WO2016199116A1

WO2016199116A1 - Method for maintenance of sterility and integrity of a drug in liquid form

Info

Publication number: WO2016199116A1
Application number: PCT/IL2015/050578
Authority: WO
Inventors: Oded Grinstein; Alan-Shaun WILKINSON; Mark Harold KESSLER
Original assignee: Teva Medical Ltd
Priority date: 2015-06-08
Filing date: 2015-06-08
Publication date: 2016-12-15

Abstract

A method for handling a drug in liquid form including providing a drug in liquid form in a vial, the drug being sterile, attaching a vial adaptor to the vial, attaching at least one syringe adaptor to the vial adaptor, at least one of the syringe adaptor and the vial adaptor being vented in a manner which prevents communication of micro-organisms and other particulates from the outside atmosphere to an interior of the vial, withdrawing a first quantity, but not all, of the drug from the vial at a first point in time and thereafter withdrawing at least one second quantity of the drug at at least one second point of time following the first point of time by at least 12 hours, the at least one second quantity of the drug maintaining the sterility of the drug.

Description

METHOD FOR MAINTENANCE OF STERILITY AND INTEGRITY OF A DRUG IN LIQUID FORM

FIELD OF THE INVENTION

The present invention relates to liquid drug handling and more particularly to methodology for maintenance of sterility and integrity of a drug in liquid form in a vial during and following removal of some, but not all, of the drug from the vial.

BACKGROUND OF THE INVENTION

Pharmacy compounding is covered under US Public Law 105-115: Section 503A states that the compounding should comply with standards of an applicable United States Pharmacopoeia or National Formulary monograph, if a monograph exists, and the United States Pharmacopoeia chapter on pharmacy compounding. The relevant USP chapter is USP <797>: Pharmaceutical Compounding - Sterile Preparations.

FDA Draft Guidance Pharmacy Compounding of Human Drug Products Under Section 503 A of the Federal Food, Drug and Cosmetics Act, December 2013 states that drug product is compounded using ingredients (other than bulk drug substance) that comply with the standards of an applicable USP or NF (National Formulary) monograph, if one exists and the USP chapters on pharmacy compounding.

United States Pharmacopoeia Chapter <797>: Pharmaceutical Compounding - Sterile Preparations states that opened or needle -punctured single-dose containers, such as bags, bottles, syringes, and vials of sterile products and Compounded Sterile Products (CSPs) shall be used within 1 hour if opened in worse than ISO Class 5 air quality, and any remaining contents must be discarded. Single-dose vials exposed to ISO Class 5 or cleaner air may be used up to 6 hours after initial needle puncture.

In the introduction, United States Pharmacopoeia Chapter <797> states: "The use of technologies, techniques, materials, and procedures other than those described in this chapter is not prohibited so long as they have been proven to be equivalent or superior with statistical significance to those described herein."

SUMMARY OF THE INVENTION

The present invention seeks to provide an improved methodology for handling of drugs in liquid form, which enables a drug in liquid form in a vial to be used effectively and safely over a time duration following initial partial drug removal from the vial, which duration substantially exceeds the currently mandated maximum duration.

There is thus provided in accordance with a preferred embodiment of the present invention, a method for handling a drug in liquid form including providing a drug in liquid form in a vial, the drug in liquid form being sterile, attaching a vial adaptor to the vial, attaching at least one syringe adaptor to the vial adaptor, at least one of the at least one syringe adaptor and the at least one vial adaptor being vented in a manner which prevents communication of micro-organisms and other particulates from the outside atmosphere to an interior of the vial, withdrawing a first quantity, but not all, of the drug in liquid form from the vial at a first point in time and thereafter withdrawing at least one second quantity of the drug in liquid form in the vial at at least one second point of time following the first point of time by at least 12 hours, the at least one second quantity of the drug in liquid form maintaining the sterility of the drug in liquid form.

Preferably, the at least one second point of time follows the first point of time by at least 24 hours. Additionally or alternatively, the at least one second point of time follows the first point of time by at least 2 days. Alternatively or additionally, the at least one second point of time follows the first point of time by at least 7 days.

In accordance with a preferred embodiment of the present invention the at least one second point of time follows the first point of time by at least 14 days. Additionally or alternatively, the at least one second point of time follows the first point of time by at least 28 days.

