Systematic Approach to Extractables/Leachables Studies. Aryo Nikopour September 11, 2014

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1 Systematic Approach to Extractables/Leachables Studies Aryo Nikopour September 11, 2014

2 Outline Definition Industry Guidance Extractables Method Development Extractables Method Validation Leachables Method Development Leachables Method Validation E/L Experience Case Studies

3 What Does Drug Packaging Do? Store and Protect The Drug Product

4 What Does Drug Packaging Do? Manufactures the Drug Delivers the Drug

5 Suitability of Container Closure System Compatibility Performance Suitability for Use Safety Protection

6 Extractables Extractable: A chemical compound (volatile, semi-volatiles non-volatile and elemental impurities) that gets extracted from a packaging component in a suitable solvent by utilizing optimum extraction conditions (time and temperature). Extractable profile for a given packaging component, typically can be a chromatogram (GC-MS, LC-MS and ICP-MS) representing all possible extractables. Extractable profile is established for all packaging components (resin, vial, foil-laminate) for their consistent quality assurance. FDA DSaRMAC, May 2004

7 Leachables Leachable is any chemical compound (volatile, non-volatile) that leaches into the drug product formulation either from a packaging component or local environment on storage (time and temperature) through expiry of the drug product. An extractable can be a leachable. Extractables Leachables

8 Leachables The identity and concentration of recurring leachables in the drug product or placebo formulation should be determined through the end of the drug products shelf life. In general, the levels of extractables should be greater than the levels of leachables for the correlation to be considered valid. Evaluation of leachables in the drug product formulation in future routine stability studies may not be needed when such a correlation exists. Guidance for Industry: Container Closure System for Packaging Human Drugs and Biologics. May 1999

9 Approaches to E/L Studies

10 FDA Guidance for Industry MDI and DPI Drug Products: Chemistry, manufacturing, and controls documentation. Suggested Testing: The drug product should be evaluated for compounds that leach from elastomeric, plastic components or the coating of the container and closure system, such as. polynuclear aromatics, nitrosamines, monomers, plasticizers, accelerators, antioxidants and vulcanizing agents

11 FDA Guidance for Industry Determine the identification and concentration profile through the end of the drug product shelf life. Correlate with the extractables profile of the container and closure components (determined under the various control extraction conditions).

12 FDA Guidance for Industry Degree of Concern Associate with Route of Administration Highest High Low Likelihood of Packaging Component-Dosage Form Interaction High Medium Low Inhalation Aerosols Injections and injectable Suspension Ophthalmic Solutions Transdermal Ointments and Oatches Nasal Aerosols and Sprays Topical Solutions and Suspensions Topical and Lingual Aerosols Oral Solution and Suspensions Sterile Powders and Powders for Injection Inhalation Powder Topical Powders; Oral Powders Oral Tablets Oral Capsules

13 Approaches to E/L Studies Determine critical components Preform a Risk assessment Extractable studies (establish extractable profile) - Control Extraction Studies - Model Extraction Studies - Routine Extraction Studies Leachable studies - Method Development - Method Validation - Stability Studies Establish correlation between extractables and leachables Toxicity Evaluation

14 Step 1. Toxicity Evaluation Consult with a toxicologist for the Toxicity Evaluation of the major extractable. Determine the Permitted Daily Exposure (PDE). Finalize the target leachable compounds based on the quantitative data of extractable and PDE data. Risk Assessments: Dose Duration Route of Administration Patient Population & Indication Special Population Women of Childbearing Potential

15 Step 2a. What Are The Critical Components? Is the container closure in contact with the formulation? Identify Material (i.e., HDPE, LDPE, etc.) Is the container closure in contact the patient s mouth or mucosa? Secondary Container/Closure? Extractables/Leachables Potential?

