In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody.

Characterization of antibody charge heterogeneity is an important task for antibody drug development. Recently, a correlation between acidic charge heterogeneity and metal-catalyzed oxidation has been observed for antibody drugs. However, to date, the acidic variants induced by metal-catalyzed oxidation have not been elucidated. In addition, it is challenging to satisfactorily explain the induced acidic charge heterogeneity, as the existing analytical workflows, which relied on either untargeted or targeted peptide mapping analysis, could lead to incomplete identification of the acidic variants. In this work, we present a new characterization workflow by combining untargeted and targeted analyses to thoroughly identify and characterize the induced acidic variants in a highly oxidized IgG1 antibody. As a part of this workflow, a tryptic peptide mapping method was also developed for accurate determination of the relative extent of site-specific carbonylation, where a new hydrazone reduction procedure was established to minimize under-quantitation artifacts caused by incomplete reduction of hydrazones during sample preparation. In summary, we identified 28 site-specific oxidation products, which are located on 26 residues and of 11 different modification types, as the sources of the induced acidic charge heterogeneity. Many of the oxidation products were reported for the first time in antibody drugs. More importantly, this study provides new insights to understanding acidic charge heterogeneity of antibody drugs in the biotechnology industry. Additionally, the characterization workflow presented in this study can be applied as a platform approach in the biotechnology industry to better address the need for in-depth characterization of antibody charge variants.

Investigation of Metal-Catalyzed Antibody Carbonylation With an Improved Protein Carbonylation Assay.

Protein carbonylation is a posttranslational modification referring to the occurrence of aldehydes and ketones in proteins. The current understanding of how carbonylation, in particular, metal-catalyzed carbonylation, occurs in recombinant mAbs during production and storage is very limited. To facilitate investigations into mAb carbonylation, we developed a protein carbonylation assay with improved assay robustness and precision over the conventional assays. We applied this assay to investigate mAb carbonylation under production, storage, and stress conditions and showed that iron, hydrogen peroxide, and polysorbate 20 at pharmaceutically relevant levels critically influence the extent of mAb carbonylation. In addition, we found that while carbonylation correlates with mAb aggregation in several cases, carbonylation cannot be used as a general indicator for aggregation. Furthermore, we observed that mAb carbonylation level can decrease during storage, which indicates that carbonylation products may not be stable. Finally, we report for the first time a positive correlation between carbonylation and acidic charge heterogeneity of mAbs that underwent metal-catalyzed oxidation. This finding shows that the impact of protein carbonylation on product quality for mAbs is not limited to aggregation but also extends to charge heterogeneity.

Phase 2 Randomized, Double-Blind, Placebo-Controlled Trial of RG7667, a Combination Monoclonal Antibody, for Prevention of Cytomegalovirus Infection in High-Risk Kidney Transplant Recipients.

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.).

Establishing a control system using QbD principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the elements and tools used to establish acceptance criteria and an attribute testing strategy (ATS) for product variants and process related impurities. The acceptable ranges for CQAs are set based on their potential impact on efficacy and safety/immunogenicity. This approach is focused on the management of patient impacts, rather than simply maintaining a consistent analytical profile. The ATS tools were designed to identify quality attributes that required process and/or testing controls, or that could be captured in a monitoring system to enable lifecycle management of the control strategy.

Characterization of oxidative carbonylation on recombinant monoclonal antibodies.

In the biotechnology industry, oxidative carbonylation as a post-translational modification of protein pharmaceuticals has not been studied in detail. Using Quality by Design (QbD) principles, understanding the impact of oxidative carbonylation on product quality of protein pharmaceuticals, particularly from a site-specific perspective, is critical. However, comprehensive identification of carbonylation sites has so far remained a very difficult analytical challenge for the industry. In this paper, we report for the first time the identification of specific carbonylation sites on recombinant monoclonal antibodies with a new analytical approach via derivatization with Girard's Reagent T (GRT) and subsequent peptide mapping with high-resolution mass spectrometry. Enhanced ionization efficiency and high quality MS(2) data resulted from GRT derivatization were observed as key benefits of this approach, which enabled direct identification of carbonylation sites without any fractionation or affinity enrichment steps. A simple data filtering process was also incorporated to significantly reduce false positive assignments. Sensitivity and efficiency of this approach were demonstrated by identification of carbonylation sites on both unstressed and oxidized antibody bulk drug substances. The applicability of this approach was further demonstrated by identification of 14 common carbonylation sites on three highly similar IgG1s. Our approach represents a significant improvement to the existing analytical methodologies and facilitates extended characterization of oxidative carbonylation on recombinant monoclonal antibodies and potentially other protein pharmaceuticals in the biotechnology industry.

Compatibility and stability of pertuzumab and trastuzumab admixtures in i.v. infusion bags for coadministration.

