Quantitation and characterization of serotype 6A activation for pneumococcal conjugate vaccine by cryo-EM and SEC methods.

Pneumococcal conjugate vaccines (PCV) typically consist of capsular polysaccharides from different S. pneumoniae serotypes which are covalently attached to carrier protein. A well-established process to manufacture PCV is through activating polysaccharide by oxidation of vicinal diols to aldehydes, followed by protein conjugation via reductive amination. Polysaccharide activation is a crucial step that affects vaccine product critical attributes including conjugate size and structure. Therefore, it is highly desired to have robust analytical methods to well characterize this activation process. In this study, using pneumococcal serotype 6A as the model, we present two complimentary analytical methods for characterization of activated polysaccharide. First, a size exclusion chromatography (SEC) method was developed for quantitative measurement of polysaccharide activation levels. This SEC method demonstrated good assay characteristics on accuracy, precision and linearity. Second, a gold nanoparticle labeled cryo-electron microscopy (Cryo-EM) technique was developed to visualize activation site distribution along polysaccharide chain and provide information on activation heterogeneity. These two complimentary methods can be utilized to control polysaccharide activation process and ensure consistent delivery of conjugate vaccine products.

Development and validation of a GC method for a key nemtabrutinib intermediate: Implementation of an in-situ free base sample preparation protocol.

A novel synthetic approach to nemtabrutinib (MK-1026) was recently developed in our laboratories. The chemistry goes through a cyrene amine intermediate which does not contain any chromophore. As a result, analysis of this key chiral intermediate by HPLC-UV is not feasible. Initial attempts to develop a HPLC-CAD method were unfruitful; therefore, a gas chromatography method was developed and optimized to effectively monitor the cyrene amine free base and related impurities generated during the process. As the synthetic process continued to be optimized, the toluene sulfonic acid salt (p-TsOH) of the cyrene amine intermediate was later identified by our process chemistry group to be beneficial in terms of ease of isolation and purity upgrade. However, repeated injections of the cyrene amine p-TsOH intermediate resulted in rapid GC column deterioration. After identifying p-TsOH as the main cause of the issue, we developed a straightforward and practical procedure that involves using a resin to remove the p-TsOH counterion in-situ, which converts cyrene amine salt to its neutral form in sample solutions. This protocol was successfully demonstrated and proven to be an efficient solution. This methodology may find applications with other analytes containing counterions that need to be neutralized prior to analysis.

Antibody enhanced HPLC for serotype-specific quantitation of polysaccharides in pneumococcal conjugate vaccine.

Bacterial infection remains as one of the major healthcare issues, despite significant scientific and medical progress in this field. Infection by Streptococcus Pneumoniae (S. Pneumoniae) can cause pneumonia and other serious infectious diseases, such as bacteremia, sinusitis and meningitis. The pneumococcal capsular polysaccharides (CPS) that constitute the outermost layer of the bacterial cell are the main immunogens and protect the pathogen from host defense mechanisms. Over 90 pneumococcal CPS serotypes have been identified, among which more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Multivalent pneumococcal vaccines have been developed to prevent diseases caused by S. Pneumoniae. These vaccines employ either purified pneumococcal CPSs or protein conjugates of these CPSs to generate antigen-specific immune responses for patient protection. Serotype-specific quantitation of these polysaccharides (Ps) antigen species are required for vaccine clinical dosage, product release and quality control. Herein, we have developed an antibody-enhanced high-performance liquid chromatography (HPLC) assay for serotype-specific quantitation of the polysaccharide contents in multivalent pneumococcal conjugate vaccines (PCVs). A fluorescence-labeled multiplex assay format has also been developed. This work laid the foundation for a serotype-specific antigen assay format that could play an important role for future vaccine research and development.

Identification and quantitation of isomeric pneumococcal polysaccharides by partial chemical degradation followed by mass spectrometry.