16 MDI Device Actuator Contaminant

17 Typical Foil-laminate Components A = Exterior layer Polyester/PP/PE ( inch) B = Inks C = Adhesive1 D = Aluminum Foil ( inch) E = F = G = H = Adhesive2 Nylon/Polyester/ PP/PE(0.001 inch) Adhesive3 Interior layer Polyester/PP/PE (0.003 inch) DSaRMAC, May 05,2004

18 Step 2b. Literature Search Review DMF (Type III) Review CDER Guidance for Container Closure Systems for Packaging of Human Drug and Biologics Review Leachables and Extractables Testing Points to Consider form ITFG/IPAC-RS Review Guidance for MDI/DPI Review USP <661> and <381> Leachables and Extractables Handbook, Wiley 2012

19 USP General Chapters USP <381> Elastomeric closure USP <660> Containers-Glass USP <661> Containers-Plastic Plastic Materials of Construction Plastic Packaging for Pharmaceutical Use Plastic Systems Used for Manufacturing Pharmaceutical Products Plastic Medical Devices to Deliver or Administer Pharmaceutical Products USP <671> Container-Performance Testing USP <87> Biological Reactivity Tests- In vitro USP <88> Biological Reactivity Tests- In vivo USP <1663> Assessment of Extractables Associated with Pharmaceutical Packaging USP<1664> Assessment of Leachables Associated with Pharmaceutical Packaging

20 Irvine Approach USP <381> Physicochemical Tests Elastomers USP <381> Heavy Metal ph Reducing Agent Total Extractable Turbidity

21 USP <661> Physiochemical Testing Procedures Plastics Extract by water at 70 C Non-volatile Residues Residue on Ignition Heavy Metals Buffer Capacity

22 Step 3. Establish Extractable Profile (Control Extraction Studies) A controlled extraction study, is a qualitative and quantitative investigation of critical components of CCS. Establish a basis for development and validation of analytical method in support of routine QC testing. Establish a basis for development of Leachable studies. Allow for the Correlation of extractables and leachables. Reference: Leachables and Extractables Handbook, Wiley 2012

23 Step 3. Establish Extractable Profile (Control Extraction Studies) 1. Extraction studies of critical component using solvents with different strengths: a. IPA (Soxhlet, Reflux) b. Water (Pressurized Vessel) c. Heptane (Soxhlet, Reflux) 2. Determine total organic carbon in extract by TOC (for Water 1b) 3. Employ HS-GC/MS to determine the volatile Extractables 4. Employ LC-MS, IC, ICP-MS methods to Screen for Extractables 5. Establish AET level 6. Identify target compounds

24 Step 3. Establish Extractable Profile (Control Extraction Studies) An Optimized Extraction method, is defined as one that yields a high number and concentration of extractables, achieves steady-state levels, that is, Asymptotic levels.

25 Step 3. Establish Extractable Profile The Proposed Specification for Extractables Extractable Level in Component Tasks > 100 ppm Structure confirmed ppm Tentatively identified < 20 ppm Reported as unknown Lower detection limits required for nitrosamine, PAHs, and MBTs, they are consider as special case Sources: ITFG/IPAC-RS Leachables and Extractables Testing: Points to Consider

26 Toxicological Threshold Level Class I Class II Class III Class IV Sensitizer Class IV Irritant Class V Genotoxicant Threshold Level (µg/day) PQRI The leachables as provided by the Chemistry, is evaluated based on individual leachables, concentration and estimated total daily dose, relative to the propose thresholds for safety evaluation

27 Level of Detection Requirement Establishment of Analytical evaluation threshold (AET) for extractables encountered in material screening: Safety Concern Threshold (SCT) needs to be known. Weight of container closure system (CCS) must be known. Maximum number of dose per day a patient will be taking.

28 Level of Detection Requirement (cont.) AET (µg/g)= [ (SCT(µg)/day) (max# of doses/day) (wt of component (g)/dose)] x (UF) Where, AET: SCT: Max # Wt. of component UF Analytical Evaluation threshold Safety Concern threshold Maximum number of doses a patient would take per day Weight of the component per dose (g/unit dose) Uncertainly factor used to correct for source of uncertainty with the method Leachables and Extractables Handbook Wiley publishing 2012

29 Level of Detection Requirement (cont.) Example Application of AET Component: Glass with Elastomeric Closure Delivery System: Injectable SCT: 1.5 µg/day No. of Doses: 1/day Vial Volume: 1 ml Max # of Dose: 1 vials/day AETproduct= [1.5 µg/day] *[1day/1vials]* [1 vial/1 ml] * [0.5]= 0.75 µg/ml

