Carola W.N. Damen

Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab.

Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied.

Bioanalytical methods for the quantification of therapeutic monoclonal antibodies and their application in clinical pharmacokinetic studies

Monoclonal antibodies (mAbs) are an upcoming group of therapeutic compounds used to treat various types of diseases. Currently more than 100 mAbs are in clinical development. To establish the pharmacokinetic (PK) properties of mAbs many (pre-)clinical studies are executed. Samples of these studies are most often analysed using immunoassays. Although immunoassays are very fast and sensitive, there are also some limitations. Alternative assays using e.g. liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), therefore, are also being developed. This paper reviews methods for the bioanalysis of the therapeutic mAbs that are currently registered, and their application in (pre-) clinical PK studies. We will focus on immunoassays and will make the comparison with other analytical techniques. We conclude that immunoassays are still the analytical technique of first choice for the analysis of mAbs, but that, unfortunately, the provided information in the literature about method performance is scarce.

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.

The bioanalysis of the monoclonal antibody trastuzumab by high-performance liquid chromatography with fluorescence detection after immuno-affinity purification from human serum

For the quantification of therapeutic monoclonal antibodies in biological specimens, enzyme-linked immunosorbent assay (ELISA) is the most widely used technique. ELISAs have some limitations and therefore alternative analytical techniques are being explored. In this study we describe the development of a bioanalytical assay using high-performance liquid chromatography (HPLC) coupled with fluorescence detection for the bioanalysis of the monoclonal antibody trastuzumab. Different extraction procedures were explored, like isolation using protein A and protein G. Finally a method using immuno-affinity purification has been developed. Trastuzumab is isolated from human serum using sepharose coupled with anti-trastuzumab idiotype antibodies. After extraction samples are injected onto a Zorbax 300SB C8 column at 75 degrees C using the organic solvents isopropanol and acetonitrile with high eluotropic strengths. The assay quantifies trastuzumab from 5 to 40 microg/mL in human serum with accuracies <20%. Samples with concentrations above the upper limit of quantification (>ULOQ; >40 microg/mL) can be diluted 5 times with control human serum prior to sample pre-treatment. The assay can now be used to analyse serum samples of patients treated with trastuzumab. The obtained results are comparable to those obtained using ELISA. This is the first report describing a bioanalytical assay using HPLC and fluorescence detection for the quantification of a monoclonal antibody at the intact protein level in human serum. This unique approach has the advantage compared to ELISA that a HPLC separation step is introduced to improve the selectivity. This method is a potential alternative to ELISA to support pharmacokinetic evaluations. However, for purification of trastuzumab from serum anti-idiotype antibodies are necessary. These anti-idiotype antibodies are also used in ELISA and as ELISA is more sensitive and less labor-intensive, ELISA probably remains the analytical technique of first choice.

P-Glycoprotein, Multidrug-Resistance Associated Protein 2, Cyp3a, and Carboxylesterase Affect the Oral Availability and Metabolism of Vinorelbine

We investigated the interactions of the anticancer drug vinorelbine with drug efflux transporters and cytochrome P450 3A drug-metabolizing enzymes. Vinorelbine was transported by human multidrug-resistance associated protein (MRP) 2, and Mrp2 knockout mice displayed increased vinorelbine plasma exposure after oral administration, suggesting that Mrp2 limits the intestinal uptake of vinorelbine. Using P-glycoprotein (P-gp), Cyp3a-, and P-gp/Cyp3a knockout mice, we found that the absence of P-gp or Cyp3a resulted in increased vinorelbine plasma exposure, both after oral and intravenous administration. Surprisingly, P-gp/Cyp3a knockout mice displayed markedly lower vinorelbine plasma concentrations than wild-type mice upon intravenous administration but higher concentrations upon oral administration. This could be explained by highly increased formation of 4′-O-deacetylvinorelbine, an active vinorelbine metabolite, especially in P-gp/Cyp3a knockout plasma. Using wild-type and Cyp3a knockout liver microsomes, we found that 4′-O-deacetylvinorelbine formation was 4-fold increased in Cyp3a knockout liver and was not mediated by Cyp3a or other cytochrome P450 enzymes. In vitro incubation of vinorelbine with plasma revealed that vinorelbine deacetylation in Cyp3a and especially in P-gp/Cyp3a knockout mice but not in P-gp-deficient mice was strongly up-regulated. Metabolite formation in microsomes and plasma could be completely inhibited with the nonspecific carboxylesterase (CES) inhibitor bis(4-nitrophenyl) phosphate and partly with the CES2-specific inhibitor loperamide, indicating that carboxylesterase Ces2a, which was appropriately up-regulated in Cyp3a and especially in P-gp/Cyp3a knockout liver was responsible for the 4′O-deacetylvinorelbine formation. Such compensatory up-regulation can complicate the interpretation of knockout mouse data. Nonetheless, P-gp, Mrp2, Cyp3a, and Ces2a clearly restricted vinorelbine availability in mice. Variation in activity of their human homologs may also affect vinorelbine pharmacokinetics in patients.

