer technologies (or process changes more broadly) and how such studies can inform process robustness and manufacturing control strategies.
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Microfluídica , Nanopartículas , Liposomas , Nanopartículas/química , ARN MensajeroRESUMEN
Accurate sequencing of single guide RNAs (sgRNAs) for CRISPR/Cas9 genome editing is critical for patient safety, as the sgRNA guides the Cas9 nuclease to target site-specific cleavages in DNA. An approach to fully sequence sgRNA using protective DNA primers followed by ribonuclease (RNase) T1 digestion was developed to facilitate the analysis of these larger molecules by hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-HRMS). Without RNase digestion, top-down mass spectrometry alone struggles to properly fragment precursor ions in large RNA oligonucleotides to provide confidence in sequence coverage. With RNase T1 digestion of these larger oligonucleotides, however, bottom-up analysis cannot confirm full sequence coverage due to the presence of short, redundant digestion products. By combining primer protection with RNase T1 digestion, digestion products are large enough to prevent redundancy and small enough to provide base resolution by tandem mass spectrometry to allow for full sgRNA sequence coverage. An investigation into the general requirements for adequate primer protection of specific regions of the RNA was conducted, followed by the development of a generic protection and digestion strategy that may be applied to different sgRNA sequences. This middle-out technique has the potential to expedite accurate sequence confirmation of chemically modified sgRNA oligonucleotides.
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Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Humanos , Ribonucleasa T1/genética , Cartilla de ADN , Oligonucleótidos , DigestiónRESUMEN
The chemistry of guide RNA (gRNA) affects the performance of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing technique. However, the literature is very scarce about the study of gRNA degradation and in particular at the single nucleotide level. In this work, we developed a workflow to characterize the impurities of large RNAs at the single nucleotide level and identified the residues prone to degradation. Our strategy involves (i) the reduction of RNA length, (ii) a chromatographic mode able to capture subtle changes in impurity polarity, and (iii) a streamlined data treatment. To illustrate the approach, stressed gRNA samples were analyzed by coupling an immobilized ribonuclease T1 cartridge to a hydrophilic interaction liquid chromatography (HILIC) column hyphenated with tandem mass spectrometry (MS/MS). Critical findings were made possible by the presented technology. In particular, the desulfurization of phosphorothioate (PS) linkages was the major degradation observed at the single nucleotide level while no change in purity profile could be observed when using conventional ion-pairing reversed-phase (IPRP) liquid chromatography. To our knowledge, this is the first time that several impurity types are screened for a large RNA molecule using an automated online digestion analysis approach.
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ARN Guía de Kinetoplastida , Espectrometría de Masas en Tándem , ARN Guía de Kinetoplastida/genética , Nucleótidos , Edición Génica/métodos , Cromatografía LiquidaRESUMEN
In this study, for the first time, the automated digestion and sequencing of an RNA molecule via the use of immobilized RNase cartridges attached to a multidimensional liquid chromatography (LC)-mass spectrometry (MS) system are presented. We first developed an on-line digestion-HILIC two-dimensional (2D)-LC-MS method in order to sequence CRISPR guide RNAs for gene editing. Three RNases (T1, A, and U2) were immobilized on polyetheretherketone cartridges, and their performance was evaluated. Ultrafast digestions were performed within 2.3 min with the on-line approach versus 30 min via the conventional off-line approach. The higher sequence coverage was achieved by the RNase T1 (71%), which is the same as the off-line mode. A 20-fold reduction in the gRNA sample amount was achieved with the on-line digestion approach (6.5 µg) in comparison to that with the off-line approach (130 µg). In the second step, a three-dimensional (3D)-LC-MS method was developed for the sequencing of fractions collected on-line across the main peak and the partially separated tail by the reference ion-pairing RPLC method. Additional insights were gained in order to better understand the cause of the main peak tailing.
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ARN Guía de Kinetoplastida , Ribonucleasas , Cromatografía Liquida/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Espectrometría de Masas en TándemRESUMEN
CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA. Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. To our knowledge, it is the first time that (i) RNA digests are separated and identified by HILIC-HRMS and (ii) chemically modified sgRNAs are directly sequenced via a bottom-up approach.