Preferably, the sterility includes sterility as measured by test performed under USP <71> sterility test. Additionally or alternatively, the second quantity of drug in liquid form also maintains physical and chemical properties of the drug in liquid form. In accordance with a preferred embodiment of the present invention the at least one of the at least one syringe adaptor and the at least one vial adaptor is vented to the atmosphere in a manner which prevents communication of micro-organisms and other particulates from the outside atmosphere to an interior of the vial.

Preferably, the drug in a liquid form is used in the preparation of a high risk level compound sterile preparation (CSP).

In accordance with a preferred embodiment of the present invention the method is performed within a Class 5 laminar air flow hood (LAF). Additionally, the method does not require a cleanroom environment as defined in USP <797>.

Preferably, the withdrawing at least one second quantity of the drug in liquid form in the vial includes withdrawing multiple quantities at multiple respective points of time following the first point of time by at least 12 hours.

In accordance with a preferred embodiment of the present invention the providing a drug in liquid form in a vial includes providing a non-liquid drug in a first vial, attaching a first vial adaptor to the first vial, providing a liquid for reconstituting the non-liquid drug in a second vial, attaching a second vial adaptor to the second vial, attaching at least one syringe to at least one syringe adaptor, attaching the at least one syringe adaptor, with the syringe attached, to the second vial adaptor, transferring at least a quantity of the liquid for reconstituting the non-liquid drug from the second vial into the at least one syringe via the at least one syringe adaptor and the second vial adaptor, thereafter, transferring the at least a quantity of the liquid for reconstituting the non-liquid drug from the at least one syringe into the first vial via the at least one syringe adaptor and the first vial adaptor. Additionally, the method for handling a drug in liquid form also includes reconstituting the non-liquid drug by moving the first vial.

In accordance with a preferred embodiment of the present invention the non-liquid drug includes Trastuzumab.

In accordance with a preferred embodiment of the present invention the drug in liquid form is Cisplatin. Alternatively, the drug in liquid form is Methotrexate.

In accordance with a preferred embodiment of the present invention the drug in liquid form includes Trastuzumab that has been reconstituted. In accordance with a preferred embodiment of the present invention, the drug in liquid form in a vial includes a non-liquid drug in a vial that has been reconstituted.

Preferably, the vial adaptor includes a spike adapted for penetrating the vial, a mechanical lock for locking the vial adaptor to the vial once the spike penetrates the vial and an element operative to vent the interior of the vial in a manner which prevents communication of micro-organisms and other particulates from the outside atmosphere to an interior of the vial. Additionally, the vial adaptor also includes a septum equipped syringe port.

In accordance with a preferred embodiment of the present invention the mechanical lock includes at least one locking element, operative to irreversibly lock the vial adaptor to the vial. Additionally, the at least one locking element includes at least one radially extending portion and at least one transversely extending portion.

In accordance with a preferred embodiment of the present invention the vial adaptor includes at least one locking element, operative to irreversibly lock the vial adaptor to the vial. Additionally, the at least one locking element includes at least one radially extending portion and at least one transversely extending portion.

In accordance with a preferred embodiment of the present invention the at least one syringe adaptor includes a septa housing, at least two septa enclosed in the septa housing defining a space therebetween and a needle, including a tip located in the space when the syringe adaptor is not connected to the vial adaptor. Additionally, the septa housing is movable relative to the needle, thereby to expose the tip.

In accordance with a preferred embodiment of the present invention at least a portion of the needle is protected by a needle protector. Additionally, the needle protector includes an elastomeric tubing element.

In accordance with a preferred embodiment of the present invention the vial adaptor is a TEVADAPTOR® vial adaptor.

