RESUMO
The concentration of macromolecules in solution is a crucial property in many areas of research, including the development and commercialization of biological therapeutics. For proteins in particular, none of the reported methods for measuring concentration detect a molecular property that is known a priori; rather, they rely on ligand binding, degradation and derivitization, or an intrinsic property that must be determined experimentally. The purpose of this report is to describe (1) a diffusion-filtered qNMR experiment (DF-qNMR) for quantitating macromolecules in complex matrices and (2) an overall method for measuring absolute protein concentration based on this DF-qNMR experiment. This method combines protein denaturation with the diffusion filter to produce clean spectra of the protein with well-resolved resonances, regardless of the matrix complexity. The concentration is then obtained by comparing the peak area of the valine/isoleucine/leucine methyl groups to an external, certified, small-molecule quantitation standard. The method, which is referred to as VILMHA (valine isoleucine leucine methyl hydrogen analysis), was tested on three proteins of various sizes. In all cases, the measured concentration was within 1.8% of the labeled value for the undiluted standard reference material evaluated. In addition, the RSD's were less than 1.25% in all cases and less than 1% in most cases. The accuracy, precision, and ease of use make this method superior to existing absolute protein concentration methods. Furthermore, VILMHA is ideally suited to serve as the basis for converting the relative protein concentration methods into absolute methods or establishing molecular-specific parameters. Finally, DF-qNMR has the potential to quantitate other types of macromolecules (e.g., such as polymers, surfactants, etc.) in the presence of small-molecule contaminants.
Assuntos
Anticorpos Monoclonais/análise , Ressonância Magnética Nuclear Biomolecular , Difusão , Substâncias Macromoleculares/análiseRESUMO
1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}azetidine-3-carboxylic acid (MRL-A) is a potent sphingosine-1-phosphate-1 receptor agonist, with potential application as an immunosuppressant in organ transplantation or for the treatment of autoimmune diseases. When administered orally to rats, radiolabeled MRL-A was found to undergo metabolism to several reactive intermediates, and in this study, we have investigated its potential for protein modification in vivo and in vitro. MRL-A irreversibly modified liver and kidney proteins in vivo, in a dose- and time-dependent manner. The binding was found to occur selectively to microsomal and mitochondrial subcellular fractions. Following a nonspecific proteolytic digestion of liver and kidney proteins, a single major amino acid adduct was observed. This adduct was characterized with LC/MS/UV and NMR spectroscopy and was found to be the product of an unprecedented metabolic activation of the azetidine moiety leading to the formation of a ring-opened α,ß-unsaturated imine conjugated to the ε-amino group of a lysine residue. The formation of this adduct was not inhibited when rats were pretreated with 1-aminobenzotriazole, indicating that P450 enzymes were not involved in the metabolic activation of MRL-A. Rather, our findings suggested that MRL-A underwent bioactivation via a ß-oxidation pathway. Several other minor adducts were identified from protein hydrolysates and included lysine, serine, and cysteine conjugates of MRL-A. These minor adducts were also detected in microsomal incubations fortified with the cofactors for acyl-CoA synthesis and in hepatocytes. Trypsin digestion of crude liver homogenates from rats treated with radiolabeled MRL-A led to the identification of a single radioactive peptide. Its sequence, determined by LC/MS analysis, revealed that the target of the major reactive species of MRL-A in vivo is Lys676 of long chain acyl-CoA synthetase-1 (ACSL1). This lysine residue has been found to be critical for ACSL1 activity, and its modification has the potential to lead to biological consequences such as cardiac hypertrophy or thermogenesis dysregulation.
Assuntos
Azetidinas/metabolismo , Proteínas/metabolismo , Receptores de Lisoesfingolipídeo/agonistas , Tiofenos/metabolismo , Administração Oral , Animais , Azetidinas/toxicidade , Biotransformação , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Marcação por Isótopo , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Espectrometria de Massas , Microssomos Hepáticos/metabolismo , Peptídeos/análise , Proteínas/química , Ratos , Receptores de Lisoesfingolipídeo/metabolismo , Tiofenos/toxicidadeRESUMO
OBJECTIVE: Ultra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog® and Lyumjev® formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. METHODS: We employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. RESULTS: Through experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. CONCLUSIONS: This study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future.