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Sistemas CRISPR-Cas , ARN Guía de Kinetoplastida , Cromatografía Liquida , Digestión , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas , RibonucleasasRESUMEN
Accelerated development of new therapeutics in an increasingly competitive landscape requires the use of high throughput analytical platforms. In addition, the complexity of novel biotherapeutic formats (e.g. fusion proteins, protein-polymer conjugates, co-formulations, etc.) reinforces the need to improve the selectivity and resolution of conventional one-dimensional (1D) liquid chromatography (LC). Liquid chromatography-mass spectrometry (LC-MS)-based technologies such as native LC-MS for intact mass analysis or peptide mapping (also called bottom-up approach)-based multi-attribute methods (MAM) have already demonstrated their potential to complement the conventional analytical toolbox for monoclonal antibody (mAb) characterization. Two-dimensional liquid-chromatography (2D-LC-MS) methods have emerged in the last ten years as promising approaches to address the increasing analytical challenges faced with novel antibody formats. However, off-line sample preparation procedures are still required for conventional 1D and 2D-LC-MS methods for the in-depth variant characterization at the peptide level. Multi-dimensional LC-MS (mD-LC-MS) combine sample preparation and multi-level (i.e. intact, reduced, middle-up and peptide) analysis within the same chromatographic set-up. This review presents an overview of the benefits and limitations of mD-LC-MS approaches in comparison to conventional chromatographic methods (i.e. 1D-LC-UV methods at intact protein level and 1D-LC-MS methods at peptide level). The current analytical trends in antibody characterization by mD-LC-MS approaches, beyond the 2D-LC-MS workhorse, are also reviewed, and our vision on a more integrated multi-level mD-LC-MS characterization platform is shared.
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Inmunoconjugados , Espectrometría de Masas en Tándem , Anticuerpos Monoclonales , Cromatografía Liquida , Inmunoconjugados/análisis , PéptidosRESUMEN
Determination of phosphorothioate oligonucleotide purity and impurity profile is commonly performed by ion-pairing reversed-phase liquid chromatography (IPRP) with a mobile phase containing triethylamine (TEA) and hexafluoro-2-propanol (HFIP). However, ion-suppressing effects of TEA hamper mass spectrometry (MS) instrumentation sensitivity and HFIP can affect the robustness of the mass spectrometer due to its corrosive nature. Anion exchange chromatography (AEX) is an orthogonal separation mode to IPRP but typically cannot be directly coupled to MS. In this work, we developed a multiple heart-cutting IPRP-, AEX-hydrophilic interaction liquid chromatography(HILIC)/MS method for quantification and high sensitivity identification of antisense oligonucleotide (ASO) impurities using a Q-Exactive mass spectrometer. Notably, both AEX-HILIC and IPRP-HILIC modes could be operated on a versatile two-dimensional liquid chromatography (2D-LC) setup including several column selectors. The HILIC mobile phase contained 25 mM ammonium acetate and allowed identifying impurities at levels down to 0.3%. Careful selection of the sample loop volume and the 2D HILIC column dimension allowed straightforward coupling of HILIC for both IPRP and AEX without the need to use any solvent modulation. Overall, the 2D HILIC allowed online desalting of AEX and IPRP modes and further separation of additional impurities.
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Automated high-throughput experimentation (HTE) is a powerful tool for scientists to explore and optimize chemical transformations by simultaneously screening yield, stereoselectivity, and impurity profiles. To analyze the HTE samples, high-throughput analysis (HTA) platforms must be fast, accurate, generic, and specific at the same time. A large amount of high-quality data is critical for the success of machine learning models in the era of big data. Conventional chiral liquid chromatography-mass spectrometry (LC/MS) HTE methods are hampered by compound co-eluting, possible ion suppression, and limited chiral column lifetime in the presence of crude reaction mixtures or complex sample matrices. To overcome these limitations, a generic and fast achiral-chiral heart-cutting two-dimensional (2D)-LC method has been developed to determine both the yield and stereoselectivity of chemical transformations within a 10 min run time. Successful implementation of the 2D-LC HTA platform in a routine drug development environment was achieved for real-world project support, with the analysis so far of over 2000 reaction mixtures prepared in the 96-well plate format. Excellent performance of the method was demonstrated by relative standard deviation (RSD) lower than 0.83% for the 1D and 2D retention times, and determination coefficients higher than 0.99. The presented HTA 2D-LC platform has had a significant impact on drug development by analyzing the HTE samples rapidly with unambiguous peak tracking and providing a robust approach for accurately generating a large amount of high-quality data in a short time.
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Cromatografía Liquida/métodos , Desarrollo de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Aprendizaje Automático , Estereoisomerismo , Factores de TiempoRESUMEN
This study describes the use of a multidimensional HPLC (2D and 4D) system for a faster and more effective characterization of an antibody-drug conjugate (ADC) product, compared to the standard off-line approach of fraction collection and off-line variant characterization. The size variants of an interchain cysteine-linked ADC were characterized to understand the effect of the different drug-to-antibody ratio (DAR) species on aggregate formation. For this purpose, the ADC product and a full panel of stressed samples were analyzed. The dimeric ADC species were baseline resolved from the main peak (Rs = 2.7) by UHP-SEC (ultra-high-performance size exclusion chromatography) under nondenaturing conditions using a buffered mobile phase containing 5% 2-propanol. A 2D-LC (SEC-HIC) method was then developed to compare the average DAR values of the main peak species vs the aggregates. A 4D-LC/MS method (SEC-reduction-digestion-RPHPLC) was also developed to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation, in a 2 h run. An average DAR value of 3.5-3.6 was found for the main peak using both 2D-LC and 4D-LC methods, and these values were consistent with DAR determined by the in-house reference hydrophobic interaction chromatography (HIC) method. The multidimensional LC approaches also showed an increase in the content of high-DAR species in the SEC fractions containing the aggregates. Overall the entire workflow of data acquisition is completed within a day using the multidimensional on-line approach, in comparison to multiple days required with the traditional off-line approaches.