Quantitation of isomeric pneumococcal polysaccharides in vaccines is a challenging task due to mixture complexity, their low quantities, and identical monosaccharide compositions. Differentiation and quantitation of isomeric pneumococcal polysaccharides were investigated here based on a partial chemical degradation mass spectrometry approach to generate an oligosaccharide marker for one isomer, and not the other. Mild base conditions were successful at generating unique ions for the isomers with the weakest glycosidic bonds, while strong base and acid conditions were successful at generating unique ions for the more stable isomers. Linear relationships between the ion abundance of the oligosaccharide marker and the starting pneumococcal polysaccharides concentration were established for all isomers. Furthermore, precision measurements for each method were below 12% demonstrating good robustness. Therefore, partial chemical degradation followed by mass spectrometry was successful at differentiating and quantifying isomeric pneumococcal polysaccharides and may be adopted for other bacterial types.

Molecular interaction mechanisms in reverse micellar extraction of microbial transglutaminase.

Reverse micellar extraction is an efficient and economical alternative for protein purification. In this study, microbial transglutaminase (MTGase) from crude materials was purified using reverse micellar extraction, and the molecular interaction mechanism in reverse micellar extraction of MTGase was explored. By using a molecular simulation study, the interaction mechanism of forward extraction was investigated. The molecular simulation results reveal the interaction of MTGase-water-surfactant is the major driving force for the forward extraction. Further, the effect of ionic strength on molecular interactions in backward extraction was investigated using 1H low-field nuclear magnetic resonance (LF-NMR) and circular dichroism (CD) spectra. In backward extraction, the interactions between water and the other two molecules (MTGase and surfactant molecules) are enhanced while the interactions between target molecules (MTGase) and the other two molecules (water and surfactant molecules) are weakened as the ionic strength increases. Moreover, the effect of size exclusion on backward extraction was also investigated. The results demonstrate size exclusion has limit effect at high ionic strength, and the weakened interaction of MTGase-water-surfactant is the main reason causing the release of the target molecules in backward extraction. This work might provide valuable reference to the MTGase purification and downstream processing.

Prediction of the Reverse Micellar Extraction of Papain Using Dissipative Particle Dynamics Simulation.

Reverse micellar extraction is a promising technology for large-scale protein purification, but its molecular interaction mechanisms have not been thoroughly characterized. In this study, a dissipative particle dynamics (DPD) molecular simulation method was employed to study the interactions among the surfactant, organic phase, water, and proteins on the mesoscopic scale. This study simulated the self-assembly process of the reverse micelle extraction of papain. The results showed that the papain could be extracted by a CTAB/isooctane/n-hexanol system, which was validated by extraction experiments. The optimized extraction recovery was 76.9 %. This study elucidates the molecular process of the reverse micellar extraction of proteins and provides a method to predict its efficacy.

Using chromatogram averaging to improve quantitation of minor impurities.

Averaging of chromatograms can lead to enhancement of signal to noise ratio (S/N) in proportion to the square root of the number of measurements. Although the general principle has been known for decades, chromatogram averaging is almost never used in current pharmaceutical research. In this study we explore the utility of this approach, showing it to be a simple and easily accessible method for boosting sensitivity for quantification of minor components and trace impurities, where current techniques deliver insufficient S/N.

A novel 6.3 kb deletion and the Rare 27.6 kb Deletion Causing α+-Thalassemia in two Chinese Patients.

We report a novel -α6.3 deletion and a rare -α27.6 deletion causing α+-thalassemia (α+-thal), in two Chinese patients. One patient was a 35-year-old Chinese man with a mild α+-thal phenotype [mean corpuscular volume (MCV) 83.6 fL] and the Hb A2 level (2.5%) was close to borderline of the normal range. Multiplex ligation-dependent probe amplification (MLPA) revealed a novel 6344 bp deletion involving the entire HBA1 gene. Mapping by gap-polymerase chain reaction (gap-PCR) defined the exact breakpoint of this deletion to be NG_000006.1: g.31022_37366del6344. It was unique relative to other forms of α-thalassemia (α-thal) reported in the literature, and was designated as -α6.3 deletion. The other patient, a 41-year-old woman had Hb H (ß4) disease [hemoglobin (Hb) level of 8.9 g/dL] with a compound heterozygosity for the - -SEA (NG_000006.1: g.26264_45564del19301) deletion. The MLPA and gap-PCR methodologies confirmed the breakpoint (NG_000006.1: g.9079_36718del27640) and identified it as the rare -α27.6 deletion.