30 Step 3. Control Extraction Documentation Protocol Introduction about Container/Closure Extraction Procedure Type of Solvents Temperature/Duration Sample Size Method of Analysis Report Introduction about Container/Closure Extraction Procedure Type of Solvents Temperature/Duration Sample Size Method of Analysis Identify Surrogate Standards Results AET Evaluation Tentative ID of Peaks above AET level

31 Step 4. Routine Extraction Based on the analytical and toxicological evaluation of the extractables from the control extraction studies, the applicant should establish discriminatory test procedures and set appropriate acceptance criteria for the extractable profiles for routine testing for each critical component of the container closure system. This testing will provide continued assurance of the batch-tobatch consistency of the composition and purity of the container and closure components. An extraction test should be performed on every incoming component batch, using water and other suitable solvents selected from the control extraction studies, to determine the individual and total extractables. *Guidance for industry: Nasal and Inhalation Solution

32 Step 5. Model Extraction Studies Conduct Model extraction studies by choosing up to 3 different mediums representative of final formulation (Placebo), e.g., buffer at various ph. Container closure will be filled with three formulation and stored at accelerated conditions, e.g. 40ºC/75%, 50ºC. Samples are analyzed at initial, 1 week, 3 and 6 weeks. Representative methods from Controlled Extraction studies will be employed to analyze the samples (LC/MS, GC/MS,..). Evaluate data and determine the potential Leachables. Model Extraction Studies will expedite the risk assessment for determination of leachable studies.

33 Step 6. Leachable Studies Risk Assessments: Dose Duration Route of Administration Patient Population & Indication Special Population Women of Childbearing Potential

34 Step 6. Leachable Studies Apply extractable analytical methods to drug formulation and aged stability samples through shelf-life. Optimize the methods to avoid interference from active and excipients. Report leachable 0.2 µg total daily intake (TDI), and identify leachable 2 µg (TDI).* *Proposed by IPAC-RS

35 Step 6. Leachable Studies Method Development Specificity Linearity QL Accuracy At Level of Concern Precision Repeatability System Suitability Robustness Method Validation Specificity Linearity QL Accuracy At Level of Concern Precision Repeatability Intermediate Precision Solution Stability System Suitability Robustness Follow ICH Q2(R1) for Quantitative Impurities

36 Step 7. Leachable Stability Studies Include Leachable Studies as part of Official Stability Studies in support of NDA and ANDA submission. Employ validated methodologies which could detect and accurately quantitate the potential Leachables. Method(s) also need to monitor and screen for unexpected leachables.

37 Step 8. Establish a Correlation Between Leachables and Extractables Based on the chromatographic and spectroscopic data, a correlation between leachables and extractables is established when each leachable can be linked qualitatively to a corresponding extractable, directly or indirectly.

38 Regulatory Requirements Nasal Spray MDI DPI Nebulizer FDA Nebulizer Guidance FDA Draft MDI/DPI FDA Nasal Spray and Inhalation Solution NA NA NA None NA ABC ABC NA ABC NA NA NA EU BDE BDE None BDE A=Controlled Extraction, B= Routine Extraction, C= Leachables, D = extraction profile for non compendial materials, E= routine extractables for testing to monitor for leachables of safety concern.

39 Irvine Pharmaceutical Services Experience pmdi Valve Nasal Spray Actuator and Cap/Liner Injectable Bags PETE Bottles LDPE in support of Inhalation Solution HDPE Bottles PVC/Foil Laminate in Support of Solid Dose Blister Pack Disposable Holding Bags (SUS) C/C for Parenteral Products (Small Molecules and mab) PFS (mab)

40 Case Studies

41 Case Study I E/L Studies in Support of PFS Pre-Filled Syringe Components Tip Caps Plunger Tip Glass Barrel

42 Case Study I E/L Studies in Support of PFS Controlled Extraction Studies for Rigid Tip Cap In Support of Glass Syringe Syringe with Tip Cap and Shield Rigid Shield Tip Cap Glass Syringe Image of Syringe with Rigid Shield and Tip Cap

43 Case Study I E/L Studies in Support of PFS Controlled Extraction Studies for Rigid Tip Cap In Support of Glass Syringe Controlled extraction studies are to systematically identify and quantify potential Extractables from the Rigid Tip Cap. Controlled extraction studies were performed for the heatinduced extraction and solvent extraction and analyzed by GC/MS, LC/MS and ICP/MS for volatile, semi-volatile, nonvolatile and elemental impurities, respectively.

