RESUMO
Cytomegalovirus (CMV) infection is a significant complication after kidney transplantation. We examined the ability of RG7667, a combination of two monoclonal antibodies, to prevent CMV infection in high-risk kidney transplant recipients in a randomized, double-blind, placebo-controlled trial. CMV-seronegative recipients of a kidney transplant from a CMV-seropositive donor (D+R-) were randomized to receive RG7667 (n = 60) or placebo (n = 60) at the time of transplant and 1, 4, and 8 weeks posttransplant. Patients were monitored for CMV viremia every 1 to 2 weeks posttransplant for 24 weeks. Patients who had seroconverted (D+R+) or withdrawn before dosing were excluded from the analysis (n = 4). CMV viremia occurred in 27 of 59 (45.8%) patients receiving RG7667 and 35 of 57 (61.4%) patients receiving placebo (stratum-adjusted difference, 15.3%; P = 0.100) within 12 weeks posttransplant and in 30 of 59 (50.8%) patients receiving RG7667 and 40 of 57 (70.2%) patients receiving placebo (stratum-adjusted difference, 19.3%; P = 0.040) within 24 weeks posttransplant. Median time to CMV viremia was 139 days in patients receiving RG7667 compared to 46 days in patients receiving placebo (hazard ratio, 0.53; P = 0.009). CMV disease was less common in the RG7667 than placebo group (3.4% versus 15.8%; P = 0.030). Adverse events were generally balanced between treatment groups. In high-risk kidney transplant recipients, RG7667 was well tolerated, numerically reduced the incidence of CMV infection within 12 and 24 weeks posttransplant, delayed time to CMV viremia, and was associated with less CMV disease than the placebo. (This study has been registered at ClinicalTrials.gov under registration no. NCT01753167.).
Assuntos
Anticorpos Monoclonais/uso terapêutico , Infecções por Citomegalovirus/prevenção & controle , Transplante de Rim , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Infecções por Citomegalovirus/tratamento farmacológico , Feminino , Humanos , Transplante de Rim/efeitos adversos , Masculino , Pessoa de Meia-Idade , Placebos , Resultado do Tratamento , Viremia/prevenção & controle , Viremia/virologiaRESUMO
Tryptic peptide mapping is routinely used in the biotech industry to confirm primary sequence, cell line stability, and to analyze posttranslational modifications. Peptide analysis is generally done by reverse phase liquid chromatography with UV or mass spectrometric detection. This method provides excellent resolution and sequence coverage. However, traditional methods are slow, and generally cannot detect small, hydrophilic peptides due to coelution with the column void volume. In this work, complementary CE-MS peptide analysis methods have been developed. The analyses are performed on a traditional CE-MS instrument with a sheath interface, and also on a novel sheathless interface that promises improved resolution and limit of detection. The methods were performed on a tryptic digest of a therapeutic monoclonal antibody for which LC-MS detects 97% sequence coverage. The 3% not covered consists of 11 peptides containing three amino acids or fewer, including two in the critical complementarity binding domain. Without further processing, the same tryptic digest was analyzed by CE-MS. Separation and detection of the 11 small peptides was achieved on CE-MS systems with both interfaces. The sheathless system produced better peak capacity and gave mass spectra with significantly less noise, while the sheath system proved to have better repeatability.
Assuntos
Anticorpos Monoclonais/química , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/metabolismo , Limite de Detecção , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Reprodutibilidade dos Testes , Tripsina/metabolismoRESUMO
In the biotechnology industry, highly sensitive and accurate methods are required for monitoring glycosylation of therapeutic recombinant monoclonal antibodies (rMAbs) due to possible effects on bioactivity. At Genentech, a method employing PNGase F digestion, fluorescent labeling of released glycans, and analysis by capillary electrophoresis (CE) is used for routine monitoring of N-linked glycosylation during process development and quality control of therapeutic glycoproteins. In our laboratory, capillary electrophoresis-mass spectrometry (CE-MS) technology was developed to identify minor glycan species in assay and it revealed several unidentified isomeric species. Additional studies indicate that these species (1-10% total glycans) are sample preparation artifacts caused by base-catalyzed epimerization of N-acetylglucosamine (GlcNAc) at the reducing terminus by following the use of commercially available PNGase F and the supplied incubation buffer (pH 7.5). As these isomeric species directly impact the accuracy of the reported results, an optimized PNGase F release step is presented which minimizes and/or eliminates the formation of these artifacts. We have found that PNGase F incubation at pH 5.5 for IgG(1) rMAbs shows no significant decrease in enzyme activity while minimizing GlcNAc epimerization. Implementation of this change has resulted in a more accurate and robust CE-laser-induced fluorescence (LIF) assay and is generally applicable to any analysis requiring PNGase F digestion of rMAbs.