Aryl or heteroaryl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

Herein we describe our research efforts around the aryl and heteroaryl substitutions at the aminal carbon of the tetracyclic indole-based HCV NS5A inhibitor MK-8742. A series of potent NS5A inhibitors are described, such as compounds 45-47, 54, 56, and 65, which showed improved potency against clinically relevant and resistance associated HCV variants. The improved potency profiles of these compounds demonstrated an SAR that can improve the potency against GT2b, GT1a Y93H, and GT1a L31V altogether, which was unprecedented in our previous efforts in NS5A inhibition.

Development and fit-for-purpose validation of a LC-MS/MS assay for fibrinogen peptide A quantitation in human plasma.

BACKGROUND: Fibrinopeptide A (FPA) is a plasma peptide, formed by the action of thrombin on fibrinogen during clog formation. FPA represents a direct indicator of thrombin activity and could potentially be used as a biomarker for anti-thrombotic therapy development. Results/Methodology: A LC-MS/MS assay with a high throughput solid phase extraction procedure was developed and validated to measure FPA in plasma. The lower limit-of-quantitation (LLOQ) of this assay was determined to be 0.16 nM. The inter- and intra-day%CV was <15%. Freeze-thaw stability of FPA was ±30% up to 3 cycles and linear response of FPA was observed for plasma dilution up to 16-fold. CONCLUSION: The assay was validated and the biological variability of FPA in plasma was established (1-30 nM).

Quantitation of human peptides and proteins via MS: review of analytically validated assays.

Since the development of monoclonal antibodies in the 1970s, antibody-based assays have been used for the quantitation of proteins and peptides and, today, they are the most widely used technology in routine laboratory medicine and bioanalysis. However, in the last couple of decades, liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) techniques have been adopted in the quantitation of small molecules, and more recently have made significant contributions in the quantitation of proteins and peptides. In this article, we will review clinical MS-based assays for endogenous peptides, proteins, and therapeutic antibodies, for which validated methods exist. We will also cover the measurement of protein turnover and the unique solutions that MS can offer in this field.

Development of novel benzomorpholine class of diacylglycerol acyltransferase I inhibitors.

Diacylglycerol acyltransferase 1 (DGAT1) presents itself as a potential therapeutic target for obesity and diabetes for its important role in triglyceride biosynthesis. Herein we report the rational design of a novel class of DGAT1 inhibitors featuring a benzomorpholine core (23n). SAR exploration yielded compounds with good potency and selectivity as well as reasonable physical and pharmacokinetic properties. This class of DGAT1 inhibitors was tested in rodent models to evaluate DGAT1 inhibition as a novel approach for the treatment of metabolic diseases. Compound 23n conferred weight loss and a reduction in liver triglycerides when dosed chronically in mice with diet-induced obesity and depleted serum triglycerides following a lipid challenge.

Discovery of novel quinoline carboxylic acid series as DGAT1 inhibitors.

Herein we report the design and synthesis of a series of novel bicyclic DGAT1 inhibitors with a carboxylic acid moiety. The optimization of the initial lead compound 7 based on in vitro and in vivo activity led to the discovery of potent indoline and quinoline classes of DGAT1 inhibitors. The structure-activity relationship studies of these novel series of bicyclic carboxylic acid derivatives as DGAT1 inhibitors are described.

Lead optimization of a pyridine-carboxamide series as DGAT-1 inhibitors.

The structure-activity relationship studies of a novel series of carboxylic acid derivatives of pyridine-carboxamides as DGAT-1 inhibitors is described. The optimization of the initial lead compound 6 based on in vitro and in vivo activity led to the discovery of key compounds 10j and 17h.