Validated assay for the simultaneous quantification of total vincristine and actinomycin-D concentrations in human EDTA plasma and of vincristine concentrations in human plasma ultrafiltrate by high-performance liquid chromatography coupled with tandem mass spectrometry.

A sensitive, specific and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the simultaneous determination of total vincristine and actinomycin-D concentrations in human plasma and an assay for the determination of unbound vincristine are presented. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and heated electrospray ionization (H-ESI) were tested as ionization interfaces. For reasons of robustness ESI was chosen followed by tandem mass spectrometry (ESI-MS/MS). For the plasma assay a 30 microL aliquot was protein precipitated with acetonitrile/methanol (50:50, v/v) containing the internal standard vinorelbine and 10 microL volumes were injected onto the HPLC system. To determine unbound vincristine, ultrafiltrate was produced from plasma using 30 kDa centrifugal filter units. The plasma ultrafiltrate was mixed with methanol (50:50, v/v), internal standard vinorelbine was added and 20 microL aliquots were injected onto the HPLC system. Separation was achieved on a 50x2.1 mm i.d. Xbridge C18 column using 1 mM ammonium acetate/acetonitrile (30:70, v/v) adjusted to pH 10.5 with ammonia, run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies in plasma vincristine from 0.25 to 100 ng/mL and actinomycin-D from 0.5 to 250 ng/mL using plasma sample volumes of only 30 microL. Vincristine in plasma ultrafiltrate can be quantified from 1 to 100 ng/mL. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human plasma and plasma ultrafiltrate with the presented methods. The assays are now in use to support clinical pharmacological studies in children treated with vincristine and actinomycin-D.

The bioanalysis of vinorelbine and 4-O-deacetylvinorelbine in human and mouse plasma using high-performance liquid chromatography coupled with heated electrospray ionization tandem mass–spectrometry

A sensitive, specific and efficient high-performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4-O-deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 microL plasma aliquot was protein precipitated with acetonitrile-methanol (1:1, v/v) containing the internal standard vinorelbine-d3 and 20 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 x 2.1 mm i.d. Xbridge C(18) column using isocratic elution with 1 mm ammonium acetate-ammonia buffer pH 10.5-acetonitrile-methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4-O-deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 microL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4-O-deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre-)clinical pharmacologic studies with vinorelbine in humans and mice.

High-performance liquid chromatography coupled with mass spectrometry for the quantitative analysis of vinca-alkaloids in biological matrices: a concise survey from the literature.

The bioanalysis of vinca-alkaloids has been investigated extensively. High-performance liquid chromatography coupled to ultraviolet, fluorescence or electrochemical detection have been described. During recent years liquid chromatography coupled with mass spectrometry (LC-MS) has become the first choice for the quantitative bioanalysis of the vinca anticancer agents. This paper reviews recent methods for the bio-analysis of vinca-alkaloids using LC-MS, supplemented with our own experience. We will focus on sample pre-treatment, chromatography and MS detection and pay attention to problems which can occur during the bioanalysis of vinca-alkaloids. These problems encounter carry-over and absorption effects and solutions will be provided how to circumvent these problems.