In accordance with a preferred embodiment of the present invention the at least one syringe adaptor is a TEVADAPTOR® syringe adaptor. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood and appreciated more fully from the detailed description which follows with reference to the drawings in which:

Figs. 1A-1K are simplified pictorial illustrations of steps in a preferred embodiment of the method of the present invention;

Figs. 2A-2S are simplified pictorial illustrations of steps in another preferred embodiment of the method of the present invention;

Figs. 3 and 4 are graphs showing typical instrument response curves for

Trastuzumab resolution solution at 214 nm and 280 nm, respectively;

Fig. 5 is a graph showing a typical Chromatogram for Resolution Solution Trastuzumab;

Fig. 6 is a graph showing a typical Chromatogram for the formulation buffer for the Trastuzumab resolution solution under identical chromatographic separation conditions as Fig. 5;

Fig 7 is a graph showing a typical Chromatographic profile for Trastuzumab reference solution following protein digest and peptide mapping using Reverse Phase high performance liquid chromatographic separation according to the USP summary validation procedure;

Fig. 8 is a graph showing a Typical Chromatogram for Resolution Solution Trastuzumab without carboxypeptide treatment on Day 28 at a detection wavelength of 280nm;

Fig. 9 is a graph showing a Typical Chromatogram for the treatment of Trastuzumab formulation buffer with caroxypeptidase under identical CEX chromatographic separation conditions as used for Trastuzumab samples at a detection wavelength of 280nm;

Fig. 10 is a graph showing a typical Electropherogram from the separation of intact Trastuzumab (HHLL) under denaturing, Non Reducing conditions performed as part of the validation for the Capillary Electrophoresis separation method for testing of loss of a light chain peptide fragment (low molecular weight impurity test); Fig. 11 is a graph showing a typical Electropherogram from the separation of the Non-Glycosylated form of Trastuzumab peptide heavy chain (NGHC) and the Glycosylated form of the Trastuzumab peptide heavy chain (HC). Separation is performed under denaturing, Reducing conditions performed as part of the validation for the Capillary Electrophoresis separation method for: Limit of NGHC Impurities: CE-SDS;

Fig. 12 is a graph showing a Typical Binding Curve for Resolution Solution Trastuzumab prepared at concentrations between 0 and 500ng/ml on Day 0 at a detection wavelength of 450nm;

Fig. 13 is a graph showing a Typical Binding Curve for Resolution Solution Trastuzumab prepared at concentrations between 0 and 500ng/ml on Day 28 at a detection wavelength of 450nm; and

Fig. 14 is a graph showing a typical drug dose response curve for Trastuzumab resolution solution (RS).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Reference is now made to Figs. 1A-1K, which generally illustrate the methodology of embodiments of the present invention.

The following examples illustrate the present invention:

Example I - Cisplatin

The following protocol was carried out:

General:

The study was carried out pursuant, inter alia, to the following standard operating procedures:

1. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Quality Guideline Q2 (Rl): Validation of Analytical Procedures: Text and Methodology, available on the ICH website.

2. ICH Quality Guideline Q1E, Evaluation of Stability Data implemented as CPMP/ICH/420/02, available on the ICH website.

3. ICH Harmonized Guidelines for Internal Quality Control in Analytical Chemistry Laboratories, available on the ICH website.

4. Standard Protocol for Deriving the Chemical Stability of Aseptic Preparations, edition 2, 2011, National Health Service (NHS) Pharmaceutical Quality Assurance Committee.

5. All growth promotion testing was in accordance with current British Pharmacopoeia standards, available on the British Pharmacopoeia website.

6. All manipulations and inoculation conditions were in accordance with standard microbiological procedures, as seen in Collins, C. H.; Microbiological methods, 1964.

7. Validation of the Environmental Bioburden was undertaken prior to conducting the main sterility study by active air sampling. Aliquots were removed from 40 vials 100 containing Cisplatin and used to inoculate vials 102 containing microbial growth medium on a total of three test time points, at days 0, 7 and 14. On each test day, one sample aliquot was withdrawn for inoculation of Tryptic Soy Broth Growth Medium (TSB) and a second aliquot for the inoculation of Fluid Thioglycollate Growth Medium (FTM).

All growth media vials required for the study were prepared by the Royal Derby Hospital in the UK in a single batch and batch released as part of their standard QC batch release process. All media were growth promotion tested (GPT) and quarantined for at least 2 weeks prior to use to demonstrate sterility of all media.

Sterility of the growth media vials was tested in accordance with the procedure set forth in Table 1.

Time Points nil Methods Specifications/Limits

Tested

Visual inspection for Media remains clear and evidence of growth within free from contamination

Visual

each growth media vial by microorganisms. All

Appearance

(TSB & FTM) during and Standard microbiology after incubation. procedures.

TABLE 1 Between samplings, the drug vials were stored according to the storage conditions listed on the drug label. All sampling took place in an "uncontrolled" preparation area which was tested for environmental bioburden prior to the sterility study.