Assuntos
Insulina de Ação Curta , Técnicas Fotoacústicas , Animais , Excipientes , Hipoglicemiantes/química , Insulina , Insulina Lispro , Camundongos , SuínosRESUMO
As the number of therapeutic proteins produced by mammalian cell cultures in the pharmaceutical industry continues to increase, the need to improve productivity and ensure consistent product quality during process development activities becomes more significant. Rational medium design is known to improve cell culture performance, but an understanding of nutrient consumption and metabolite accumulation within the medium is required. To this end, we have developed a technique for using 1D (1)H NMR to quantitate nonprotein feed components and metabolites in mammalian cell cultures. We refer to the methodology as "Fermentanomics" to differentiate it from standard metabolomics. The method was found to generate spectra with excellent water suppression, signal-to-noise, and resolution. More importantly, nutrient consumption and metabolite accumulation was readily observed. In total, 50 media components have been identified and quantitated. The application of Fermentanomics to the optimization of a proprietary CHO basal medium yielded valuable insight regarding the nutrient levels needed to maintain productivity. While the focus here is on the extracellular milieu of CHO cell cultures, this methodology is generally applicable to quantitating intracellular concentrations and can be extended to other mammalian cell lines, as well as platforms such as yeasts, fungi, and Escherichia coli.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Meios de CulturaRESUMO
MRL-1, a cannabinoid receptor-1 inverse agonist, was a member of a lead candidate series for the treatment of obesity. In rats, MRL-1 is eliminated mainly via metabolism, followed by excretion of the metabolites into bile. The major metabolite M1, a glutathione conjugate of MRL-1, was isolated and characterized by liquid chromatography/mass spectrometry and NMR spectroscopic methods. The data suggest that the t-butylsulfonyl group at C-2 of furopyridine was displaced by the glutathionyl group. In vitro experiments using rat and monkey liver microsomes in the presence of reduced glutathione (GSH) showed that the formation of M1 was independent of NADPH and molecular oxygen, suggesting that this reaction was not mediated by an oxidative reaction and a glutathione S-transferase (GST) was likely involved in catalyzing this reaction. Furthermore, a rat hepatic GST was capable of catalyzing the conversion of MRL-1 to M1 in the presence of GSH. When a close analog of MRL-1, a p-chlorobenzenesulfonyl furopyridine derivative (MRL-2), was incubated with rat liver microsomes in the presence of GSH, p-chlorobenzene sulfinic acid (M2) was also identified as a product in addition to the expected M1. Based on these data, a mechanism is proposed involving direct nucleophilic addition of GSH to sulfonylfuropyridine, resulting in an unstable adduct that spontaneously decomposes to form M1 and M2.
Assuntos
Biocatálise , Glutationa Transferase/metabolismo , Piridinas/farmacocinética , Compostos de Enxofre/farmacocinética , Animais , Bile/química , Biotransformação/fisiologia , Cromatografia Líquida , Citosol/metabolismo , Cães , Glutationa/metabolismo , Haplorrinos , Humanos , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , Masculino , Microssomos Hepáticos/enzimologia , Estrutura Molecular , NADP/metabolismo , Piridinas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/antagonistas & inibidores , Especificidade da Espécie , Compostos de Enxofre/metabolismo , Espectrometria de Massas em TandemRESUMO
The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.
Assuntos
Anticorpos Monoclonais/química , Biofarmácia/normas , Laboratórios/normas , Espectroscopia de Ressonância Magnética/métodos , Humanos , Reprodutibilidade dos TestesRESUMO
Diffusion-ordered NMR spectroscopy, while quite powerful, is limited by its inability to resolve signals that are severely overlapped in the proton spectrum. We present here a DOSY experiment that uses selective TOCSY as an editing/preparation period. With this method, well-resolved signals of the analytes are selectively excited and the magnetization subsequently transferred by isotropic mixing to resonances buried in the matrix background, which are then resolved by the ensuing DOSY sequence. Key to the success of our proposed method is the incorporation of a highly effective zero-quantum filter into the selective TOCSY preparation period, which prevents zero-quantum coherence from being carried into the DOSY part of the pulse sequence. Further improvement in spectral resolution can be obtained by expanding the proposed experiment into a 3D sequence and utilizing the homonuclear decoupling feature of the BASHD-TOCSY technique. Both pulse sequences were found to greatly simplify the DOSY spectrum of a 'dirty' sucrose/raffinose mixture, as the complex matrix background is no longer present to obscure or overlap with the signals of interests. Furthermore, complete resolution of the relevant signals was achieved with the 3D sequence.
