Note #1. Heparin manufacturing process (Supplementary Fig. 1) Note #4. Molecular weight determinations (Supplementary Table 1)

Transcription

1 Supplementary Notes Accompanying information to Szajek et al.: U.S. Regulatory and Compendial Response to the Global Heparin Contamination Crisis Table of Contents: Note #1. Heparin manufacturing process (Supplementary Fig. 1) Note #2. NMR analysis (Supplementary Fig. 2) Note #3. Capillary electrophoresis (CE) analysis Note #4. Molecular weight determinations (Supplementary Table 1) Note #5. Adoption of a new potency assay (Supplementary Fig. 3 & Table 2) Note #6. Impurities (Supplementary Fig. 4 & Fig. 5) References 1

2 Note #1. Heparin (HP) Manufacturing process Supplementary Fig. 1. HP Manufacturing Process Step 1. Extraction: Heparin is released and solubilized from animal intestinal mucosa by proteolysis with enzyme e.g. alcalase or by hydrolysis in basic aqueous milieu at elevated temperatures and chloride concentration. Potential impurities: Water soluble entities from mucosa. Examples: related substances (dermatan sulfate (DS), chondroitin sulfate (CS), heparan sulfate (HS), peptides and proteins, nucleotides and polynucleotides, various inorganic salts. Step 2: a) Formation of insoluble complex with positively charged ammonium salts or binding to anion exchange resin b) Isolation of complex or resin with heparin by filtration c) Dissolution of complex or elution of resin by solutions with high concentrations of chloride salts d) Precipitation with methanol, ethanol or acetone e) Drying of crude heparin Potential impurities: Solvents, co-precipitated chloride salts. Entities binding to the positively charged ammonium salt or anion exchange resin. Examples of the impurities are related substances (DS, CS, HS), peptides and proteins, nucleotides and polynucleotides. Step 3: a) Oxidation with agents like agent like CH 3 CO 3 H, H 2 O 2 or KMnO 4 b) Precipitation with methanol, ethanol or acetone c) Filtration d) Drying, e.g. vacuum drying, tray drying, freeze drying, spray drying etc. Potential impurities: Co-precipitated chloride salts, Mn 2+, EDTA, oxalate, solvents, related substances (DS, CS, HS), peptides and proteins, nucleotides and polynucleotides 2

3 The polysaccharide molecules found in the final HP products might differ from their original forms present in mucosa due to these chemical processes. Those molecular changes, including molecular weight distribution profile, reduction of molecular weight, and peroxide formation, are associated with specific manufacturing processes, and thus are often seen as process fingerprints. Note #2. NMR Analysis of HP NMR spectroscopy has been shown to be a valuable tool for the characterization of low molecular weight HPs, but not for routine analysis of unfractionated HP. At the height of the HP contamination crisis, the FDA identified 1 H-NMR as a useful tool for identifying the contaminant in heparin, particularly through the presence of a signal at ~2.15 ppm 1. In the context of development of a compendial NMR method, initial experiments were performed on a 300 MHz instrument. Further studies then demonstrated a 2-fold enhancement of sensitivity at 500 MHz field strength. Based on these data and an expected improvement in signal resolution, the Panel recommended implementation of the FDA protocol, but required the use of a field strength of 500 MHz or greater. At 500 MHz it is possible to detect OSCS in heparin with a limit of detection of approximately 0.25% (w/w). The implementation of the 1 H-NMR technique with the Stage 1 and Stage 2 revision to the HP monographs in USP demonstrated the value of this technique. Challenges with interpretation of the 1 H-NMR technique were identified during laboratory studies of the Stage 1 and Stage 2 monograph revisions. Many times, these challenges were associated with small, spurious signals occurring close to the OSCS signal in the 1 H-NMR spectrum of HP (Supplementary Fig. 2), some of which resulted 3