44 Case Study I E/L Studies in Support of PFS Volatile Extractable Profile for Tip Cap Propene Tip Cap Sample Isobutylene Isopropanol Carbon Disulfide Thiirane AET = 1.05 µg/g Headspace-GC/MS Total Ion Chromatogram of Tip Cap A Sample

45 Case Study I E/L Studies in Support of PFS Semi-Volatile Extractable Profile for Tip Cap Unknown 1 Unknown 2 Tip Cap [20% EtOH Extract] Thiodiglycol 2,2 -Dithiobis ethanol 2-[(1-Phenylethyl)-phenol 4-[(1-Phenylethyl)-phenol AET = 1.05 µg/g GC/MS Total Ion Chromatogram of Tip Cap Solution for 20% EtOH Solvent Extraction

46 Case Study I E/L Studies in Support of PFS Extractable Profile The identities of all extractables were verified against authentic reference standards except for 2- and 4-(1-Phenylethyl)-phenol, which were verified only by matching to NIST library.

47 Case Study I E/L Studies in Support of PFS AET Value In Support of Controlled Extraction Studies for Tip Cap Proposed SCT (Safety Concern Threshold) = 1.5 µg/day Max# of Dose = 1 Wt. of Component = 0.8 g UF (uncertainty factor) = 0.5 SCT ( µg) max# of dose wt AET (µg/g) = day day = = 0.94 ( µg / g) of component ( g) dose ( UF) Parameter AET (µg/g) Tip Cap 0.94 (µg/g)

48 Case Study I E/L Studies in Support of PFS Leachable Method Development & Validation

49 Case Study I E/L Studies in Support of PFS Tungsten (W): Originates from the tungsten pins used to produce the channel through which the needle in the syringe is mounted. Silicone Oil (Polydimethylsiloxane, PDMS): Widely used as a lubricant for the plunger in pre-filled syringes. Both silicone oil and tungsten have a tendency to interact with proteins leading to aggregation. The quality of a protein drug product is therefore affected by the presence of tungsten or silicone oil from the syringe.

50 Case Study I E/L Studies in Support of PFS Mechanism of silicone oil-induced protein aggregation: A protein can adsorb onto the hydrophobic silicone oil/water interface, which may or may not be reversible. Over time, protein can lose its conformation (denaturation), which is mostly irreversible. Denatured species can revert to the bulk and form aggregates with similar molecules in solution. (Source: Concise Encyclopedia of High Performance Silicones (2014), pp )

51 Case Study I E/L Studies in Support of PFS Approach for Silicone Oil Method Development: Develop and optimize a Liquid-Liquid Extraction technique to obtain silicone oil from the Protein Drug Product Develop an HPLC method to separate out silicone oil Use Evaporative Light Scattering Detection (ELSD) to identify and quantify the silicone oil present.

52 Case Study I E/L Studies in Support of PFS Sample Preparation by Liquid-Liquid Extraction: Two-phase extraction using a variety of non-polar solvents resulted in low recoveries of silicone oil from Protein Drug Product Poor recoveries presumed to be due to Silicone Oil tightly binding to protein. Recoveries of above 80% were obtained using a complex onephase extraction technique optimized for extracting silicone oil from protein. (Recoveries 83-96% at 6 µg/ml level).

53 Case Study I E/L Studies in Support of PFS HPLC-ELSD Chromatogram of Protein Drug Product Spiked with 25 ppm Silicone Oil (PDMS)

54 Case Study I E/L Studies in Support of PFS Summary: A method for determination of Silicone Oil in Protein Drug Product was developed with a quantitation limit (QL) of 6.0 µg/ml. An optimized extraction procedure was required to obtain good recoveries of silicone oil from drug product. Free Silicone Oil was found in various placebo-filled syringes at levels from less than QL to 73.2 µg/ml. Tungsten was found in the placebo-filled syringes from 0.10 to 2.51 µg/ml. Other compounds found as extractables, were not detected as leachable in the Protein Drug Product.

55 Case Study I E/L Studies in Support of PFS Non-GMP Study of Tungsten and Silicone Oil in Placebo-filled Syringe Tungsten results are for the contents of each of nine syringes for each syringe type. Silicone oil results are for the contents of each of nine syringes for Syringe Type 2, and nine preparations with the combined contents of two syringes each for the other syringe types. QL is 2.0 µg/ml for placebo study.