All inoculated media vials were incubated according to the microbiological testing protocol described hereinbelow.

Microbiological integrity testing of the TEVADAPTOR® Closed- System Drug Transfer system in an "uncontrolled" environment when used with preservative free single dose Cisplatin vials Overview

Preparation of the 40 vials containing Cisplatin for sterility testing on days: 0, 7 and 14. Materials

100 ml Type I glass vials 102, with a 20mm vial cap size and a volume of 50 ml, of Tryptic soy broth (TSB). The TSB was made at an external lab. All batches were tested for sterility and positive control tested to meet the United States Pharmacopoeia and European Pharmacopoeia Growth Promotion test. The tests were carried out by an external contract Quality Control Laboratory.

TEV ADAPTOR® vial adaptor 110, suitable for use with a 20mm vial cap.

TEV ADAPTOR® vial adaptor aseptic cap 120 as supplied with the TEVADAPTOR® vial adaptor 110, for use with a 20mm vial cap, for each TEVADAPTOR® vial adaptor 110

TEVADAPTOR® syringe adaptor 130

5 ml Sterile luer lock syringes 140

Sterile 70% IMS solution prepared from 70 ml isopropyl alcohol and 30 ml sterile water for infusion (WFI)

Sterile 70% IMS prep pads Sterile wipes containing a mixture of 70%

Isopropyl alcohol and 30% water made from 70 ml isopropyl alcohol mixed with 30 ml sterile water for infusion (WFI).

Equipment

Validated 20-25°C (+/- 1°C) and 30-35°C (+/- 1°C) incubators continuously temperature monitored using a validated Comark temperature monitoring system.

Calibrated SAS active air sampling equipment.

Background environmental microbiological assessment

The study was performed in a single location identified to simulate a realistic hospital ward location in terms of viable airborne and surface microbial contaminants. The location was an uncontrolled environment. The room construction and air supply were standard.

The location did not contain the four levels of increasing protective measures of HEPA filtered air quality (ISO Class 7 or 8 Ante Area, ISO Class 7 Buffer Area, ISO Class 5 Primary Engineering Control Area and the ISO 5 laminar air flow hood in the Direct Compounding Area), air pressure cascades and gowning defined in

USP Chapter <797> for sterile compounding.

The preparation location was tested for bioburden prior to performing the study. Air samples from this location were tested using a calibrated SAS air sampler with Tryptic Soy Agar (TSA) and Sabourand Dextrose Agar (SDA) growth media plates. The sampling was carried out before the main study to enable an accurate assessment of the microbiological background of the environments selected to use in the main study. Sampling was performed on three separate days. On each day, lm³ air sampling with TSA and SDA was performed in the morning, middle of day and afternoon.

On the day of the main study, lm³ samples with TSA and SDA were collected at the following time points: in the morning, the middle of the day and the afternoon. A total of six lm³ air samples were collected and subjected to testing. Surface viable counts on the work bench where sampling was performed were measured by lifting five Tryptic Soy Agar (TSA) contact plates prior to testing but after decontaminating the work bench surface using 70% isopropanol (IPA) spray and laboratory paper towel wipes. The five contact plates were incubated immediately after sampling. Aseptic Transfer Procedure

All aseptic manipulation work was carried out in the "uncontrolled" preparation environment that mirrors that used at a clinical preparation level. The Bioburden of the preparation environment was confirmed in terms of suitability based upon the results from the pre- study environmental background testing described hereinabove.

The aseptic transfers each took place as illustrated generally in Figs. 1A-

1K. First, a previously unused and sterile TEVADAPTOR® vial adaptor 110, commercially available from Teva Medical Ltd. of Israel, and including a TEVADAPTOR® vial adaptor cap 120, was initially locked onto each vial 100, as seen at Fig. 1A. A preferred embodiment of the TEVADAPTOR® vial adaptor 110 is fully described in U.S. Patent 8,122,923, the disclosure of which is hereby incorporated by reference.

Thereafter, a previously unused and sterile TEVADAPTOR® syringe adaptor 130, commercially available from Teva Medical Ltd. of Israel, and including a

TEVADAPTOR® syringe adaptor cap 135, was attached to a luer lock syringe 140, as seen in Fig. IB. A preferred embodiment of the TEVADAPTOR® Syringe Adaptor 130 is fully described in U.S. Patent 8,122,923, the disclosure of which is hereby incorporated by reference.

