Edward K. Chess

The US regulatory and pharmacopeia response to the global heparin contamination crisis

The contamination of the widely used lifesaving anticoagulant drug heparin in 2007 has drawn renewed attention to the challenges that are associated with the characterization, quality control and standardization of complex biological medicines from natural sources. Heparin is a linear, highly sulfated polysaccharide consisting of alternating glucosamine and uronic acid monosaccharide residues. Heparin has been used successfully as an injectable antithrombotic medicine since the 1930s, and its isolation from animal sources (primarily porcine intestine) as well as its manufacturing processes have not changed substantially since its introduction. The 2007 heparin contamination crisis resulted in several deaths in the United States and hundreds of adverse reactions worldwide, revealing the vulnerability of a complex global supply chain to sophisticated adulteration. This Perspective discusses how the US Food and Drug Administration (FDA), the United States Pharmacopeial Convention (USP) and international stakeholders collaborated to redefine quality expectations for heparin, thus making an important natural product better controlled and less susceptible to economically motivated adulteration.

Collisionally activated dissociations of aminocyclitol-aminoglycoside antibiotics and their application in the identification of a new compound in tobramycin samples.

Several aminocyclitol-aminoglycoside antibiotics have been studied by tandem mass spectrometry. Glycosidic bond cleavages were the major reactions in the low energy collisionally activated decomposition (CAD) of the protonated antibiotics. Only the glycoside residing on the C6—O of the 2-deoxystreptamine was observed to undergo significant decomposition at the C2—C3 and O—C1 bonds. The comprehension of the CAD of known aminoglycosides aided in the identification of an unknown impurity in tobramycin. The unknown compound was initially detected by reverse phase high-performance liquid chromatography following dinitrofluorobenzene derivatization of the amino groups. The molecular weight of the dinitrobenzene derivative measured by LC mass spectrometry (MS) led to the detection of two isomeric impurities in tobramycin by LC-MS using an amino column. Their CAD spectra were subsequently acquired by LC-MS/MS. One of the two compounds was determined to be a known compound, 6″-0-carbamyltobramycin with the carbamyl group substituted on the glycoside residing on the C6—O of 2-deoxystreptamine. The fragmentation pattern of the other compound was interpreted as that the unknown was also a carbamyltobramycin. The carbamyl group was, however, substituted on 2-deoxystreptamine. It was speculated that the carbamyl group was substituted at the C1 amino group. This compound, to our knowledge, has not been reported before.

Model for estimating the accumulation of solutes leaching from polymeric containers into parenteral solutions

Abstract A model allowing for the prediction of the thermodynamically constrained accumulation of leachables migrating from polymeric containers has been developed. The model predicts accumulation based on three accumulation-limiting mechanisms: total available pool, solute solubility and solute partitioning. Equations relating a solutes solvent-water partition coefficients ( P o - w and P h - w ) and its aqueous solubility and partitioning properties have been developed. Thus, one can predict leachable accumulations from the physical dimensions of the system being studied (container weight and solution volume) and from the solutes partition coefficients. The maximal accumulation of the leachable in solution will be the lowest value predicted via the three accumulation-limiting mechanisms. The model has been successfully applied to the migration of 2-ethylhexanoic acid out of a styrene-butadiene-styrene block co-polymer and accurately predicts the accumulation behavior of this species as a function of solution pH.

Characterization of pertussis toxoid by two-dimensional liquid chromatography–tandem mass spectrometry

Pertussis toxoid, an acellular pertussis vaccine prepared by hydrogen peroxide treatment in the presence of Fe(3+), has not been well characterized. Because the toxoid has been a part of the DTaP vaccine for infants, it is of interest and significance to have a clear understanding of its structure. The five subunits of pertussis toxin (PT) have a combined molecular weight of approximately 95,000Da. The peroxide treatment in toxoid formation introduces additional complexity into the protein sequence. To maximize sequence coverage, a two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) approach was used to analyze the tryptic digest of toxoid as a whole. An analytical-scale high-performance liquid chromatography (HPLC) instrument using a pentafluorophenyl (PFP) column was used as the first-dimensional LC for fraction collection. The fractions were then analyzed by nanoLC-MS/MS using a C18 column to acquire collision-activated dissociation (CAD) spectra of the tryptic peptides. It is shown that a PFP column has a different peptide retention specificity from a C18 column. A combination of a PFP column and a C18 column is a viable approach for dispersing peptides in a complex mixture. From the structures of 65 peptides that represented approximately 50% of its sequence, PT was found to have sustained heavy oxidative damages during toxoid preparation. Nearly all methionine, cysteine, and (likely) tryptophan residues were oxidized. Evidence of histidine and tyrosine oxidation was also observed. In addition, a large percentage of asparagine was found hydrolyzed to aspartic acid. These findings corrrelate well with the reduction of PT toxicity by peroxide treatment.