Gas-phase formation of protonated benzene during collision-induced dissociation of certain protonated mono-substituted aromatic molecules produced in electrospray ionization.

Protonated benzene, C(6)H(7) (+), has been studied extensively to understand the structure and energy of a protonated organic molecule in the gas phase. The formation of C(6)H(7) (+) is either through direct protonation of benzene, i.e., chemical ionization, or through fragmentation of certain radical cations produced from electron ionization or photon ionization. We report a novel observation of C(6)H(7) (+) as a product ion formed in the collision-induced dissociation (CID) of protonated benzamide and related molecules produced via electrospray ionization (ESI). The formation of C(6)H(7) (+) from these even-electron precursor ions during the CID process, which has not been previously reported, is proposed to occur from the protonated molecules via a proton migration in a five-membered ring intermediate followed by the cleavage of the mono-substituent C--C bond and concurrent formation of an ion-molecule complex. This unique mechanism has been scrutinized by examining some deuterated molecules and a series of structurally related model compounds. This finding provides a convenient mean to generate C(6)H(7) (+), a reactive intermediate of considerable interest, for further physical or chemical investigation. Further studies indicate that the occurrence of C(6)H(7) (+) in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) appears to be a rather common phenomenon for many compounds that contain 'benzoyl-type' moieties. Hence, the observation of the C(6)H(7) (+) ion in LC/ESI-MS/MS can be used as an informative fragmentation pathway which should facilitate the identification of a great number of compounds containing the 'benzoyl-type' and similar structural features. These compounds are frequently present in food and pharmaceutical products as leachable impurities that require strict control and rapid elucidation of their identities.

Use of liquid chromatography/tandem mass spectrometric molecular fingerprinting for the rapid structural identification of pharmaceutical impurities.

Use of liquid chromatography/tandem mass spectrometric (LC/MS(n)) molecular fingerprinting is systematically demonstrated as a very effective tool for rapid structural elucidation of pharmaceutical impurities through a case study in which three isomers of betamethasone sodium phosphate (BSP) were rapidly identified as degradants formed due to the D-homoannular ring expansion of the steroid core structure of BSP in the solid state. The rapid structural elucidation of these degradants was achieved by matching or closely matching the UV profiles, molecular weights, and more importantly the fragmentation patterns obtained from the LC/MS(n) (n = 1 to 3) analysis of their enzyme-catalyzed hydrolytic products, respectively, with those of a D-homoannular isomer of betamethasone available in our LC/MS(n) molecular fingerprint database. This strategy of using LC/MS(n) molecular fingerprinting to obtain high-confidence structures of unknown species is then validated by structure verification through one- (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) experiments.

Rapid structure elucidation of drug degradation products using mechanism-based stress studies in conjunction with LC-MS(n) and NMR spectroscopy: identification of a photodegradation product of betamethasone dipropionate.

Betamethasone dipropionate is an active pharmaceutical ingredient (API) that is used in various dosage forms of finished products for the treatment of inflammatory disorders. An unknown degradant was observed during a solution stability study of betamethasone dipropionate. An approach that combines LC-MS(n), mechanism-based stress studies, semi-preparative HPLC purification and structure elucidation by NMR spectroscopy was used to identify the unknown species. The key step of this approach is the design of relevant stress studies based on the plausible degradation mechanism that is revealed by the informative LC-MS(n) analysis. The appropriately designed mechanism-based stress studies not only verify the degradation mechanism but also produce enough quantities of the unknown species for further structure elucidation/confirmation by NMR spectroscopy. With this strategy, the unknown degradant was rapidly identified as lumibetametasone dipropionate, a photodegradation product of betamethasone dipropionate.

A comparative study of enol aldehyde formation from betamethasone, dexamethasone, beclomethasone and related compounds under acidic and alkaline conditions.