Thereafter, following removal of TEVADAPTOR® vial adaptor cap 120 and TEVADAPTOR® syringe adaptor cap 135, as seen in Fig. 1C, the previously unused and sterile TEVADAPTOR® syringe adaptor 130, having attached thereto the luer lock syringe 140, was operatively engaged with the TEVADAPTOR® vial adaptor

110, as seen in Fig. ID.

On each of days 0, 7 and 14, two aseptic transfers from each drug vial were performed using TEVADAPTOR® syringe adaptors 130 and 5 ml luer lock syringes 140, in which only 2.5 ml of Cisplatin was withdrawn from each vial 100, as seen in Fig. IE.

Thereafter, the TEVADAPTOR® syringe adaptor 130, having syringe 140, containing 2.5 ml of Cisplatin, connected thereto, was disconnected from the TEVADAPTOR® vial adaptor 110, which remained locked onto vial 100 and was covered by TEVADAPTOR® vial adaptor cap 120, as seen in Fig IF.

An additional previously unused and sterile TEVADAPTOR® vial adaptor, designated by reference numeral 150, commercially available from Teva Medical Ltd. of Israel, and including a TEVADAPTOR® vial adaptor cap designated by reference numeral 160, was initially locked onto each growth medium vial 102, as seen in Fig. 1G.

Thereafter, following removal of TEVADAPTOR® vial adaptor cap 160, as seen in Fig. 1H, the TEVADAPTOR® syringe adaptor 130, having syringe 140 connected thereto, was connected in one continuous movement to the designated growth medium vial 102 (TSB or FTM) via TEVADAPTOR® vial adaptor 150, as seen in Fig II.

Thereafter, a 2.5 ml aliquot of Cisplatin from each drug vial was added to one TSB vial 102 and one FTM vial 102 from the syringe 140 via the TEVADAPTOR® syringe adaptor 130 and the TEVADAPTOR® vial adaptor 150, as seen in Fig 1J.

Thereafter, the TEVADAPTOR® syringe adaptor 130 was disconnected from the TEVADAPTOR® vial adaptor 150 of growth medium vial 102 and discarded after use. Aseptic TEVADAPTOR® vial adaptor cap 160 was fitted onto the TEVADAPTOR® vial adaptor 150, as seen in Fig IK.

All of the thus inoculated growth medium vials 102 were sent for 14 day incubation.

The following sequence of actions took place:

Day 0: Preparation of Test Cisplatin vials 100 for testing

All aseptic work was carried out within the designated "uncontrolled" preparation area.

The bench was wiped down using 70% IMS spray and sterile 70% IMS prep pads.

The protective cap was removed from each vial 100 and the bung of each vial 100 was swabbed with sterile IPA 70% prep pad and left to dry for 2 minutes.

Each TEVADAPTOR® vial adaptor 110 was aseptically removed from its packaging and attached to a vial 100 leaving TEVADAPTOR® vial adaptor cap 120 in place.

Day 0: Preparation of Growth Media vials 102 for testing

The bench was wiped down using 70% IMS spray and sterile 70% IMS prep pads.

The cap was removed from a TSB growth medium vial 102, the bung was swabbed with a sterile IPA 70% prep pad and left to dry for 2 minutes. A TEVADAPTOR® vial adaptor 150 was aseptically removed from its packaging and attached to the TSB vial 102 leaving the TEVADAPTOR® vial adaptor cap 160 in place.

The cap was removed from a FTM growth medium vial 102, the bung was swabbed with a sterile IPA 70% prep pad and left to dry for 2 minutes.

A TEVADAPTOR® vial adaptor 150 was aseptically removed from its packaging and attached to the FTM growth medium vial 102 leaving the TEVADAPTOR® vial adaptor cap 160 in place.

Two TEVADAPTOR® syringe adaptors 130 were aseptically removed from their packaging and each attached to a 5 ml syringe 140.

The TEVADAPTOR® vial adaptor cap 120 was removed from each TEVADAPTOR® vial adaptor 110 and the septum of the TEVADAPTOR® vial adaptor 110 was swabbed with a sterile IPA 70% prep pad and left to dry for 2 minutes.