Assuntos
Carboidratos/química , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Rafinose/química , Processamento de Sinais Assistido por Computador , Sacarose/químicaRESUMO
We describe a unique band-selective method that utilizes a selective composite gradient to simultaneously achieve band selection and coherence pathway selection. This element is similar to the composite gradient known as the CLUB sandwich except the original broadband pulses have been replaced with selective pulses and the strengths of the antipolar gradients have been unbalanced. In this way, only the signals within the inversion band will continue to dephase throughout the duration of the element and satisfy the proper encoding-to-decoding gradient ratio necessary for coherence selection. Apart from the inverted polarity and asymmetry of the gradients, the band-selective CLUB sandwich is identical to the DPFGSE sequence and provides many of its desirable characteristics. We have successfully incorporated the band-selective CLUB into the DQF-COSY pulse sequence to create a band-selective experiment that offers the selectivity desired for resolution enhancement while maintaining excellent phase behavior. This is demonstrated on the congested aliphatic region of the ionophorous antibiotic Lasalocid A.
Assuntos
Antibacterianos/química , Lasalocida/química , Espectroscopia de Ressonância Magnética/métodos , Algoritmos , Estrutura MolecularRESUMO
Compared to its 2D counterpart, the selective 1D NOESY experiment offers greatly simplified spectral interpretation and is invaluable to the structure elucidation of small-to-medium sized molecules, although its application is limited to well-resolved resonances only. The doubly selective 1D TOCSY-NOESY experiment allows the 1D NOESY experiment to be extended to resonances within overlapped spectral regions. However, existing methods do not address the critical issue of zero-quantum interference, which leads to severe anti-phase distortions to the line shape of scalar coupled spins and often complicates the identification of weak NOE enhancements. In this communication, we describe an improved selective TOCSY edited preparation (STEP) function and its application to the selective 1D NOESY experiment. The STEP function incorporates a novel zero-quantum filter introduced by Thrippleton and Keeler [Angew. Chem. Int. Ed. 42 (2003) 3938], which permits essentially complete suppression of zero-quantum coherence in a single scan. Residual anti-phase distortions due to spin-state mixing are removed using the double difference methodology reported by Shaka et al. [45th Experimental NMR Conference, Pacific Grove, USA, 2004]. The combined use of these techniques ensures that the final spectra are free of distortions, which is crucial to the reliable detection of weak NOE enhancements. Although employed as an additional preparation period in the example demonstrated here, the STEP function affords a general editing tool for spectral simplification and can be applied to a range of experiments.
Assuntos
Ionóforos/química , Lasalocida/química , Ressonância Magnética Nuclear Biomolecular/métodosRESUMO
Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools.
Assuntos
Aminoácidos/metabolismo , Anticorpos/metabolismo , Biotecnologia/métodos , Animais , Reatores Biológicos , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Fermentação , Hibridomas/metabolismo , Camundongos , Polissacarídeos/metabolismoRESUMO
This letter provides the first pharmacological proof of principle that the sst3 receptor mediates glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. To enable these studies, we identified the selective sst3 antagonist (1R,3R)-3-(5-phenyl-1H-imidazol-2-yl)-1-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-ß-carboline (5a), with improved ion channel selectivity and mouse pharmacokinetic properties as compared to previously described tetrahydro-ß-carboline imidazole sst3 antagonists. We demonstrated that compound 5a enhances GSIS in pancreatic ß-cells and blocks glucose excursion induced by dextrose challenge in ipGTT and OGTT models in mice. Finally, we provided strong evidence that these effects are mechanism-based in an ipGTT study, showing reduction of glucose excursion in wild-type but not sst3 knockout mice. Thus, we have shown that antagonism of sst3 represents a new mechanism with potential in treating type 2 diabetes mellitus.
RESUMO
A structure-activity relationship study of the imidazolyl-ß-tetrahydrocarboline series identified MK-4256 as a potent, selective SSTR3 antagonist, which demonstrated superior efficacy in a mouse oGTT model. MK-4256 reduced glucose excursion in a dose-dependent fashion with maximal efficacy achieved at doses as low as 0.03 mg/kg po. As compared with glipizide, MK-4256 showed a minimal hypoglycemia risk in mice.