4 from variations in the HP manufacturing process, especially in the oxidation steps 2, 3. Through collaboration with heparin manufacturers, cases were identified where the addition of ethylenediaminetetraacetic acid (EDTA) improved the resolution of the HP 1 H-NMR spectrum 4, 5. Therefore, to finalize the overall revisions, the Stage 3 heparin 1 H-NMR procedure modifications focused on the method for allowing a lower detection limit for OSCS through proper sample preparation, optimization of instrument parameters and to allow use of EDTA 6. A B Supplementary Fig H-NMR spectra of heparin, full width. A: OSCS contaminated heparin API (~17 mole percent OSCS), contains dermatan as well. B: heparin API, no evidence of OSCS (control), some dermatan present. Expansion region around the N-methyl signal for each spectrum on the right. The ppm range in the 1 H-NMR spectrum of particular interest, at ~ 2 ppm, contains 13 C satellite signals, which can complicate spectral interpretation and need be removed by 13 C decoupling. The coupling constant for the J CH of the main HP methyl is 129 Hz. 4

5 This results in a signal at 129/2ν, where ν is the operating frequency of the spectrometer. Thus, at 400, 500, 600, 700, and 800 MHz there will be a resonance with an intensity of 0.55% of the main heparin signal at 2.21, 2.18, 2.16, 2.14, and 2.13 ppm, respectively. 13 C-decoupling is a routine procedure on any modern spectrometer and was added as a guideline to laboratories performing this test. Note #3. Capillary electrophoresis (CE) analysis Capillary electrophoresis (CE) was introduced in the Stage 1 USP heparin monograph for OSCS detection. In March 2008, during the heparin contamination crisis, the FDA published an 1 H-NMR and a CE method developed by Baxter Healthcare as orthogonal techniques to screen heparin samples for the presence of an adulterant, OSCS 7. At the time that the Stage 2 revision was conducted, the separation between OSCS and HP was not adequate to precisely quantitate the OSCS. In addition, the detection limit of the early CE method was between 1-5% w/w OSCS in heparin, far above the required 0.1% w/w level required by FDA s guidelines. CE analysis is less commonly performed in manufacturing quality control labs in compared with high performance liquid chromatography (HPLC) analysis. Given these limitations of the initial CE method, the Panel considered other separation techniques to replace the CE method. In the Stage 2 revision of USP heparin monograph, the CE method was replaced by a strong-anion exchange high performance liquid chromatography (SAX-HPLC) method. It should be noted that a new CE method with improved sensitivity for OSCS was later reported by a research team from Baxter Healthcare after the Stage 2 revision was completed 8. 5

6 Note #4. Molecular weight determinations Supplementary Table 1. Sodium Parameter 1 M w 2 M M /M USP acceptance criteria for molecular weight distribution of HP Criterion Da Not more than 20% Not less than 1 1. Weight average molecular weight 2. Proportion of material with molecular weight greater than Da 3. Ratio of the proportion of material with molecular weight of Da to the proportion of material with molecular weight of Da. Note #5. Adoption of a New Potency Assay Historically, the HP potency determination in the USP monograph was based on the time to prevent 50% clot gelation of sheep plasma 1, 9. However, the assay was not specific for HP and could be influenced by other substances 10. One major contributing factor to the HP crisis of 2008 was that the OSCS contaminant was found to mimic the anticoagulant activity of HP in this test, thereby allowing contaminated batches to pass acceptance criteria. As shown in Supplementary Table 2, there was an increase in specific activity of HP by comparison with potencies obtained by anti-factor-iia or antifactor Xa assays with increasing amounts of OSCS. Purified OSCS was found to have very low anti-xa and anti-iia activity, but have comparable potency to heparin as measured by sheep plasma (Supplementary Table 2). Namely, OSCS has approximately 40-fold higher activity in the sheep plasma assay than in the chromogenic anti-factor IIa and anti-factor Xa assays. Therefore, OSCS can increase 6

7 the apparent activity of HP in sheep plasma assays due to the non-specific nature of the assay. Supplementary Table 2. Influence of oversulfated chondroitin sulfate (OSCS) on heparin activity assays: Sample EP sheep plasma assay IU/mg (95% CL) Heparin ( ) Heparin + 1% OSCS ( ) Heparin + 5% OSCS ( ) Heparin + 10% OSCS ( ) Heparin + 15% OSCS ( ) OSCS ( ) Anti- IIa assay IU/mg (95% CL) ( ) ( ) ( ) ( ) ( ) 4.89 ( ) Anti- Xa Assay IU/mg (95% CL) Anti- Xa: sheep plasma Anti- Xa: anti- Iia ( ) ( ) ( ) ( ) ( ) ( ) In light of these concerns, the Panel considered it important to implement an antifactor IIa potency assay using a chromogenic substrate in the Stage 2 HP monograph 11, 12, replacing the less specific assay. The anti-factor IIa potency assay and the antifactor Xa assay utilize purified reagents [antithrombin, factor IIa (FIIa), or factor Xa (FXa)] and chromogenic substrates that are specific to either FIIa or FXa. Because unfractionated heparin, low molecular weight (LMW) heparins, and heparan sulfate are the only agents that are known to potentiate the inhibitory activity of antithrombin, these chromogenic assays are therefore specific to heparin and LMW heparins, and thus they reduce the risk of adulteration. 7