56 Case Study II Leachable Studies in Support of Single-Use Bioprocess Bag (SUS) Introduction: Single-Use BioProcess Bags are made of an advanced medical-grade film. This film is a five-layer, 14 mil cast film and coextruded polyolefin film. The outer layer is a polyester elastomer coextruded with an EVOH barrier layer and an ultra-low density polyethylene product contact layer. The layers of film also include: co-polyester ether (COPE), a blend of maleic anhydride modified and unmodified polyethylene, ethylene vinyl alcohol, and an ethylene alpha olefin material. The commercial Bag sizes can very from less than 1 liter to greater than 1,500 liters. The volume of the Single-Use BioProcess Bags used for this study is 100 ml.

57 Case Study II Leachable Studies in Support of Single-Use Bioprocess Bag (SUS) Techniques: 1. Ion Chromatography was used to quantitate levels of acids in bags 2. An Agilent GC/MS system was used to analyze the Headspace volatile organic compounds and the Soxhlet solvent extractables for semi-volatile organics. 3. An API 4000 TM LC/MS/MS system was used to analyze Soxhlet IPA/Heptane/H 2 O extractions for polar, non-polar and aqueous extractables. Model Extraction Conditions: 1. All samples stored at 60ºC for 0, 1, 2, 3 and 4 weeks. 2. Model extraction solution were analyzed by IC, GC/MS and LC/MS.

58 Case Study II Leachable Studies in Support of Single-Use Bioprocess Bag (SUS) Results Ion Chromatography Bag Boric Acid (µg/ml) Formic Acid (µg/ml) Citric Acid (µg/ml) Type I Type II

59 Case Study II Leachable Studies in Support of Single-Use Bioprocess Bag (SUS) 21 Potential Leachables found from Controlled extraction Study: Low molecular acids: Acetic acid, Boric acid, Formic acid, and Citric acid Three long chain aliphatic carboxylic acids Palmitic acid, Stearic acid, and Myristic acid Five low MW, non-antioxidant Aromatic compounds Dibutylphthalate, Dibutyl phthalate, 2, 4-Di-tert-butylphenol, 2, 5-Di-tertbutylphenol, 1, 3-Di-tert-butylbenzene, and 2, 2 -Bipyridyl Amides: Erucamide, Hexadecanamide, Octadecanamide, Oleamide Three Antioxidants: Irganox 1010, Irganox 1076, and Irganox 168 Siloxane: Decamethylcyclopentasiloxane

60 Case Study II Leachable Studies in Support of Single-Use Bioprocess Bag (SUS) Unknown Peak LC/MS/MS ESI Negative Scan RT= min Structure of Unknown (Irgafos 168 degradant) Irgafos 168 Determination of the Structure of an Unknown Peak by MS/MS

61 Case Study II Leachable Studies in Support of Single-Use Bioprocess Bag (SUS) O O P O OH The unknown peak is an Irgafos 168 Degradant Synthesis Characterization Method Development Method Validation Quantitation of the Sample in Bioprocess Bag

62 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions Purpose: Quantitation of Extractables from MDI Solution Extractables: MBT, MBTS, Acenaphthene, Phenanthene, Anthracene, Fluoranthene, Pyrene T.J. Deng & A. Nikopour

63 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions

64 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions Extraction Profile: 300 mg nitrile rubber closure in MDI valves is extracted in 5 ml ETOH for 60 minutes by sonication. Sample Preparation: Add the content of an inhaler (Albuterol MDI) to 25 ml 0.01 N HCl.