Enol aldehydes are one type of key degradation and metabolic intermediates from a group of corticosteroids containing the 1,3-dihydroxyacetone side chain on their D-rings, such as betamethasone, dexamethasone, beclomethasone, and related compounds. The formation of enol aldehydes from these corticosteroids is via acid-catalyzed beta-elimination of water from the side chain, a process known as Mattox rearrangement. It was recently reported by our group that enol aldehydes could also be formed directly from the corresponding 17,21-diesters of these corticosteroids but only under alkaline condition, which was proposed to follow a variation pathway of the original Mattox rearrangement. In this paper, we report the results of a comparative study of enol aldehyde formation from these structurally similar corticosteroids (under the original acidic Mattox condition) and their 17,21-diesters (under the alkaline Mattox variation condition), respectively. In general, enol aldehydes were found to be formed under both conditions; however, the ratios of the E- and Z-isomers of the enol aldehyde were different in each case. The only exception was beclomethasone 17,21-diester under the alkaline condition, where a competing elimination of HCl from the 9,11-positions became predominant. These results can be explained by their structural differences with regard to the Mattox mechanism and its variation pathway. Lastly, solvent effect under acidic condition was studied between an aprotic and a protic solvent and the result suggests that enol aldehyde formation is greatly favored in an aprotic environment.

Application of LC-MS(n) in conjunction with mechanism-based stress studies in the elucidation of drug impurity structure: rapid identification of a process impurity in betamethasone 17-valerate drug substance.

Through a case study, the use of LC-MS(n) technique in conjunction with a mechanism-based stress study is shown to be a very effective way in the rapid elucidation of unknown drug impurities. In this case, the drug substance sample was first analyzed using LC-MS(n) through which the unknown species was found to be a valeryl-containing, isomeric impurity of the active pharmaceutical ingredient (API), betamethasone 17-valerate, based on its molecular ion and major fragments. Since a substantial knowledge regarding a large number of isomeric impurities of betamethasone has been accumulated in the literature as well as in our laboratory, a hydrolytic stress study (forced degradation) of the isolated unknown species was then designed and carried out accordingly in order to remove the valeryl group from the unknown species. During the stress study, a betamethasone isomer was generated as expected. However, a new unknown species isomeric to betamethasone 17-valerate was also formed unexpectedly. By comparing the UV spectra and more importantly MS(n) fragmentation patterns of the two newly formed species with those of betamethasone, dexamethasone, betamethasone 17-valerate, and betamethasone 21-valerate, these two unknown species generated in the stress study were identified as dexamethasone and dexamethasone 21-valerate, respectively. Based on the plausible reaction mechanism of the forced degradation, the original impurity present in betamethasone 17-valerate drug substance was then identified as dexamethasone 17-valerate; the structure assignment was later confirmed by various 1D and 2D NMR experiments. The efficient conversion from dexamethasone 17-valerate to dexamethasone 21-valerate was also observed during a 2D NMR acquisition of the isolated dexamethasone 17-valerate sample.

Identification of an unknown extraneous contaminant in pharmaceutical product analysis.

During the content uniformity test of a drug product (tablet formulation), an unknown peak was observed in the HPLC chromatograms. Upon further investigation, it was determined that the unknown peak was originated from an external source and, therefore, the drug product is free of this unknown peak. The next step was to identify the structure of this unknown peak in order to determine the source of this contaminant species. In this paper, we wish to present the strategy and the results of the experiments that led to the identification of this unknown peak. LC-PDA/UV and LC-MS(n) analyses were conducted to obtain the UV spectrum, molecular weight and MS(n) fragmentation pathways of the unknown peak. The MS analysis revealed certain structural features of the unknown species and a number of model compounds that contain such features were then examined for their UV absorbance profiles in an attempt to establish the functional group connectivity within the unknown species. A careful examination of these results in conjunction with the determination of the high-resolution molecular weight led to a short list of potential candidates for the unknown species, among which the most likely one was 1,3-diphenylguanidine. The identification of the unknown contaminant was confirmed by spiking experiments using the authentic compound. The potential source of this contaminant was also identified as derived from the safety filler of the pipette bulb used to prepare the sample solutions during the drug analysis.