Cynthia D. Sommers

Quality Assessment of U.S. Marketplace Vancomycin for Injection Products Using High-Resolution Liquid Chromatography-Mass Spectrometry and Potency Assays

ABSTRACT In response to a published concern about the potency and quality of generic vancomycin products, the United States Food and Drug Administration investigated a small sampling of the vancomycin products available in North America with regard to purity, content, and potency. To facilitate identification of impurities, a new liquid chromatography method was developed using high-resolution mass spectrometry in addition to diode array detection to characterize impurities in several commercial products. Furthermore, a microbiological assay was utilized to link the analytical profiles with an in vitro potency. All products tested met the quality specifications outlined in the United States Pharmacopeia (USP) (vancomycin hydrochloride for injection monograph) for impurities and potency (USP, Vancomycin hydrochloride for injection. United States Pharmacopeia and National Formulary, vol USP 34-NF 29, 2011).

Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-I digest patterns

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (Mw) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A2) for each sample prior to molecular weight assessment. For UFH, a meanu2009±u2009standard deviation value for Mw of 16,773u2009±u2009797 was observed with a range of 15,620 to 18,363 (nu2009=u200920, run in triplicate). For LMWHs by SEC-MALLS, we measured mean Mw values for dalteparin, tinzaparin, and enoxaparin of 6,717u2009±u200971 (nu2009=u20094), 6,670u2009±u2009417 (nu2009=u20093), and 3,959u2009±u2009145 (nu2009=u20093), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135u2009±u2009643 (nu2009=u200920), 5,845u2009±u200945 (nu2009=u20094), 6,049u2009±u200995 (nu2009=u20093), and 4,772u2009±u200969 (nu2009=u20093), respectively. These orthogonal measurements are the first Mw results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10xa0years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.

Sensitive Detection of Oversulfated Chondroitin Sulfate in Heparin Sodium or Crude Heparin with a Colorimetric Microplate Based Assay

In this work we describe a 96-well microplate assay for oversulfated chondroitin sulfate A (OSCS) in heparin, based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. The assay takes advantage of several unique properties of heparin, OSCS, and LPTP, including OSCS inhibition of heparinase I and II activity, the molecular weight dependence of heparin-LPTP spectral shifts, and the distinct association of heparin fragments and OSCS to LPTP. These factors combine to enable detection of the presence of 0.003% w/w spiked OSCS in 10 μg of heparin sodium active pharmaceutical ingredient (API) using a plate reader and with visual detection to 0.1% levels. The same detection limit for OSCS was observed in the presence of 10% levels of dermatan sulfate (DS) or chondroitin sulfate A (CSA) impurities. In addition, we surveyed a selection of crude heparin samples received by the agency in 2008 and 2009 to determine average and extreme DS, CSA, and galactosamine weight percent levels. In the presence of these impurities and the variable heparin content in the crude heparin samples, spiked OSCS was reliably detected to the 0.1% w/w level using a plate reader. Finally, authentically OSCS contaminated heparin sodium API and crude samples were distinguished visually by color from control samples using the LPTP/heparinase test.

Characterization of currently marketed heparin products: Key tests for LMWH quality assurance ☆

During the 2007-2008 heparin crisis it was found that the United States Pharmacopeia (USP) testing monograph for heparin sodium or low molecular weight heparins did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS). In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to detect not only the contaminant OSCS, but also to improve assurance of quality and purity of these drug products. The USP monographs for the low molecular weight heparins (LMWHs) approved for use in the United States (dalteparin, tinzaparin and enoxaparin) are also undergoing revision to include many of the same tests used for heparin sodium, including; one-dimensional (1D) 500 MHz (1)H NMR, SAX-HPLC, percent galactosamine in total hexosamine and anticoagulation time assays with purified Factor IIa or Factor Xa. These tests represent orthogonal approaches for heparin identification, measurement of bioactivity and for detection of process impurities or contaminants in these drug products. Here we describe results from a survey of multiple lots from three types of LMWHs in the US market which were collected after the 2009 heparin sodium monograph revision. In addition, innovator and generic versions of formulated enoxaparin products purchased in 2011 are compared using these tests and found to be highly similar within the discriminating power of the assays applied.