The physical/chemical stability and potential interactions after diluting two immunoglobulin G1 monoclonal antibodies (mAb), pertuzumab (Perjeta®) and trastuzumab (Herceptin®), in a single intravenous (i.v.) infusion bag containing 0.9% saline (NaCl) solution was evaluated. As commercial products, pertuzumab and trastuzumab are administered through i.v. infusion to patients sequentially, that is, one drug after the other. To increase convenience and minimize the in-clinic time for patients, the compatibility of coadministering pertuzumab (420 and 840 mg) mixed with either 420 or 720 mg trastuzumab, respectively, in a single 250 mL polyolefin or polyvinyl chloride i.v. bag stored for up to 24 h at 5°C or 30°C was determined. The controls (i.e., pertuzumab alone in an i.v. bag, trastuzumab alone in an i.v. bag) and the mAb mixture were assessed using color, appearance, and clarity, concentration and turbidity by ultraviolet spectroscopy, particulate analysis by light obscuration, size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate, analytical ultracentrifugation, and ion-exchange chromatography. Additionally, capillary zone electrophoresis, imaged capillary isoelectric focusing, and potency were utilized to measure the stability of the admixtures containing 1:1 mixtures of pertuzumab/trastuzumab and their respective controls (420 mg pertuzumab alone and 420 mg trastuzumab alone). No observable differences were detected by the above methods in the pertuzumab/trastuzumab mixtures stored up to 24 h at either 5°C or 30°C. The physicochemical methods as listed above were able to detect both molecules as well as the minor variants in the drug mixture, even though some overlap of mAb species were seen in the chromatograms and electropherograms. Furthermore, biophysical analysis also did not show any interactions between the two mAbs or any physical instability under these conditions. Additionally, the drug mixture tested by the pertuzumab-specific inhibition of cell proliferation bioassay showed comparable potency before and after storage. On the basis of these results, pertuzumab and trastuzumab admixture in a single i.v. bag is physically and chemically stable for up to 24 h at 5°C or 30°C and can be used for clinical administration.

Capillary electrophoresis-mass spectrometry methods for tryptic peptide mapping of therapeutic antibodies.

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.

Characterization of deamidated peptide variants by micro-preparative capillary electrophoresis and mass spectrometry.

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.

Investigation of sample preparation artifacts formed during the enzymatic release of N-linked glycans prior to analysis by capillary electrophoresis.

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.

On-line CE-LIF-MS technology for the direct characterization of N-linked glycans from therapeutic antibodies.

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.

Removal of C-ring from the CD-ring skeleton of 1alpha,25-dihydroxyvitamin D3 does not alter its target tissue metabolism significantly.

It is now well established that 1alpha,25(OH)2D3 is metabolized in its target tissues through the modifications of both side chain and A-ring. The C-24 oxidation pathway is the side chain modification pathway through which 1alpha,25(OH)2D3 is metabolized into calcitroic acid. The C-3 epimerization pathway is the A-ring modification pathway through which 1alpha,25(OH)2D3 is metabolized into 1alpha,25(OH)2-3-epi-D3. During the past two decades, a great number of vitamin D analogs were synthesized by altering the structure of both side chain and A-ring of 1alpha,25(OH)2D3 with the aim to generate novel vitamin D compounds that inhibit proliferation and induce differentiation of various types of normal and cancer cells without causing significant hypercalcemia. Previously, we used some of these analogs as molecular probes to examine how changes in 1alpha,25(OH)2D3 structure would affect its target tissue metabolism. Recently, several nonsteroidal analogs of 1alpha,25(OH)2D3 with unique biological activity profiles were synthesized. Two of the analogs, SL 117 and WU 515 lack the C-ring of the CD-ring skeleton of 1alpha,25(OH)2D3. SL 117 contains the same side chain as that of 1alpha,25(OH)2D3, while WU 515 contains an altered side chain with a 23-yne modification combined with hexafluorination at C-26 and C-27. Presently, it is unknown how the removal of C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 would affect its target tissue metabolism. In the present study, we compared the metabolic fate of SL 117 and WU 515 with that of 1alpha,25(OH)2D3 in both the isolated perfused rat kidney, which expresses only the C-24 oxidation pathway and rat osteosarcoma cells (UMR 106), which express both the C-24 oxidation and C-3 epimerization pathways. The results of our present study indicate that SL 117 is metabolized like 1alpha,25(OH)2D3, into polar metabolites via the C-24 oxidation pathway in both rat kidney and UMR 106 cells. As expected, WU 515 with altered side chain structure is not metabolized via the C-24 oxidation pathway. Unlike in rat kidney, both SL 117 and WU 515 are also metabolized into less polar metabolites in UMR 106 cells. These metabolites displayed GC and MS characteristics consistent with A-ring epimerization and were putatively assigned as C-3 epimers of SL 117 and WU 515. In summary, we report that removal of the C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 does not alter its target tissue metabolism significantly.

Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins.

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.

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1.

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.

Selective digestion and novel cleanup techniques for detection of benzo[a]pyrene diol epoxide-DNA adducts by capillary electrophoresis/mass spectrometry.