8 To ensure the purity of HP, the Stage 2 revision also included a change to the potency specification to not less than 180 USP Units per mg. In 2000, Mulloy et al. 13 found that the specific activity of the majority of the HP samples tested was over 180 International Unit/mg. This finding further documented that the original potency test which had an acceptance criteria of 140 USP Units/ per mg, was outdated. During the revision process, the manufacturers and other stakeholders assayed a number of representative HP batches using the chromogenic potency assays and found that the majority of batches passed the criterion of 180 USP Units per mg. Supplementary Fig. 3 illustrates the potency results for aiia and axa from 90 batches tested in one laboratory. Close to 80% of the batches were found to pass the potency specification, and in addition, only 5 batches fell outside the axa to aiia ratio of 0.9 to 1.1. The outliers were poor quality products resulted from poor manufacturing processes. These batches were not contaminated with OSCS as confirmed by NMR analysis. Use of the ratio of anti-fxa to anti-fiia activity ( ) as an identity test also decreases potential contamination and improves the purity of HP. Although both LMW heparin and heparan sulfate can potentiate the activity of antithrombin, both substances give a higher anti- FXa to anti-fiia ratio than heparin. Concurrent with the implementation of the new potency assay, the definition for the USP Unit of heparin activity was adjusted to make it equivalent to the International Unit (IU) for HP. Over the past 30 years, there has been an approximately 10% drift in the USP Heparin Unit from the IU of HP. Re-alignment of the USP Unit and the IU was achieved by calibration of the current USP Heparin for Bioassays RS against the 5 th International Standard for UFH, using the USP anti-factor IIa chromogenic potency 8

9 assay ( It was determined that a 10% change in the USP Heparin Unit would have no significant clinical effects based on how heparin is used in the clinical setting and how patients are monitored using the activated partial thromboplastin time (aptt) to ensure that the heparin therapy is safe and efficacious 14. Supplementary Fig. 3. Anti-factor IIa and anti-factor Xa activity of 90 batches of heparin sodium API from 8 manufacturers. The activities were measured using USP monograph methods against the 6 th International Standard for Unfractionated Heparin (6 th IS). The USP unit for Heparin Sodium is equivalent to the International Unit as defined by the 6 th IS. The green dash line indicates the specification by anti-iia potency assay of 180 U/mg. The black line represents anti-xa to anti-iia ratio of 1, with the red lines defining ratios of 0.9 and 1.1. The majority of the batches tested (71/90) passed the specification of not less than 180 U/mg and only 5 batches were found outside the identity anti-xa to anti-iia ratios of 0.9 to

10 Note #6. Impurities Test Galactosamine Analysis-The analytical method for galactosamine measurement in total hexosamine consists of a sample preparation where galactosamine and glucosamine are completely released from the GAG backbone by acid hydrolysis. Subsequently, galactosamine is separated from glucosamine by high performance ion chromatography (HPIC), and is quantified by pulsed amperometric detection (PAD). The result is calculated as the percent peak area of the galactosamine peak to the sum of the galactosamine and glucosamine peak areas in the sample solution where the galactosamine response is corrected by the response ratio of galactosamine to glucosamine, calculated from the chromatogram of the hydrolyzed standard solution. A typical chromatogram of a sample solution containing 5% galactosamine of total hexosamine is shown in Supplementary Fig #2 nc Glucosamine Galactosamine