65 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions HPLC Method: Symmetry C18, 2 mm x 50 mm UV at 325 nm for MBT and PAHs UV at 280 nm for MBT Flow rate 0.4 ml/min Gradient mode

66 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions LOD/LOQ of HPLC-UV Method Compound LOD (ppm) LOQ (ppm) MTB MTBS Pyrene Anthracene Acenaphthene Phenathene Fluoranthene

67 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions mau MBT pyrene Time (min) Extractable Profile of Elastomeric Closure

68 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions mau MBT Pyrene Time (min) Leachables Found in Albuterol Sulfate MDI Solution (expired)

69 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions MBT and PAHs by LC-MS (SIM) method

70 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions ng/ml (ppb) in solution Compound LOD LOQ Mercaptobenzothiazole (MBT) 5 16 acenaphthene phenathrene anthrecene fluoranthene pyene LOD/LOQ of MBT and PAHs by LC-MS (SIM) method

71 Case Study III HPLC and LC/MS Determination of MBT/MBTS and PAHs in Metered Dose Inhalation Solutions Conclusion: HPLC/UV and LC-MS methods were developed to simultaneously quantify MBT/MBTs and PAHs extractables/leachables in MDI device and drug formulation. The detection limits of all compounds ranged from 70 ppb to 0.7 ppm for the UV method, and 0.5 ppb to 0.4 ppm for the LC-MS method. MBT and pyrene were detected and identified in Albuterol MDI solution.

72 Case Study IV Quantitation of N-Nitrosamines by LC/MS/MS N-Nitrosamines are potential carcinogens. N-Nitrosamines are common extractables in pharmaceutical elastomeric packaging. FDA expects the detection limit (DL) of N-Nitrosamines to reach ppb level.

73 Case Study IV Quantitation of N-Nitrosamines by LC/MS/MS A Reverse Phase APCI(+) MS/MS method Method Detection/Quantification Limit, ng/ml (ppb) in Solution Compound LOD LOQ N-Nitrosodimethylamine (NDMA) N-Nitrosodiethylamine (NDEA) N-Nitrosomethylethylamine (NMEA) N-Nitrosodi-n-propylamine (NDPA) N-Nitrosodi-n-butylamine (NDBA) N-Nitrosodiphenylamine (NDFA) N-Nitrosomorpholine (NMOR) N-Nitrosopiperidine (NPIP) N-Nitrosopyrrolidine (NPYR)

74 Case Study IV Quantitation of N-Nitrosamines by LC/MS/MS Example: NDEA, NPIP, and NDPA at 0.1 ng Injection

75 Case Study IV Quantitation of N-Nitrosamines by LC/MS/MS Conclusion: Sub-ppb quantitation limits (QL) of N-Nitrosamines are achieved by an LC/MS/MS method. The improved method sensitivity facilitates the extractable and leachable sample preparation. The characteristic MS/MS transition of each N-Nitrosamine eliminates false positive detection likely to occur in other detection methods (TEA and UV).

76 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Purpose: To Evaluate Extraction Profile of Elastomeric Closure Used in Support of the Lyophilized and Solution Product.

77 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS 80ºC for 30 min Empty Vial (Blank) AET = 113 µg/g 2-Methyl-pentane 3-Methyl-pentane Hexane Methylcyclopentane Tetrahydrofuran Cyclohexane Heptane XYZ 13-mm Lyo Stoppers Headspace Volatile Extractables Profile of Company XYZ 13-mm LYOPHILIZATION STOPPER

78 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Headspace Volatile Extractable Profile of ZYX 20-mm SERUM STOPPERS 80ºC for 30 min Empty Vial (Blank) AET = 42 µg/g 2-Methyl-pentane 3-Methyl-pentane Hexane Methylcyclopentane Cyclohexane ZYX 20-mm Serum Stoppers

79 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Headspace Volatile Extractable Results by GC/MS No. Extractable Name XYZ 13-mm Lyo Stopper Extractable Conc. (µg/g) ZYX 20-mm Serum Stopper Extractable Conc. (µg/g) 1 2-Methyl-pentane Methyl-pentane Hexane Methylcyclopentane Tetrahydrofuran Cyclohexane Heptane

80 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Soxhlet Semi-volatile Extractable Profile Rubber Oligomer XYZ 13-mm Lyo Stoppers & ZYX 20-mm Serum Stoppers BHT Octacosane C 28 H 58 Hexacosane C 26 H 54 Tetracosane C 24 H 50 Docosane C 22 H 46 Eicosane C 20 H 42 Octadecane C 18 H 38

81 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Soxhlet Semi-volatile Extractable Results for XYZ 13-mm LYOPHILIZATION STOPPERS (Eight Semi-volatile Extractables found by GC/MS) Semi-volatile Extractable Name Water Extraction Conc. (µg/g) Ethanol Extraction Conc. (µg/g) Heptane Extraction Conc. (µg/g) AET (µg/g) BHT Rubber Oligomer Octadecane Eicosane Docosane Tetracosane Hexacosane Octacosane

