Characterization of protein therapeutics by mass spectrometry: recent developments and future directions.

Mass spectrometry (MS) has become a powerful technology in the discovery and development of protein therapeutics in the biopharmaceutical industry. This review article describes recent developments and future trends in the characterization of protein therapeutics using MS. We discuss top-down MS for the characterization of protein modifications, hydrogen/deuterium exchange MS and ion mobility MS methods for higher order protein structure studies. Quantitative analysis of protein therapeutics (in vivo) by MS as an orthogonal approach to immunoassay for pharmacokinetics studies will also be illustrated.

A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins.

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.

Periodic, partial inhibition of IkappaB Kinase beta-mediated signaling yields therapeutic benefit in preclinical models of rheumatoid arthritis.

We have previously shown that inhibitors of IkappaB kinase beta (IKKbeta), including 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline (BMS-345541), are efficacious against experimental arthritis in rodents. In our efforts to identify an analog as a clinical candidate for the treatment of autoimmune and inflammatory disorders, we have discovered the potent and highly selective IKKbeta inhibitor 2-methoxy-N-((6-(1-methyl-4-(methylamino)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-7-yl)pyridin-2-yl)methyl)acetamide (BMS-066). Investigations of its pharmacology in rodent models of experimental arthritis showed that BMS-066 at doses of 5 and 10 mg/kg once daily was effective at protecting rats against adjuvant-induced arthritis, despite showing only weak inhibition at 10 mg/kg against a pharmacodymanic model of tumor necrosis factor alpha production in rats challenged with lipopolysaccharide. The duration of exposure in rats indicated that just 6 to 9 h of coverage per day of the concentration necessary to inhibit IKKbeta by 50% in vivo was necessary for protection against arthritis. Similar findings were observed in the mouse collagen-induced arthritis model, with efficacy observed at a dose providing only 6 h of coverage per day of the concentration necessary to inhibit IKKbeta by 50%. This finding probably results from the cumulative effect on multiple cellular mechanisms that contribute to autoimmunity and joint destruction, because BMS-066 was shown to inhibit a broad spectrum of activities such as T cell proliferation, B cell function, cytokine and interleukin secretion from monocytes, T(H)17 cell function and regulation, and osteoclastogenesis. Thus, only partial and transient inhibition of IKKbeta is sufficient to yield dramatic benefit in vivo, and this understanding will be important in the clinical development of IKKbeta inhibitors.

Development and implementation of a stereoselective normal-phase liquid chromatography-tandem mass spectrometry method for the determination of intrinsic metabolic clearance in human liver microsomes.

The stereoselective determination of stereoisomers in biological samples provides vital information on stereospecific metabolism and pharmacokinetic profiles of the drugs. Despite the unique advantage and the great success of normal-phase (NP) HPLC for the separations of drug stereoisomers using polysaccharide-type chiral stationary phases (CSPs), the technique is rarely applied to quantitative HPLC-MS-MS bioanalysis. This is, at least in part, due to the incompatibility between the usual mobile phase (n-hexane or n-heptane) in normal-phase HPLC and the MS ionization sources which poses a potential detonation hazard. An environmentally friendly and nonflammable alternative solvent, ethoxynonafluorobutane (ENFB), was reported previously to potentially provide an ideal solution for combining the powers of stereoselective NP chromatographic separation and MS-MS detection. In this study, a stereoselective NP-HPLC-MS-MS method was developed using ENFB to quantify a pair of Bristol Myers Squibb (BMS) proprietary drug stereoisomers and their ketone metabolite for an in vitro study, which demonstrated, for the first time, the practical applicability and utility of ENFB for bioanalysis in pharmaceutical industry. The effects of different organic modifiers and temperature, as well as the comparison between ENFB and the usual solvent, heptane, for the separation, are discussed. The resolution of the stereoisomers was achieved using 63% of 3:1 mixture of ethanol and methanol with 37% ENFB on a Chiralpak AD-H column at 50 degrees C. High sensitivity was obtained using the MS-MS detection in the positive ion atmospheric pressure chemical ionization (APCI) mode. The lower limit of quantitation (LLOQ) for the first stereoisomer and the ketone metabolite was 5 ng/mL, and was 10 ng/mL for the second isomer in the human liver microsome-potassium phosphate buffer matrix. The linear dynamic range of 5-1000 ng/mL for both isomers and 10-1000 ng/mL for the metabolite were demonstrated with R2 > or =0.997. The precision of the analysis was <5% R.S.D. at or above the nominal concentration of 80 ng/mL, and <20% R.S.D. at 8 ng/mL. The mean bias was less than 15%. Extraction recovery and acceptable matrix interference were demonstrated using one isomer and the ketone, and better than 75% recovery and less than 25% ion suppression or interference were found. The method was successfully implemented for an in vitro intrinsic metabolic clearance study.