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.

Reversed-phase ion-pairing liquid chromatography/ion trap mass spectrometry for the analysis of negatively charged, derivatized glycans.

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.

1Alpha,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of 1alpha,25-dihydroxy vitamin D3: production and biological activity studies in pulmonary alveolar type II cells.

Pulmonary alveolar type II cells have been shown to be a possible target for the secosteroid hormone, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], during perinatal transition. At present, there is great interest to isolate and identify the metabolites of 1alpha,25(OH)2D3 produced in its target tissues and to determine the contribution of each individual metabolite of 1alpha,25(OH)2D3 to the final expression of the pleiotropic actions attributed to 1alpha,25(OH)2D3. Of all the known metabolites of 1alpha,25(OH)2D3, 1alpha,25(OH)2-3-epi-D3 has gained most attention as it is produced only in specific tissues and possesses significant activity in tissues in which it is produced. Furthermore, in vivo studies indicate that this metabolite when compared to 1alpha,25(OH)2D3 is less calcemic. Therefore, we performed the present study to identify production of 1alpha,25(OH)2-3-epi-D3 in alveolar type II cells, and to evaluate its effect on surfactant synthesis. We incubated NCI-H441 cells, an alveolar type II cell line, with 1alpha,25(OH)2D3 and demonstrated that these cells metabolize 1alpha,25(OH)2D3 to various previously well-characterized polar metabolites, and to a less polar metabolite which was unequivocally identified as 1alpha,25(OH)2-3-epi-D3 by GC/MS and HPLC analysis. Further, biological activity studies in H441 cells indicated that 1alpha,25(OH)2-3-epi-D3 possesses significant activity in terms of its ability: (i) to increase surfactant phospholipid synthesis, (ii) to induce surfactant SP-B mRNA gene expression, and (iii) to increase surfactant SP-B protein synthesis. However, the activity of 1alpha,25(OH)2-3-epi-D3 when compared to 1alpha,25(OH)2D3 in generating VDR-mediated transcriptional activity in ROS 17/2.8 cells transfected with human osteocalcin VDRE/growth hormone gene construct, was significantly reduced. The high metabolic stability of 1alpha,25(OH)2-3-epi-D3, as previously proposed by us, may be a possible explanation for the high in vitro activity in spite of the reduced VDR-mediated transcriptional activity. In summary, we report for the first time the pathways of 1alpha,25(OH)2D3 metabolism in pulmonary alveolar type II cells and indicate that 1alpha,25(OH)2-3-epi-D3, a natural intermediary metabolite of 1alpha,25(OH)2D3 possesses significant activity in stimulating surfactant synthesis in alveolar type II cells.

Metabolism of 2-methyl analogs of 1alpha,25-dihydroxyvitamin D3 in rat osteosarcoma cells (UMR 106).

Several novel A-ring modified analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] have been synthesized in order to investigate the structure-function relationships of 1alpha,25(OH)2D3. We synthesized A-ring modified analogs which contain a methyl group on C-2 of the A-ring. There are eight 2-methyl diastereomers, which differ in the stereochemistry of the methyl group on C-2 and the hydroxyl groups on C-1 and C-3. Further our biological activity studies of the 2-methyl diastereomers indicated that the potency of each analog is highly dependent on the stereochemistry of the A-ring substituents [Konno et al., Biorg. Med. Chem. Letts. 8(2), 151-156 (1998); Nakagawa et al., Biochem. Pharmacol. 60(12), 1937-1947 (2000)]. For example, the VDR binding affinities exhibited by the 1alpha-isomers are significantly higher than those exhibited by the 1beta-isomers. Furthermore, out of all the 1alpha-isomers, the 2alpha-methyl isomers, when compared to the corresponding 2beta-methyl isomers, showed much higher potency in inducing cell differentiation of HL-60 cells, but failed to stimulate apoptosis. In contrast the 2beta-methyl isomers strongly stimulated apoptosis. At present it is unknown how the addition of the 2-methyl modification to the hormone, 1alpha,25(OH)2D3 alters its metabolism in target tissues. Previously, we reported that 1alpha,25(OH)2D3 is metabolized in rat osteosarcoma (UMR 106) cells via both the C-24 oxidation and the C-3 epimerization pathways. Therefore, we studied the metabolism of the four 1alpha,2-methyl diastereomers in UMR 106 cells. Our results indicated that in UMR 106 cells, all four diastereomers were metabolized into several polar metabolites via the C-24 oxidation pathway. Thus, the presence of the 2-methyl group on the A-ring did not inhibit the metabolism of the analogs via the C-24 oxidation pathway. However, it is significant to note that the 2-methyl group prevented the metabolism of the analogs via the C-3 epimerization pathway. In summary, we report that the 2-methyl group interferes with the action of the enzyme(s) involved in C-3 epimerization, but not with the enzyme 1alpha,25(OH)2D3-24-hydroxylase, which is responsible for C-24 oxidation pathway.

Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

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.