1. SPECIFIC QUALITY PROBLEMS WITH ANTIBIOTICS AND ANTITUBERCULOTICS Pr A. NICOLAS, PhD Head of the French Laboratories and Controls Directorate AFSSAPS Interregional Seminar for Quality Control Laboratories involved in WHO Prequalification Programme and/or participating in respective sampling and testing projects, Nairobi, Kenya, 23-25 September 2009 1-3

2. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 2 |2 | ANTIBIOTICS AND ANTITUBERCULOTICS Classification Origin Drug quality reports Fermentation products Impact on the composition of a monograph Antituberculosis dosage forms Conclusion

3. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 3 |3 | ANTIBIOTICS - CLASSIFICATION Very diverse class of compounds Generally grouped according chemical structures –Aminoglysides –Cephalosporins and related beta lactams –Glycopeptides –Macrolides –Penicillins –Quinolones –Sulphonamides and diaminopyrimidines –Tetracyclines

4. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 4 |4 | ANTITUBERCULOSIS - CLASSIFICATION Miscellaneous group of antibacterials Include –Cycloserine –Ethambutol –Isoniazid –Pyrazinamide –Rifampicin

5. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 5 |5 | WHO List of Prequalified Medicinal Products Prequalified antituberculosis products (june 2009) –Cycloserine –Ethambutol –Isoniazid –Ethionamide –Pyrazinamide –Ethambutol + Isoniazid –Ethambutol + Isoniazid + Rifampicin –Ethambutol + Isoniazid + Pyrazinamide + Rifampicin –Isoniazid + Rifampicin –Isoniazid + Pyrazinamide + Rifampicin

6. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 6 |6 | Drug Quality Reports Affecting USAID-assisted Countries (1) USP Drug Quality and Information Program –Updated june 2009 –Repository of publicly-available information to raise awereness of the problem of counterfeit and substandard medicines –More often cited drugs : antimalarials, antiretrovirals –But also : antibiotics, antituberculosis products

7. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 7 |7 | Drug Quality Reports Affecting USAID-assisted Countries (2) Ghana –13/08/2004 Counterfaiting of Ampicillin (root crop cassava) Nigeria –29/05/2008 Augmentin (amoxicillin/clavulanic acid) Ampicillin –15/05/2009 Amoxicillin, ciprofloxacin, ampicillin, cloxacillin, erythromycin

8. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 8 |8 | Drug Quality Reports Affecting USAID-assisted Countries (3) Sierra Leone –17/06 2008 : chloraphecol, tetracycline –9/06/2008 : amoxicillin, ampicillin South Africa –27/05/2009 Antib-4 (pyrazinamide, ethambutol, isoniazid, rifampicin) Ebsar (isoniazid + rifampicin) Tanzania –5/10/2008 : antibiotics Uganda –1/10/2005 : cloxacillin

9. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 9 |9 | ORIGIN These active substances could be obtained by : –synthetic route –semi-synthetic route derived from fermentation product by process involving at least cleavage and/or formation of covalent bonds followed by extraction/purification steps –fermentation The origin impacts the purity profile, choice of the analytical methods and their performance

10. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 10 | REFERENCES : ICH and Ph. Eur. Guideline ICH Q3A(R2) « Impurity in New Drug Substances » Ph. Eur. Chapter 5.10 «Control of Impurities in substances for pharmaceutical use» Ph. Eur. General monograph (2034) «Substances for pharmaceutical use» do not cover « fermentation products and semi-synthetic products derived therefrom » for application of reporting, identification and qualification thresholds

11. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 11 | THRESHOLDS FOR ORGANIC IMPURITIES IN ACTIVE SUBSTANCES ( General case ) Qualification threshold Identification threshold Reporting threshold Maximum daily dose Use > 0.15 per cent> 0.10 per cent> 0.05 per cent≤ 2 g/day Human use or human and veterinary use > 0.05 per cent > 0.03 per cent> 2 g/day Human use or human and veterinary use > 0.50 per cent> 0.20 per cent> 0.10 per centNot applicable Veterinary use only

12. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 12 | PRODUCTS OF FERMENTATION (1) Relevant monograph from Ph. Eur. « Products of fermentation (1468) » This monograph applies to : products obtained by semi-synthesis from a product of fermentation and those obtained by biocatalytic transformation whole broth concentrates or raw fermentation products A complex class of compounds with many analytical challenges

13. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 13 | PRODUCTS OF FERMENTATION (2) Fermentation processes are biosynthetic pathways consisting of parallel reactions –Some antibiotics are mixtures of structurally related substances –Not always clear which of these substances contribute to activity (active or impurity ?) –Separation not always achievable

14. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 14 | PRODUCTS OF FERMENTATION (3) Biosynthetic pathways difficult to control (variation from batch to batch) –Different strains of same species may lead to different impurity profiles –Large differences in media components in fermentation may lead to different impurity profiles

15. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 15 | MONOGRAPH – DEFINITION (1) Substances obtained by synthetic or semi-synthetic route correspond to a major known compound Substances obtained by fermentation are often but not always defined as mixtures The name of the strain is indicated

16. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 16 | MONOGRAPH – DEFINITION (2) Synthetic : ciprofloxacin 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4- dihydroquinoline-3-carboxylic acid. Semisynthetic : rifampicin 2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)- 5,6,9,17,19-Pentahydroxy-23-methoxy-2,4,12,16,18,20,22- heptamethyl-8-[[(4-methylpiperazin-1-yl)imino]methyl]-1,11-dioxo- 1,2-dihydro-2,7-(epoxypentadeca[1,11,13]trienimino)naphto[2,1- b]furan-21-yl acetate Semisynthetic antibiotic obtained from rifamycin SV.

17. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 17 | MONOGRAPH – DEFINITION (3) Fermentation leading to a known major compound : josamycin Josamycin is a macrolide antibiotic produced by certain strains of Streptomyces narbonensis var. josamyceticus var. nova, or obtained by any other means. The main component is…… Fermentation leading to a mixture : gentamicin Mixture of the sulphates of antimicrobial substances produced by Micromonospora purpurea, the main components being gentamicins C1, C1a, C2, C2a and C2b.

18. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 18 | MONOGRAPH - IDENTIFICATION Synthetic and semisynthetic –Identification by IR spectroscopy (2.2.24) Mixtures obtained by fermentation –IR if structures of the individual compounds are very similar : erythromycin –Identification by examination of the chromatograms obtained in the test for composition Gentamicin : the chromatogram obtained with the test solution shows 5 principal peaks having the same retention times as the 5 principal peaks in the chromatogram obtained with reference solution (gentamicin sulphate CRS ).

19. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 19 | MONOGRAPH - TESTS Composition –Used to specify complex mixture such as some fermented polypeptides (tyrothricin) or aminoglycosides (gentamicin) Related substances –ICH impurity limits/thresholds cannot be applied because of wide variety in purity and complexity –Not possible to establish one fixed set of thresholds for identification and qualification –Specifications should be derived on a case by case basis considering batch results of various suppliers

20. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 20 | MONOGRAPH - TESTS COMPOSITION In the case of mixture a « composition test » is described Composition. Liquid chromatography (2.2.29): use the normalisation procedure taking into account only the peaks due to gentamicins C1, C1a, C2, C2a and C2b; use the chromatogram supplied with gentamicin sulphate CRS to identify the corresponding peaks. gentamicin sulphate CRS Limits –gentamicin C1 : 20.0 per cent to 40.0 per cent, –gentamicin C1a : 10.0 per cent to 30.0 per cent, –sum of gentamicins C2, C2a, and C2b: 40.0 per cent to 60.0 per cent

21. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 21 | COMPOSITION – GENTAMICIN (1) Known structures of 5 components

22. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 22 | COMPOSITION – GENTAMICIN (2) J. Pharm. Biomed. Analysis, 2007, 45, 257-262

23. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 23 | MONOGRAPH - TESTS RELATED SUBSTANCES (1) Same method as for composition but different specifications Content in impurities can be very high and chemical structures are not always known Gentamicin Limits (for related substances eluting before gentamicin C1a): –any impurity: maximum 3.0 per cent –total: maximum 10.0 per cent

24. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 24 | MONOGRAPH - TESTS RELATED SUBSTANCES (3) Threshold for « any unspecified impurity » in current antibiotics monographs from Ph. Eur. –Ciprofloxacine : 0.1 % –Cefradine : 0.25 % –Amoxicillin trihydrate : 1 % –Gentamicin sulphate : 3.0 %

25. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 25 | MONOGRAPH ASSAY – MICROBIOLOGICAL TITRATION Microbiological assay of antibiotics (2.7.2) –Two methods A. Diffusion method (19 antibiotics) B. Turbidimetric method (14 antibiotics) –Indication of the reagents, micro-organisms, pH, temperature –Chapter 5.3. Statistical analysis of results of biological assays and tests –Use of CombiStats www.edqm.eu

26. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 26 | MONOGRAPH - ASSAY HPLC (1) ASSAY – HPLC (2.2.29) –For synthetic and semi-synthetic drugs –For mixtures obtained by fermentation if similar activity of the individual compounds –Possibilty of sums of active substances HPLC/UV –For chromophoric compounds (general case) –Or after derivatization (amikacine)

27. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 27 | MONOGRAPH - ASSAY HPLC (2) Erythromycin –Content: sum of the contents of erythromycin A, erythromycin B and erythromycin C: 93.0 per cent to 102.0 per cent (anhydrous substance), erythromycin B: maximum 5.0 per cent, erythromycin C: maximum 5.0 per cent.

28. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 28 | MONOGRAPH - ASSAY HPLC (3) Other detection modes (aminosides) –HPLC/PAD (pulsed amperometric detection), Ph. Eur and US, Ph. Int. Difficult to handle, poor precision Noisy, high disregard limit (1.0 % for framycetine and neomycine sulphate) –HPLC/ELSD Gentamicin sulphate (Chinese Ph.) Simultaneous determination of content in sulphates (in place of titration)

29. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 29 | MONOGRAPH - STORAGE General conditions : in airtight container Specific conditions –Protected from light: tiamuline –Multiple conditions In an airtight container, at a temperature of 2 °C to 8 °C : potassium clavulanate Under nitrogen in an airtight container, protected from light, at a temperature not exceeding 25 °C : rifampicin

30. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 30 | MONOGRAPH - TRANSPARENCY LIST (IMPURITIES) List of specified and other detectable impurities (for definition see Ph. Eur. General monograph 2034) All cases –Absence to a very long list Nystatin, Josamycin, tyrothricin : no list Tiamuline : 6 specified impurities, 12 other detectable impurities GRAHEK R and ZUPANCIC-KRALJ J. Pharm. Biomed. Anal. XXX(2009)xxx in press Identification of gentamicin impurities by LC tandem mass spectrometry : 17 impurities

31. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 31 | ANTITUBERCULOSIS DOSAGE FORMS Ph. Int., 1st Suppl : 6 new monographs including 2, 3 and 4 component fixed-dose preparations –Doxycycline capsules –Isoniazid and ethambutol hydrochloride tablets –Rifampicin capsules and tablets –Rifampicin and isoniazid tablets –Rifampicin, isoniazid and pyrizinamide tablets –Rifampicin, isoniazid, pyrizinamide and ethambutol hydrochloride tablets Introduction of dissolution test in monographs for tablets and capsules : doxycycline, isoniazid

32. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 32 | 4 COMPONENTS ANTITUBERCULOSIS TABLETS Rifampicin (150 mg), isoniazid (75 mg), pyrizinamide (400 mg) and ethambutol hydrochloride (275 mg) tablets Rifampicin : semisynthetic antibiotic obtained from rifamycin SV. Isoniazid, pyrizinamide and ethambutol hydrochloride : synthetic compounds Monographs in Ph. Eur., USP, Ph. Int., Chinese

33. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 33 | ETHAMBUTOL, HCl ChinesePh. Int.USPPh. Eur. Min 98.5 % Titrimetry 98.0 – 100.5 % Titrimetry 98.0 – 100.5 % Titrimetry 99.0 – 101.0 % Titrimetry Content Aminobutanol TLC : 1.0 % Aminobutanol TLC : 1.0 % Aminobutanol Derivatization by fluorescamine Fluorimetry 385/485 nm : 1.0 % HPLC 215 nm Imp B : 1.0 % Other : 0.10 % Total : 1.0 % GC (2.4.24) Imp D : 5 ppm (1-2 dichloroethane) TLC Imp A : 1.0% Impurities

34. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 34 | ISONIAZID ChinesePh. Int.USPPh. Eur. Min 99.0 % Titrimetry 98.0 – 101.0 % Titrimetry 98.0 – 102.0 % HPLC 99.0 – 101.0 % Titrimetry Content TLC Hydrazine: 0.02 % TLC Hydrazine: 0.02 % NONETLC Hydrazine : 0.05 % Other : 0.2 % Impurities

35. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 35 | PYRAZINAMIDE ChinesePh. Int.USPPh. Eur. Min 99.0 % Titrimetry 98.5 – 101.0 % Titrimetry 99.0 – 101.0 % Titrimetry 99.0 – 100.5 % Titrimetry Content TLC Any : 0.2 % None TLC Any : 0.2 % Impurities

36. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 36 | RIFAMPICIN ChinesePh. Int.USPPh. Eur. Min 93.0 % HPLC 254 nm 97.0 – 102.0 % Absorbance 475 nm 95.0 – 103.0 % HPLC 254 nm 97.0 – 102.0 % Absorbance 475 nm Content HPLC 254 nm Rifampicinquino ne : 1.5 % Rifampicin N - Oxide: 0.5 % 3-formyl- rifamycin SV : 0.5 % Total other:3.0% TLC Rifampicinquinone : 1.5 % 3-formyl-rifamycin SV : 0.5 % Other : 1.0 % HPLC 254 nm Rifampicinquinone : 1.5 % Other : 1.0 % Total : 3.5 % HPLC 254 nm Rifampicinquinone : 1.5 % Other : 1.0 % Total : 3.5 % Impurities

37. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 37 | 4 COMPONENTS ANTITUBERCULOSIS TABLETS – Ph. Int. (1) Rifampicin (150 mg), isoniazid (75 mg), pyrizinamide (400 mg) and ethambutol hydrochloride (275 mg) tablets Ph. Int. (4th Edition, 1st supplement 2008) Identification –Tests A and B (retention time/HPLC for assay) or –Test C : 2 TLC systems (different stationary phases)

38. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 38 | 4 COMPONENTS ANTITUBERCULOSIS TABLETS – Ph. Int. (2) Impurities –Rifampicin-related substances (RP-HPLC-UV) Addition product : 3-formylrifamycin SV (specified impurity of rifampicin limited to 0.5% in the API) and isoniazid : 5.0 % Rifampicin quinone : 4.0 % Any other impurity : 1.5 % Total : 10.0 % Disregard limit : 0.1 % –Impurities in API 3-formylrifamycin SV : 0.5 % Rifampicin quinone : 1.5 % Any other impurity : 1.0 %

39. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 39 | 4 COMPONENTS ANTITUBERCULOSIS TABLETS - Ph. Int. (3) No dissolution test Assay –First RP-HPLC-UV system for isoniazid, pyrizinamide and ethambutol hydrochloride –Second RP-HPLC-UV system for rifampicin –Specifications : 90.0 to 110.0 % of the amounts stated on the label

40. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 40 | 4 COMPONENTS ANTITUBERCULOSIS TABLETS - USP Identification By retention time (2 HPLC systems) Dissolution Not less than 75 % of the labeled amounts of each active substance in 45 min Impurities No test Assay 2 HPLC systems : content 90.0 to 110.0 % in each component

41. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 41 | ANTIBIOTICS – COUNTERFAITING (1) Screening of some of the most conterfeited antibiotics in a single chromatographic run Gaudiano M.C. et coll, J. Pharm. Biomed. Anal., 2008, 48, 303-309 RP-HPLC method, elution by gradient, detection 230 nm Ampicillin, amoxicillin + clavulanic acid, doxycycline, cloxacillin, chloramphenicol

42. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 42 | ANTIBIOTICS – COUNTERFAITING (2)

43. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 43 | MACROLIDES – COUNTERFAITING (1) Fast chemical identification system for screening of counterfeit drugs of macrolide antibiotics CHANG-QIN HU et coll., J. Pharm. Biomed. Anal., 2006, 540, 68-74 –Two color reactions + 2 TLC –Sreening of 10 macrolides

44. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 44 | MACROLIDES – COUNTERFAITING (2)

45. Sampling and testing for Quality Control Laboratories, Nairobi, September 2009 45 | CONCLUSION Products from fermentation (antibiotics/antituberculosis) are a challenge for analysts and for the authorities –Complex mixtures –All compounds not well known (impurity profile) –Specific analytical methods (HPLC/amperometry) –Content determined by activity assay Products obtained by synthetic routes –Obey to ICHQ3A (R2) –Large place for classical HPLC methods coupled to UV detection –Impurity profile well known