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Anticuerpos/análisis , Cromatografía Líquida de Alta Presión/normas , Inmunoconjugados/análisis , Fragmentos de Péptidos/análisis , Anticuerpos/química , Cisteína/química , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoconjugados/química , Luz , Oxidación-Reducción , Mapeo Peptídico , Proteolisis , Soluciones , Tripsina/químicaRESUMEN
The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization. On the basis of the observation that limited SEC performance was observed in nondenaturing MS compatible ammonium acetate buffer compared with classical phosphate salts, a multidimensional analytical approach was proposed. It combines comprehensive online two-dimensional chromatography (SEC×SEC), with ion mobility and mass spectrometry (IM-MS) in nondenaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification, and quantitation of adalimumab size variants. Benefits of the SEC×SEC-native IM×MS were further highlighted on forced degraded pembrolizumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous identification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results emphasize the power of comprehensive 2D LC×LC setups hyphenated to IM×MS in nondenaturing conditions with unprecedented performance including: (i) maintaining optimal SEC performance (under classical nonvolatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all separated size variants.
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Anticuerpos Monoclonales Humanizados/análisis , Anticuerpos Monoclonales/análisis , Antineoplásicos Inmunológicos/análisis , Bevacizumab/análisis , Cromatografía en Gel , Espectrometría de MasasRESUMEN
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.
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Cromatografía en Gel , Inmunoconjugados/química , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de MasasRESUMEN
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.
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Anticuerpos Monoclonales/aislamiento & purificación , Electroforesis Capilar/métodos , Antineoplásicos Inmunológicos , Electroforesis Capilar/instrumentación , Concentración de Iones de Hidrógeno , Ipilimumab/análisis , Ipilimumab/aislamiento & purificación , Nivolumab/análisis , Nivolumab/aislamiento & purificación , Polietilenglicoles , Control de CalidadRESUMEN
"Switchable water" is an aqueous solution containing a water-soluble amine additive that exhibits CO2 -switchable properties, such as large changes in ionic strength, by forming an ammonium bicarbonate salt. Switchable water has been used to reversibly "salt-out" organic compounds from water. This study explores the salting out of several compounds in switchable water when CO2 is present and also explores the solubility of small molecules in switchable water, compared to pure water, when CO2 is absent. The results show that organic compounds are generally more soluble in switchable water than pure water in the absence of CO2 , but less soluble in the presence of 1â atm CO2 . Exceptions include carboxylic acids and phenols which, presumably due to their acidity, are more soluble in switchable water than in pure water, even when CO2 is applied. Kirkwood-Buff solvation theory was applied to gain insights into the effects of the amine additive on the aqueous solubility of caffeine. Furthermore, the switchable properties of the additives allow for the preparation of switchable aqueous two-phase systems.
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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.
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Ingredientes Alimentarios/análisis , Oligopéptidos/análisis , Amidas/química , Cromatografía Líquida de Alta Presión , Conformación Molecular , Dióxido de Silicio/química , Espectrometría de Masas en TándemRESUMEN
Antibody-drug conjugates (ADCs) are designed by chemically linking highly potent cytotoxic small molecule drugs to monoclonal antibodies of unique specificity for targeted destruction of cancer cells. This innovative class of molecules incurs unique developmental challenges due to its structural complexity of having both small molecule and protein components. The stability of the small molecule payload on the ADC is a critical attribute as it directly relates to product efficacy and patient safety. This study describes the use of an end-to-end automated workflow for effective and robust characterization of the small molecule drug while it is conjugated to the antibody. In this approach, online deconjugation was accomplished by an autosampler user defined program and 1D size exclusion chromatography was utilized to provide separation between small molecule and protein species. The small molecule portion was then trapped and sent to the 2D for separation and quantification by reversed-phase liquid chromatography with identification of impurities and degradants by mass spectrometry. The feasibility of this system was demonstrated on an ADC with a disulfide-based linker. This fully automated approach avoids tedious sample preparation that may lead to sample loss and large assay variability. Under optimized conditions, the method was shown to have excellent specificity, sensitivity (LOD of 0.036 µg/mL and LOQ of 0.144 µg/mL), linearity (0.04-72.1 µg/mL), precision (system precision %RSD of 1.7 and method precision %RSD of 3.4), accuracy (97.4 % recovery), stability-indicating nature, and was successfully exploited to analyze the small molecule drug on a panel of stressed ADC samples. Overall, the workflow established here offers a powerful analytical tool for profiling the in-situ properties of small molecule drugs conjugated to antibodies and the obtained information could be of great significance for guiding process/formulation development and understanding pharmacokinetic/pharmacodynamic behavior of ADCs.