Modeling of the leachables impact on the engineering of parenteral product container systems

Abstract This study examines the impact, from a leachables perspective, of charging a solution contact component of a polymeric solution container. By charging such a container component, one provides a new source of leachable solutes. The distribution of these leachables within a three phase system (the original container, the new component and the solution) will determine the impact of the container change. Extraction of a material, coupled with chromatographic and/or spectroscopic analysis of the resultant extracts, provides the materials leachable profile. The total organic carbon (TOC) content of the extracts is introduced as a species non-specific measure of the total extractable burden. Leaching models are developed for the container and its changed component by correlating these materials interaction constants with solvent/solvent partition coefficients of model solutes. The solvent-solvent partition coefficients of the members of the leachable profile, as well as the partition coefficients of the composite TOC, are determined. The leaching models are coupled with the leachables partition coefficients to allow for a prediction of leachable accumulation in a specific product design. These predictions reconcile well with actual experimental observations.

Collective sampling of intact anionic polysaccharide components and application in quantitative determination by LC-MS.

Anionic polysaccharides, such as glycosaminoglycans (GAGs) and alginate, readily undergo source-induced fragmentation when analyzed by electrospray mass spectrometry with the use of high source cone voltage. The dissociation chemistry converts all components of a polysaccharide into a small set of structurally characteristic small saccharides. This chemistry enables the collective detection of a polysaccharide through the detection of one or more small saccharides. This ability, combined with the elution of polysaccharides as relatively compact bands using ion-pairing reverse phase liquid chromatography, created a unique opportunity for the development of LC-MS methods suitable for the quantitative analysis of intact anionic polysaccharides. Feasibility of this approach is demonstrated with a mixture of heparin, chondroitin sulfate A, and alginate.

Acetonitrile Adduct Formation as a Sensitive Means for Simple Alcohol Detection by LC-MS

AbstractSimple alcohols formed protonated acetonitrile adducts containing up to two acetonitrile molecules when analyzed by ESI or APCI in the presence of acetonitrile in the solvent. These acetonitrile adducts underwent dissociation to form a nitrilium ion, also referred to as the substitution ion. Diols and triols behaved differently. In ESI, they formed only one acetonitrile adduct containing one acetonitrile. The S ion was not observed in ESI and was only weakly observed from the dissociation of the (M + ACN + H)+ ion. On the other hand, the S ion was abundantly formed from the diols in APCI. This formation of acetonitrile adducts and substitution ion from simple alcohols/diols offers an opportunity to detect simple alcohols/diols sensitively by LC-MS interfaced by ESI or APCI. The utility of this chemistry was demonstrated in a method developed for the quantification of cyclohexanol in rat plasma by monitoring the CID-induced fragmentation from the S ion to a fragment ion. Graphical Abstractᅟ

Pertussis toxoid structure: A collaboration and comparison of two-dimensional liquid chromatography–tandem mass spectrometry, ultraperformance liquid chromatography–mass spectrometryE, and capillary liquid chromatography–matrix-assisted laser desorption ionization–tandem mass spectrometry

The overall structure of pertussis toxoid has been established by analysis of its tryptic digest using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS), capillary liquid chromatography-matrix-assisted laser desorption ionization-tandem mass spectrometry (CapLC-MALDI-MS/MS), and ultraperformance liquid chromatography-mass spectrometry(E) (UPLC-MS(E)). In addition to oxidation and hydrolysis of amino acids losses of terminal peptides are observed. On-line UPLC-MS(E) generated a similar sequence coverage as the other two methods that involved off-line fraction collection. In light of recent favorable comparisons to data-dependent acquisition, UPLC-MS(E) should be the initial method of choice for analysis of a peptide mixture of moderate complexity.