82 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Soxhlet Semi-volatile Extractable Results for ZYX 20-mm SERUM STOPPERS (Eight Semi-volatile Extractables found by GC/MS) Semi-volatile Extractable Name Water Extraction Conc. (µg/g) Ethanol Extraction Conc. (µg/g) Heptane Extraction Conc. (µg/g) AET (µg/g) BHT Rubber Oligomer Octadecane Eicosane Docosane Tetracosane Hexacosane Octacosane

83 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Non-volatile Extractable Profile By LCMS/MS

84 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Elemental Extractable Profile by ICP-MS Elemental Extractable Name Heptane Extraction Conc. [XYZ 13-mm Lyo Stopper] (µg/g) Heptane Extraction Conc. [ZYX 20-mm Serum Stopper] (µg/g) Mg Ca Sr Cu Zn Ni Ta V Not Detected AET (µg/g)

85 Case Study V CONTROLLED EXTRACTION STUDIES IN SUPPORT OF COMPANY XYZ 13-MM LYOPHILIZATION STOPPERS AND COMPANY ZYX 20-MM SERUM STOPPERS Conclusions: 7 Volatile Extractables found but All are less than AET. 8 Semi-volatile Extractables found but only BHT and Rubber oligomers are greater than AET. No non-volatile Extractable found. 8 Elemental Extractables found but all are less than AET.

86 List of E/L Posters Heat-induced Extractable Profile Study In PVC films Using Headspace GC/MS Methodology ; Mai Zhou, Kareem Chehade, Aryo Nikopour; 234th American Chemical Society National Meeting, Poster ID: , Division of Analytical Chemistry, 19 August 2007, Boston, MA, Residual Solvents Screening By Utilization Headspace - GC/MS ; Aryo Nikopour, Mai Zhou, Ruggero Picci, Darren Lorenz; 2007 AAPS Annual Meeting and Exposition, Poster ID: 3451, Nov , 2007, San Diego, CA, USA Heat-Induced and Soxhlet Solvent Extraction Profile Study In Foil Laminate Film Using GC/MS Methodology ; Mai Zhou, Kareem A. H. Chehade, Aryo A. Nikopour; 2008 AAPS Annual Meeting and Exposition, Poster ID: 3225, Nov , 2008, Atlanta, Georgia, USA Testing of Biopharmaceuticals for USP Class 1, 2 and 3 Residual Solvents Using GC/MS, Mai Zhou, John Cui, Aryo Nikopour, Paul Maffuid, BIOZONA 2009, Arizona s Annual Bioscience Conference, April 7, 2009, Phoenix, Arizona Heat-Induced and Soxhlet Solvent Extraction Profile Study in Medical Rubber Component used for Container Closure System Using GC/MS Methodology, Mai Zhou, John Cui, Joseph Bordas-Nagy, Aryo A. Nikopour, 2009 AAPS Annual Meeting and Exposition, Poster ID: T2106, Nov. 10, 2009, Los Angeles, California, USA Heat-Induced and Soxhlet Solvent Extraction Profile Study in HDPE Bottle Used for Container Closure System Using GC/MS Methodology ; Mai Zhou, Dong Zhao, John Cui, Joseph Bordas-Nagy, Aryo A. Nikopour; 2010 AAPS Annual Meeting and Exposition, Poster ID: W5005; Nov. 17, 2010, New Orleans, Louisiana, USA Determination of Melamine, Ammeline, Ammelide and Cyanuric Acid in Pharmaceutical Raw Materials by LC-MS/MS ; John Cui, Mai Zhou, Aryo Nikopour; 2013 AAPS Annual Meeting and Exposition, Poster ID: AM ; Nov. 10, 2013, San Antonio, Texas, USA Heat-Induced and Soxhlet Solvent Extraction Profile Study in Elastomeric Closure Using GC/MS Methodology ; Mai Zhou, Spencer Huang, Robert Lee, Aryo Nikopour; 2013 AAPS Annual Meeting and Exposition, Poster ID: AM ; Nov. 10, 2013, San Antonio, Texas, USA

87 E/L Team

88 E/L Team

89 THANK YOU Questions? Comments?