Tam T. T. N. Nguyen

Determination of platinum drug release and liposome stability in human plasma by CE-ICP-MS.

An in vitro method for simultaneous assessment of platinum release and liposome stability of liposomal formulations in human plasma is demonstrated. The development and assessment of the method was performed on a PEGylated liposomal formulation containing cisplatin. Complete separation of free cisplatin, encapsulated cisplatin and cisplatin bound to plasma components was achieved by capillary electrophoresis (CE) separation and simultaneous monitoring of phosphorous (phospholipid) and platinum (cisplatin) by inductively coupled plasma mass spectrometry (ICP-MS). The method allows assessment of the encapsulation efficiency of the formulation, the physical stability of liposomes as well as cisplatin leakage in human plasma. The method was applied for studying the disintegration of liposomes and the interactions of leaked cisplatin with plasma components. Triggered release of the drug into plasma by sonication was also demonstrated. Analysis of liposomal formulations with alternative phospholipid compositions containing oxaliplatin showed similar results. Thus, the present in vitro method is suitable for mimicking the in vivo drug release profile in human plasma after administration of liposomal platinum formulations to patients. This approach may be of use in early drug development as well as in quality control.

Characterization of a liposome-based formulation of oxaliplatin using capillary electrophoresis: encapsulation and leakage.

A capillary electrophoresis-based method to characterize a PEGylated liposomal drug formulation of the anti-cancer agent oxaliplatin was developed. Pharmaceutical characterization in terms of determination of the free and total oxaliplatin concentrations in the liposomal formulation was successfully performed allowing calculation of the percentage of encapsulated drug and encapsulation efficiency. The trapping efficiency was likewise calculated. The capillary electrophoresis method allowed liposome characterization in the intended formulation media (sucrose solution with low electrolyte concentration), and the attained results were consistent with inductively coupled plasma mass spectrometry measurements. Accelerated drug leakage studies were initiated by the sonication of the PEGylated formulation, using an ultrasound probe, subsequently the drug leakage was determined by capillary electrophoresis. The results obtained with the PEGylated liposomes demonstrate that capillary electrophoresis may be a useful tool for the characterization of liposomal drug formulations.

Thiol-ene Monolithic Pepsin Microreactor with a 3D-Printed Interface for Efficient UPLC-MS Peptide Mapping Analyses

To improve the sample handling, and reduce cost and preparation time, of peptide mapping LC-MS workflows in protein analytical research, we here investigate the possibility of replacing conventional enzymatic digestion methods with a polymer microfluidic chip based enzyme reactor. Off-stoichiometric thiol-ene is utilized as both bulk material and as a monolithic stationary phase for immobilization of the proteolytic enzyme pepsin. The digestion efficiency of the, thiol-ene based, immobilized enzyme reactor (IMER) is compared to that of a conventional, agarose packed bed, pepsin IMER column commonly used in LC-MS based protein analyses. The chip IMER is found to rival the conventional column in terms of digestion efficiency at comparable residence time and, using a 3D-printed interface, be directly interfaceable with LC-MS.

Simultaneous measurement of phosphorus and platinum by Size Exclusion Chromatography coupled to Inductively Coupled Plasma Mass Spectrometry (SEC-ICPMS) using xenon as reactive collision gas for characterization of platinum drug liposomes

Liposomes have attracted intensive attention as drug delivery systems in anti-cancer therapy. Since liposomes are constructed by self-assembling of phospholipids, ICP-MS is a suitable method for simultaneous determination of liposomes and encapsulated metallic drug substances such as platinum-based drugs. An efficient method for simultaneous determination of phosphorus and platinum in liposome samples has been established based on the use of xenon as a collision gas in DRC-ICP-MS. Under the optimum conditions with respect to signal to noise ratio, the interferences were suppressed and the detection limits of phosphorus and platinum were 0.3 and 0.05 ng mL−1, respectively. Quality control was performed by using a certified reference material BCR 273 and biological reference materials. For the purpose of investigation of liposome stability and metallo-drug release from liposomes, a hyphenated method based on size exclusion chromatography was developed for separation of free and encapsulated platinum in a model liposome formulation of oxaliplatin. Moreover, an accelerated drug release study was performed by sonication of liposomal samples and using the developed hyphenated method to determine the drug leakage. It has been demonstrated that the SEC-DRC-ICP MS method was an efficient tool in the development and characterization of liposome based formulations of metallic drugs.

Evaluation of a membrane desolvator for LC-ICP-MS analysis of selenium and platinum species for application to peptides and proteins