Assuntos
Eletroforese Capilar/métodos , Polissacarídeos/química , Acetilglucosamina/química , Catálise , Corantes Fluorescentes , Espectrometria de Massas , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/químicaRESUMO
Glycan characterization of therapeutic proteins is of utmost importance due to the role of carbohydrates in protein stability, half-life, efficacy and mechanism of action. The primary assay for characterization and lot release of N-linked glycans on glycoprotein products at Genentech, Inc., is a capillary electrophoresis (CE) based assay, wherein PNGase F-released, APTS-labeled glycans are separated by CE with laser induced fluorescence (LIF) detection. With the growing number of new molecular entities in the pipeline, a fast and direct characterization approach is of increasing importance. This paper describes the development of CE-MS technology with on-line LIF detection that allows identification of major and minor glycan species (1-5% of total glycans) by providing accurate mass information. Data is presented for therapeutic rMAbs which presented previously unidentified, minor peaks during routine CE-LIF analysis. CE-LIF-MS was then used to provide accurate mass on these species, identifying CE peaks corresponding to sialylated (G1 + NANA, G2 + NANA), afucosylated (G0-GlcNAc-fucose) and low-level isomers of major APTS-labeled glycans G0, G1, G1' and G2.
Assuntos
Anticorpos Monoclonais/química , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Polissacarídeos/análise , Fluorescência , LasersRESUMO
Capillary electrophoresis (CE) was compared with reversed-phase liquid chromatography for its ability to separate native and deamidated peptides. CE is shown to provide superior resolution of these peptides due to its charge-based separation mechanism. Fraction collection performed using a standard CE instrument equipped with a 96-well plate permits subsequent characterization by nanospray mass spectrometric (MS) analysis. Additional in-depth analysis by MS/MS is able to provide the location of the deamidation site based on y-ion mass shifts of 1Da.
Assuntos
Eletroforese Capilar/métodos , Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , DesaminaçãoRESUMO
With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE-mass spectrometry (CE-MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE-MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE-laser-induced fluorescence (CE-LIF). For each study, CE-MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE-MS for profiling biomolecules.
Assuntos
Eletroforese Capilar/métodos , Glicoconjugados/análise , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sequência de Carboidratos , Fluorescência , Glicoconjugados/química , Glicoconjugados/metabolismo , Indicadores e Reagentes , Lasers , Dados de Sequência Molecular , Pirenos/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
The significant complexity, similar polarity and lack of ionizable sites make the analysis of glycans an analytical challenge. These compounds are often derivatized and separated by normal-phase high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) followed by UV or fluorescence detection. Due to widespread use of reversed-phase chromatography coupled to electrospray mass spectrometry as an analytical tool, our laboratory has developed this methodology for the analysis of glycans derivatized with a negatively charged tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). It is possible to exploit the ability of this negatively charged tag to interact with a mobile phase ion-pairing reagent, allowing retention on a reversed-phase C(18) column for subsequent on-line UV or MS analysis. ANTS-derivatized samples, including a maltooligosaccharide ladder and glycans released from bovine ribonuclease B, bovine fetuin, and chicken ovalbumin, were analyzed using this method. In addition to reversed-phase retention, ribonuclease B and ovalbumin derivatives displayed highly desirable isomeric separation. With the use of mass spectrometric detection for glycan identity, this allowed relative quantitation of individual components.
Assuntos
Polissacarídeos/análise , Animais , Bovinos , Galinhas , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Ovalbumina/análise , Ribonucleases/química , Espectrofotometria Ultravioleta , alfa-Fetoproteínas/análiseRESUMO
The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.
Assuntos
Eletroforese Capilar/métodos , Polissacarídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Corantes Fluorescentes , Indicadores e Reagentes , Oligossacarídeos/química , alfa-Fetoproteínas/químicaRESUMO
Benzo[a]pyrene (BP) is a ubiquitous environmental polycyclic aromatic hydrocarbon (PAH) which, upon metabolic conversion to reactive benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), has been found to attach covalently to DNA. Given the low level of DNA adducts typically present in vivo or in vitro, an essential first step prior to capillary electrophoresis/mass spectrometry (CE/MS) (or liquid chromatography/mass spectrometry (LC/MS)) analysis of the DNA digests is the removal of the bulk non-adducted nucleotides, enzymes or salts, and isolation of enriched adducts. This report focuses on the development of novel sample handling methods aimed at facilitating the analysis of BPDE-DNA adducts by CE/MS. This approach involves a simple variation on the digestion procedure, in combination with the use of metal affinity ZipTips for the more efficient cleanup of BPDE-DNA adducts formed in vitro for subsequent CE/MS analysis. The previously described digestion procedure, consisting of micrococcal nuclease, spleen phosphodiesterase and nuclease P1, allows for selective dephosphorylation of normal nucleotides, while leaving adducted nucleotides intact. Metal affinity ZipTips, typically used for selective extraction of phosphopeptides, were used here for extraction of adducted nucleotides. The utility of metal affinity SPE was tested on mixtures of dG and dGp, wherein nucleotide extracts contained no detectable nucleosides by CE/UV analysis. An in vitro BPDE-DNA incubation was then digested using the above procedure. Metal affinity solid-phase extraction (SPE) was subsequently used for the selective isolation of phosphorylated components, i.e., adducted nucleotides, from the mixture of enzymes and non-adducted nucleosides. SPE extracts were enriched in nucleotide adducts and analyzed using sample stacking and CE/MS. This method has several advantages over previously described cleanup procedures for dGp-BPDE adducts: fast, simple, uses commercially available materials, no need for excessive dilution (small scale), the suitability for use with automation, and possible applicability to other bulky hydrophobic adducts.