RESUMO
We have investigated the prospect of identifying organic reaction products directly from separated thin-layer chromatography (TLC) spots with high-resolution magic-angle spinning (HRMAS) NMR. The concept is to use the TLC spots for NMR analysis so that spectra can be obtained before the reaction is worked up, but without having to elute the product from the TLC stationary phase. Thus, the separated spot is scraped from the plate, transferred to an HRMAS sample rotor, and suspended with a deuterated solvent. Herein, we describe the effects of having the stationary phase present during NMR acquisition. Using a Varian 4 mm gHX Nanoprobe and rotenone as a test compound, we found that the presence of the stationary phase during NMR acquisition resulted in (i) a large, broad 'background' signal near 4.6 ppm and (ii) a decrease in the signal-to-noise ratio due to the adsorption of the product molecules to the adsorbent. However, both effects could be adequately and conveniently eliminated. The background signal was removed by using either a CPMG pulse sequence or chemical exchange. The adsorption was avoided by using a more polar solvent system. Finally, we found that spectra with good signal-to-noise ratio and resolution could be acquired in a matter of minutes even for cases of limited product concentration. Therefore, we believe the technique has value and provides the organic chemist with another option to obtain NMR data critical for structural elucidation or verification.
Assuntos
Cromatografia em Camada Fina/métodos , Espectroscopia de Ressonância Magnética/métodos , Acetatos , Estrutura Molecular , Sensibilidade e EspecificidadeRESUMO
We report the lateral diffusion properties of 2,2'-di-O-decyl-3,3'-di-O-(eicosanyl)-bis-(rac-glycero)-1,1'-diphosphocholine (C20BAS) using pulsed-field gradient NMR (PFG-NMR) and fluorescence recovery after photobleaching (FRAP). C20BAS membranes display a melting transition at Tm = 15.7 degrees C, as determined by differential scanning calorimetry and 31P NMR chemical shift anisotropy. The lateral diffusion coefficient of C20BAS, as determined by PFG-NMR and FRAP, at 25 degrees C, were DPFG-NMR = 1.9 +/- 0.6 x 10(-8) cm2/s and DFRAP C20BAS = 1.2 +/- 0.1 x 10(-8) cm2/s, respectively. In comparison, the lateral diffusion coefficient of the monopolar phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was 1.8 +/- 0.9 x 10(-8) and 2.5 +/- 0.9 x 10(-8) cm2/s using PFG-NMR and FRAP, respectively.
Assuntos
Glicerilfosforilcolina/análogos & derivados , Lipídeos de Membrana/química , Difusão , Recuperação de Fluorescência Após Fotodegradação , Técnica de Fratura por Congelamento , Glicerilfosforilcolina/química , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica , Termodinâmica , Thermoplasma/químicaRESUMO
CRISIS (Compensation of Refocusing Inefficiency with Synchronized Inversion Sweep) is a powerful technique for obtaining multiplicity-edited HSQC spectra without compromising sensitivity. However, the stringent requirement for the duration of the CRISIS waveforms makes them unsuitable for other functions, such as band selection or IMPRESS (IMProved REsolution using Symmetrically Shifted pulses). We report here a modified CRISIS-gHSQC pulse sequence employing time-reversed 13C pi/2 EBURP-2 pulses. This IC-bs-gHSQC (IMPRESS-CRISIS-bs-gHSQC) sequence was found to be equally useful for acquiring multiplicity-edited, band-selective spectra individually or in tandem with IMPRESS. Remarkably, the latter provides multiple spectra in significantly less time and is the preferred approach when several crowded regions need to be assigned unambiguously. The use of adiabatic sweeps and the CRISIS pulses enable IC-bs-gHSQC to give better sensitivity than the original IMPRESS sequence for band-selective spectra.
RESUMO
The DOSY analysis of dilute mixtures can be a challenge because a high signal-to-noise ratio is required for the best DOSY results. The sensitivity increase gained from new probe technologies (e.g. cold and nano probes) could enable one to acquire good DOSY spectra on sample amounts too low for conventional probes. In this article, we investigated the performance of cold and nano probes for qualitative DOSY analysis of concentrated and sample-limited mixtures, and compared the results with those of the conventional probe. We first measured the fluid flow for each probe. All three probes exhibited only relatively small levels of flow; consequently, a double-stimulated echo pulse sequence was not employed in the subsequent DOSY experiments. This decision was based on three facts: (1) flow-induced phase distortions were not observed, (2) our intentions are only to perform qualitative mixture analysis, and (3) discarding 50% of the already limited signal cannot be afforded. Although the cold and nano probes produced DOSY results for the concentrated mixture that were inferior to the conventional probe, the increase in the signal-to-noise ratio observed with these probes proved to be advantageous for the dilute three-component mixture. Furthermore, the cold probe showed slightly superior performance over the nano probe; thus, we conclude that among the probes examined the cold probe is best suited for qualitative DOSY analysis of sample-limited mixtures.