Analysis of peptides and proteins and their interactions with endogenous elements and metal-based drugs in biological systems demands highly efficient chromatographic systems with the possibility of performing gradient elution to achieve efficient separations. As the detector of choice in metal analysis, the ICP-MS, does not tolerate a high load of organic solvents, these should be removed from the chromatographic eluent prior to the entrance of the ICP-MS detector. The purpose of this study was to evaluate a membrane desolvation (MD) system (Aridus II) for its capability of removing organic solvents from the eluent prior to the ICP-MS introduction and at the same time study the influence on sensitivity and examine if the desolvator system jeopardized the inherent species independent sensitivity of ICP-MS. Selenium and platinum were used as model elements. The MD system was optimized regarding to sweep gas and nitrogen gas flow rates. Sensitivity was highly dependent on the combination of sweep gas flow and N2 addition, and the desolvator system should be optimized for each element. After optimization, 100% methanol and acetonitrile were tolerated by the ICP-MS with an eluent flow rate of 0.2 mL min−1. This opens the possibility of performing LC-ICP-MS analysis by gradient elution with 0–100% organic solvents. Sensitivities were generally increased by employment of the MD system, but the species independent sensitivity of ICP-MS was lost for selenium compounds (trimethylselenonium ion (TMSe), selenomethionine (SeMet), Se-methylselenocysteine (Se-MeSeCys), Se-methylseleno-N-acetyl-galactosamine (SeGalac), selenite and selenate). Sensitivities of the different Se compounds were highly dependent on the desolvator temperature. Different Pt compounds (inorganic Pt-salt, cisplatin and oxaliplatin) showed no species dependent behavior. Linearity was obtained for flow injection analysis of SeMet, TMSe and a selenopeptide in 50% methanol and of inorganic Pt, cisplatin and oxaliplatin in 50% acetonitrile. The optimized system was applied for gradient elution LC-ICP-MS of a cisplatin-albumin adduct and lysate and media samples from a cell uptake study of a selenopeptide.

Expression, Purification and Characterization of GMZ2’.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite

PurposeProduction and characterization of a chimeric fusion protein (GMZ2’.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2’.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite.MethodsGMZ2’.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS.ResultsCP-GMZ2’.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2’.10C. CP-GMZ2’.10C and IP-GMZ2’.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2’.10C and two reference fusion proteins encompassing similar parts of Pfs48/45.ConclusionGMZ2’.10C, combining GMZ2’ and correctly-folded Pfs48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.

Investigating the utility of minimized sample preparation and high-resolution mass spectrometry for quantification of monoclonal antibody drugs

HighlightsThree different LC–MS/MS methods using QqQ, Q‐TOF and Q‐Orbitrap were developed and compared for quantitation of a model mAb in biological fluids.The capabilities of HRMS combined with a simple sample treatment for fast and sensitive quantification of mAbs in complex matrices were revealed.The pharmacokinetic properties of Rendomab‐B1 in mice over 15 days after administration were determined. ABSTRACT Determination of the pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) is essential for their successful development as drugs. For this purpose, besides the traditional ligand binding assay (LBA), LC–MS/MS method using low resolution mass spectrometers (e.g. triple quadrupole (QqQ)) has become routinely used, however, complicated and lengthy sample pre‐treatment (employing immuno‐affinity) is often necessary for obtaining sufficient sensitivity and selectivity. In this study, we investigate the capabilities of high‐resolution MS instruments for circumventing the complex sample preparation currently needed for sensitive LC–MS/MS‐based quantification of mAbs. Employing a simple one‐step sample pre‐treatment workflow, we compare the ability of three different LC–MS platforms for absolute quantification of a representative monoclonal antibody Rendomab‐B1 in serum and plasma. The samples are subjected to protein precipitation with methanol, followed by pellet digestion with trypsin prior to LC–MS analysis. AQUA peptides based on two surrogate mAb peptides selected from an extensive in‐silico and experimental screening are used as internal standards. MS/MS acquisitions are developed and systematically examined for 1) a low‐resolution QqQ operated in selected reaction monitoring (SRM) acquisition mode, 2) a high‐resolution hybrid Quadrupole‐Orbitrap (Q‐Orbitrap) operated in parallel reaction monitoring (PRM) acquisition mode and 3) a high‐resolution hybrid Quadrupole‐Time‐of‐flight (Q‐TOF) operated in SRM acquisition mode with enhanced duty cycle (EDC) function. The sensitivity of the high‐resolution Q‐Orbitrap and Q‐TOF methods was significantly higher (LOD of 80 ng/mL) in serum/plasma samples than the low‐resolution QqQ method. Finally, the real‐world utility of the developed high‐resolution MS method with minimized sample handling was demonstrated and validated by determining the PK profile of Rendomab‐B1 in mice by a 10‐point in vivo study over 15 days.

The extraordinary thermal stability of EstA from S. islandicus is independent of post translational modifications

Enzymes from thermophilic and hyper‐thermophilic organisms have an intrinsic high stability. Understanding the mechanisms behind their high stability will be important knowledge for the engineering of novel enzymes with high stability. Lysine methylation of proteins is prevalent in Sulfolobus, a genus of hyperthermophilic and acidophilic archaea. Both unspecific and temperature dependent lysine methylations are seen, but the significance of this post‐translational modification has not been investigated. Here, we test the effect of eliminating in vivo lysine methylation on the stability of an esterase (EstA). The enzyme was purified from the native host S. islandicus as well as expressed as a recombinant protein in E. coli, a mesophilic host that does not code for any machinery for in vivo lysine methylation. We find that lysine mono methylation indeed has a positive effect on the stability of EstA, but the effect is small. The effect of the lysine methylation on protein stability is secondary to that of protein expression in E. coli, as the E. coli recombinant enzyme is compromised both on stability and activity. We conclude that these differences are not attributed to any covalent difference between the protein expressed in hyperthermophilic versus mesophilic hosts.