Detection of Possible Economically Motivated Adulterants in Heparin Sodium and Low Molecular Weight Heparins with a Colorimetric Microplate Based Assay

Recently, we described a 96-well plate format assay for visual detection of oversulfated chondroitin sulfate A (OSCS) contamination in heparin samples based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. Here, we establish the specificity of the LPTP/heparinase test with a unique set of reagents that define the structural requirements for significant LPTP chemosensor color change. For example, we observed a biphasic behavior of larger shifts to the red in the UV absorbance spectra with decreasing average molecular weight of heparin chains with a break below 12-mer chain lengths. In addition, the oversulfation of chondroitin sulfate A (CSA) to a partially (PSCS) or fully (OSCS) sulfated form caused progressively less red shift of LPTP solutions. Furthermore, glycosaminoglycans (GAGs) containing glucuronic acid caused distinct spectral patterns compared to iduronic acid containing GAGs. We applied the LPTP/heparinase test to detection of OSCS (≥0.03% (w/w) visually or 0.01% using a plate reader) in 10 μg amounts of low molecular weight heparins (LMWHs; i.e. dalteparin, tinzaparin, or enoxaparin). Furthermore, because other oversulfated GAGs are possible economically motivated adulterants (EMAs) in heparin sodium, we tested the capacity of the LPTP/heparinase assay to detect oversulfated dermatan sulfate (OSDS), heparin (OSH), and heparan sulfate (OSHS). These potential EMAs were visually detectable at a level of ∼0.1% when spiked into heparin sodium. We conclude that the LPTP/heparinase test visually detects oversulfated GAGs in heparin sodium and LMWHs in a format potentially amenable to high-throughput screening.

Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colony stimulating factor) therapeutics from different manufacturers

The FDA has approved more than 100 protein and peptide drugs with hundreds more in the pipeline (Lanthier et al. in Nat Rev Drug Discov 7(9):733–737, 2008). Many of these originator biologic products are now coming off patent and are being manufactured by alternate methods than the innovator as follow-on drugs. Because changes to the production method often lead to subtle differences (e.g., degradation products, different posttranslational modifications or impurities) in the therapeutic (Schiestl et al. in Nat Biotechnol 29(4):310–312, 2011), there is a critical need to define techniques to test and insure the quality of these drugs. In addition, the emergence of protein therapeutics manufactured by unapproved methodologies presents an ongoing and growing regulatory challenge. In this work, high-resolution mass spectrometry was used to determine the presence or absence of posttranslational modifications for one FDA-approved and three foreign-sourced, unapproved filgrastim products. Circular dichroism (CD) was used to compare the secondary structure and probe the temperature stability of these products. Native 2D 1H,15N-heteronuclear singular quantum coherence (HSQC) NMR test was applied to these samples to compare the higher-order structure of the four products. Finally, a cell proliferation assay was performed on the filgrastims to compare their bioactivity, and stressed filgrastim was tested in the bioassay to better understand the effects of changes in protein structure on activity. The results showed that orthogonal approaches are capable of characterizing the physiochemical properties of this protein drug and assessing the impact of structural changes on filgrastim purity and potency.

Modern analytics for synthetically derived complex drug substances: NMR, AFFF-MALS, and MS tests for glatiramer acetate.

Glatiramer acetate (GA) is a mixture of synthetic copolymers consisting of four amino acids (glutamic acid, lysine, alanine, and tyrosine) with a labeled molecular weight range of 5000 to 9000xa0Da. GA is marketed as Copaxone™ by Teva for the treatment of multiple sclerosis. Here, the agency has evaluated the structure and composition of GA and a commercially available comparator, Copolymer-1. Modern analytical technologies which can characterize these complex mixtures are desirable for analysis of their comparability and structural “sameness.” In the studies herein, a molecular fingerprinting approach is taken using mass-accurate mass spectrometry (MS) analysis, nuclear magnetic resonance (NMR) (1D-1H-NMR, 1D-13C-NMR, and 2D NMR), and asymmetric field flow fractionation (AFFF) coupled with multi-angle light scattering (MALS) for an in-depth characterization of three lots of the marketplace drug and a formulated sample of the comparator. Statistical analyses were applied to the MS and AFFF–MALS data to assess these methods’ ability to detect analytical differences in the mixtures. The combination of multiple orthogonal measurements by liquid chromatography coupled with MS (LC–MS), AFFF–MALS, and NMR on the same sample set was found to be fit for the intended purpose of distinguishing analytical differences between these complex mixtures of peptide chains.