Simulated moving columns technique for enantioselective supercritical fluid chromatography.

This article describes a very useful extension of an unique column switching technique called "Simulated Moving Columns" (SMC) that was previously reported for chiral high performance liquid chromatography (HPLC) (Zhang and McConnell, Journal of Chromatography A 2004;1028:227-238). SMC uses two or three short chiral columns connected in series, and enables the unresolved enantiomers to separate repeatedly and exclusively through each of the columns until sufficient resolution is attained. The technique is significantly enhanced through the use of supercritical fluid chromatography (SFC). The supercritical or near critical carbon dioxide (CO(2)) used in the mobile phase of SFC possesses the properties of a liquid as well as a gas, and usually results in much sharper peaks compared to HPLC. Consequently, by combining SMC with SFC (SMC-SFC), we were able to dramatically increase the number of SMC cycles with significantly less band broadening compared to HPLC. For the first time, an enantioselective SFC separation was demonstrated by increasing the column from the actual 20 cm length to reach a half meter virtual length with remarkably enhanced efficiency. Off-column band broadening resulting from a two-column SMC system was measured, and its impact on the enantioselectivity of SMC-SFC was found to be much less than in SMC-HPLC.

Enantioselective chromatography in drug discovery.

Molecular chirality is a fundamental consideration in drug discovery, one necessary to understand and describe biological targets as well as to design effective pharmaceutical agents. Enantioselective chromatography has played an increasing role not only as an analytical tool for chiral analyses, but also as a preparative technique to obtain pure enantiomers from racemates quickly from a wide diversity of chemical structures. Different enantioselective chromatography techniques are reviewed here, with particular emphasis on the most widespread high performance liquid chromatography (HPLC) and the rapidly emerging supercritical fluid chromatography (SFC) techniques. This review focuses on the dramatic advances in the chiral stationary phases (CSPs) that have made HPLC and SFC indispensable techniques for drug discovery today. In addition, screening strategies for rapid method development and considerations for laboratory-scale preparative separation are discussed and recent achievements are highlighted.

High-throughput chiral analysis of albuterol enantiomers in dog plasma using on-line sample extraction/polar organic mode chiral liquid chromatography with tandem mass spectrometric detection.

An automated chiral chromatography/tandem mass spectrometry bioanalytical method for the determination of albuterol in dog plasma was developed. The method employed on-line sample extraction using turbulent flow chromatography coupled to a Chirobiotic T column for chiral separation using a polar organic mobile phase consisting of methanol, 0.02% formic acid, and 0.1% ammonium formate. The analytes were detected by a tandem mass spectrometer operated in positive ion mode. The (S)- and (R)-isomers were resolved chromatographically with retention times of 5.1 and 5.6 min, respectively. The analytical run time was 8 min. The enantiomers did not interconvert either in mobile phase or in dog plasma at room temperature over the course of at least 2 h. The assay has a linear dynamic range from 2.5-2500 nM for both enantiomers. The lower limit of quantitation (LLOQ) was 2.5 nM for both enantiomers using 50 microL of plasma. The accuracy and precision of intraday validation were determined at five concentration levels of six replicates. The accuracy of the method for the (R)-isomer ranged from 94-103% of nominal concentrations, and the precision (%CV) ranged from 3.6-12%. The accuracy of the method for the (S)-isomer ranged from 94.5-108% of nominal concentrations, and the precision ranged from 3.2-9.3%. Interday accuracy and precision were evaluated for three days at five concentrations for one replicate. The accuracy of the method for the (R)-isomer ranged from 98-110% of nominal concentrations, and the precision ranged from 1.5-10.6%. The accuracy of the method for the (S)-isomer ranged from 96-104% of nominal concentrations, and the precision ranged from 1.5-8.7%. The combination of turbulent flow on-line sample extraction with polar organic mode chiral chromatography provided a specific, rugged, and high-throughput method for the chiral analysis of albuterol in biological fluids.

Molecular design and structure--activity relationships leading to the potent, selective, and orally active thrombin active site inhibitor BMS-189664.

A series of structurally novel small molecule inhibitors of human alpha-thrombin was prepared to elucidate their structure-activity relationships (SARs), selectivity and activity in vivo. BMS-189664 (3) is identified as a potent, selective, and orally active reversible inhibitor of human alpha-thrombin which is efficacious in vivo in a mouse lethality model, and at inhibiting both arterial and venous thrombosis in cynomolgus monkey models.