11 Supplementary Fig. 4. A typically chromatogram of a sample solution containing 5% galactosamine of total hexosamine. Protein Impurities Analysis-The stage 3 Lowry method is a colorimetric assay in which a 1-mL aliquot of 30 mg/ml of an aqueous solution of HP is treated with 5 ml of a Lowry reagent. This method is able to detect the concentration of protein contaminant as low as 0.01 mg/ml. The acceptance criterion is not more than 0.1% w/w protein/heparin. Some substance could interfere with protein determination using Lowry method. To remove the interfering substances, a trichloroacetic acid is used to precipitate proteins. The amount of the precipitated protein is then determined by the using the standard method 15. Supplementary Fig. 5. HPLC Chromatogram of all nucleoside peak standards of all nucleoside standards. 11

12 Nucleotidic impurities-the nonspecific UV absorbance test for nucleotidic impurities has been replaced by a quantitative test in the stage 3 USP Heparin monograph. The new assay hydrolyzes DNA to deoxynucleosides using an endonuclease enzyme from Serratia marcescens (E.C ), Benzonase 16. After the depolymerization, the samples are chromatographed on a C18 column eluted with a solvent containing ammonium acetate, acetic acid and acetonitrile. The DNA nucleosides thymidine, 2 deoxyadenosine, 2 deoxyguanonsine, and 2 -deoxycytidine, and the RNA nucleosides guanosine, adenosine, cytidine, and uridine are chromatographically separated, detected, and quantified (Supplementary Fig. 5). References 1. Pharmacopeia Official monographs: heparin sodium (US 32- NF27. ). 2. Mourier, P.A.J., Guichard, O.Y., Herman, F. & Viskov, C. Heparin sodium compliance to USP monograph: Structural elucidation of an atypical 2.18 ppm NMR signal. J. Pharm. Biomed. Anal , (2012). 3. Mourier, P.A.J., Guichard, O.Y., Herman, F. & Viskov, C. Heparin sodium compliance to the new proposed USP monograph: Elucidation of a minor structural modification responsible for a process dependent 2.10 ppm NMR signal. J. Pharm. Biomed. Anal. 54, (2011). 4. McEwen, I. Broadening of 1H NMR signals in the sectra of heparin and OSCS by paramagnetic transition metal ions. The use of EDTA to sharpen the signals. J. Pharm. Biomed. Anal. 49, (2010). 5. McEwen, I. et al. Effect of Ca2+ on the 1H NMR chemical shift of the methyl signal of oversulphated chondroitin sulphate, a contaminant in heparin. J. Pharm. Biomed. Anal. 49, (2009). 6. Pharmacopeia Official monographs: heparin sodium, (Accessed January 8, 2014). (US 37- NF32). 7. Patel, R.P., Narkowicz, J.P., Hutchinson, E.F., Hilder, E.F. & Jacobson, G.A. A simple capillary electrophoresis method for the rapid separation and determination of intact low molecular weight and unfractionated heparins. J. Pharm. Biomed. Anal. 46, (2008). 8. Wielgos, T., Havel, K., Ivanova, N. & Weinberger, R. Determination of impurities in heparin by capillary electrophoresis using high molarity phosphate buffers. J. Pharm. Biomed. Anal. 49, (2009). 9. Walenga, J.M., Fareed, J., Hoppensteadt, D. & Emanuele, R.M. In vitro evaluation of heparin fractions: old vs. new methods. Crit. Rev. Clin. Lab. Sci. 22, (1986). 10. Mousa, S.A., Fareed, J., Iqbal, O. & Kaiser, B. Tissue Factor Pathway inhibitor in thrombosis and beyond. Methods Mol. Med. 93, (2004). 11. Pharmacopeia Official monographs: heparin sodium. (US 34- NF29). 12

13 12. Mousa, S.A. Heparin and low- molecular weight heparins in the thrombosis and beyond. Methods Mol. Biol. 663, (2010). 13. Mulloy, B., Gray, E. & Barrowcliffe, T.W. Characterization of unfractionated heparin: comparison of materials from the last 50 years. Thromb. Haemost. 84, (2000). 14. Honchel, R. et al. A dose- response study in animals to evaluate the anticoagulant effect of the stage 2 unfractionated heparin USP monograph change. Regul. Toxicol. Pharmacol. 60, (2011). 15. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, (1951). 16. Quinlivan, E.P. & Gregory, J.F.I. DNA digestion to deoxyribonucleoside: a simplified one- step procedure. Anal. Biochem. 373, (2008). 13