Analyses of marketplace tacrolimus drug product quality: Bioactivity, NMR and LC–MS

Tacrolimus (FK506) is a potent, narrow therapeutic index, immunosuppressive drug used to avoid organ rejection in patients that have undergone organ transplantation. Recent clinical reports suggested a significant reduction in the tacrolimus concentration/dose ratio in the plasma of liver and kidney recipients when the reference listed drug was substituted with a generic drug. In response to these concerns about switching between tacrolimus from different approved manufacturers during treatment, the FDA initiated purity, potency and quality studies of the innovator and generic tacrolimus products available in the US marketplace. A combination of analytical methods, including mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and bioactivity assay were developed and validated to assess the quality of tacrolimus. These tests measured the identity, impurities and activity of tacrolimus from active pharmaceutical ingredient (API) sources and with formulated drug product from five different approved manufactures. In addition, some testing was performed on tacrolimus capsules obtained from a non US approved Indian source. The data obtained showed no discernible difference in the impurity profiles and potency between the generic and innovator tacrolimus products.

Characterization of currently marketed heparin products: adverse event relevant bioassays.

The polyanion oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in heparin products and was associated with severe hypotensive responses and other symptoms in patients receiving the drug. The OSCS associated adverse reactions were attributed to activation of the contact system via the plasma mediator, activated factor XII (FXIIa), which triggers kallikrein (KK) activity. Unlike heparin alone, OSCS, is able to activate FXII in plasma and stably bind to FXIIa enhancing plasma KK activity and the induction of vasoactive mediators such as bradykinin (BK), C3a and C5a. Similarly OSCS can interfere with heparin neutralization by the polycationic drug protamine. Here, we assess heparin (heparin sodium, dalteparin, tinzaparin or enoxaparin)-protamine complex formation and plasma based bioassays of KK, BK and C5a in a 96-well plate format. We establish the normal range of variation in the optimized bioassays across multiple lots from 9 manufacturers. In addition, because other oversulfated (OS) glycosaminoglycans (GAGs) besides OSCS could also serve as possible economically motivated adulterants (EMAs) to heparin, we characterize OS-dermatan sulfate (OSDS), OS-heparan sulfate (OSHS) and their native forms in the same assays. For the protamine test, OS-GAGs could be distinguished from heparin. For the KK assay, OSCS and OSDS were most potent followed by OSHS, and all had similar efficacies. Finally, OSDS had a greater efficacy in the C5a and BK assays followed by OSCS then OSHS. These data established the normal range of response of heparin products in these assays and the alteration in the responses in the presence of possible EMAs.

PF4-HIT antibody (KKO) complexes activate broad innate immune and inflammatory responses

INTRODUCTIONnHeparin-induced thrombocytopenia (HIT) is an immune-mediated complication of heparin anticoagulation therapy resulting in thrombocytopenia frequently accompanied by thrombosis. Current evidence suggests that HIT is associated with antibodies developed in response to multi-molecular complexes formed by platelet factor 4 (PF4) bound to heparin or cell surface glycosaminoglycans. These antibody complexes activate platelets and monocytes typically through FcγRIIA receptors increasing the production of PF4, inflammatory mediators, tissue factor and thrombin. The influence of underlying events in HIT including complex-induced pro-inflammatory cell activation and structural determinants leading to local inflammatory responses are not fully understood.nnnMETHODSnThe stoichiometry and complex component requirements were determined by incubating fresh peripheral blood mononuclear cells (PBMC) with different concentrations of unfractionated heparin (H), low molecular weight heparin (LMWH), PF4- and anti-PF4-H complex antibodies (KKO). Cytokine mRNA or protein were measured by qRT-PCR or Meso Scale Discovery technology, respectively. Gene expression profile analysis for 594 genes was performed using Nanostring technology and analyzed using Ingenuity Pathway Analysis software.nnnRESULTS AND CONCLUSIONSnThe data show that antibodies magnify immune responses induced in PBMCs by PF4 alone or in complex with heparin or LMWH. We propose that following induction of HIT antibodies by heparin-PF4 complexes, binding of the antibodies to PF4 is sufficient to induce a local pro-inflammatory response which may play a role in the progression of HIT. In vitro assays using PBMCs may be useful in characterizing local inflammatory and innate immune responses induced by HIT antibodies in the presence of PF4 and different sources of heparins.nnnFDA DISCLAIMERnThe findings and conclusions in this article are solely the responsibility of the authors and are not being formally disseminated by the Food and Drug Administration. Thus, they should not be construed to represent any Agency determination or policy.