The TEVADAPTOR® syringe adaptor 130, having the syringe 140 attached thereto, was connected to the TEVADAPTOR® vial adaptor 110 of vial 100 and 2.5 ml of Cisplatin was withdrawn from the vial 100 by the syringe 140 via the TEVADAPTOR® syringe adaptor 130 and the TEVADAPTOR® vial adaptor 110.

The TEVADAPTOR® syringe adaptor 130 was thereafter disconnected from the TEVADAPTOR® vial adaptor 110 of vial 100 by depressing the wings of the TEVADAPTOR® syringe adaptor 130. The TEVADAPTOR® vial adaptor cap 120 of the TEVADAPTOR® vial adaptor 110 was reattached to the TEVADAPTOR® vial adaptor 110 of vial 100.

The TEVADAPTOR® syringe adaptor 130, having the syringe 140, containing 2.5 ml of Cisplatin, attached thereto, was thereafter attached via TEVADAPTOR® vial adaptor 150 to the TSB growth medium vial 102.

The plunger of syringe 140 was depressed, thereby inoculating the TSB growth medium vial 102 with 2.5 ml of Cisplatin via the TEVADAPTOR® vial adaptor 150, while the TSB growth medium vial 102 was in an upright position and not inverted.

Following inoculation of the TSB growth medium vial 102, the wings of the TEVADAPTOR® syringe adaptor 130 were depressed and the TEVADAPTOR® syringe adaptor 130 was disconnected from the TEVADAPTOR® vial adaptor 150 and from the TSB growth media vial 102. The TEVADAPTOR® syringe adaptor 130 and syringe 140 were discarded.

A second aliquot of 2.5 ml of Cisplatin was then withdrawn from the same Cisplatin drug vial 100 for inoculating the FTM growth media vial 102. The TEVADAPTOR® vial adaptor cap 120 was removed from the TEVADAPTOR® vial adaptor 110 and the septum of the TEVADAPTOR® vial adaptor 110 was swabbed with a sterile IP A 70% prep pad and left to dry for 2 minutes.

The TEVADAPTOR® syringe adaptor 130, having the syringe 140, containing 2.5 ml of Cisplatin, attached thereto, was thereafter attached via TEVADAPTOR® vial adaptor 150 to the FTM growth medium vial 102.

The plunger of syringe 140 was depressed, thereby inoculating the FTM growth medium contained in vial 102 with 2.5 ml of Cisplatin via the TEVADAPTOR® vial adaptor 150, while the FTM growth medium vial 102 was in an upright position and not inverted.

Following inoculation of the FTM growth medium vial 102, the wings of the TEVADAPTOR® syringe adaptor 130 were depressed and the TEVADAPTOR® syringe adaptor 130 was disconnected from the TEVADAPTOR® vial adaptor 150 and from the FTM growth medium vial 102. The TEVADAPTOR® syringe adaptor 130 and syringe 140 were discarded.

The above procedure was followed for each of the 40 vials of Cisplatin. A total of 80 samples were withdrawn from the vials 100 and used to inoculate 40 vials 102 of TSB and 40 vials 102 of FTM. All of the inoculated TSB growth medium vials 102 and FTM growth medium vials 102 were sent for incubation, as described in the microbiological procedure hereinbelow.

The surfaces of the bench were cleaned and all other materials were removed.

The vials 100 of Cisplatin were stored at room temperature, in accordance with the storage conditions listed on the drug label, within the "uncontrolled" preparation area.

Days 7 and 14

All Cisplatin vials 100 were sampled on each of days 7 and 14 according to the same sampling process as described above.

Microbiological Procedure

The TSA contact plates were incubated at 0-25 °C for 3 days and then at 30-35°C for 2 days. The results are set forth in the final report below.

The TSA air sample plates were incubated for 5 days at 30-35°C.

The SDA air sample plates were incubated at 20-25°C for 7 days.

The three most abundant air sample environmental organisms were identified to genus level post incubation of SDA and TSA air sampling. Each of these most abundant organisms was then transferred into a sterile TSB using aseptic technique within a laminar air flow hood (LAF).

Isolated mold or yeast TSB samples were incubated at 20-25 °C for 14 days. Isolated bacterial TSB samples were incubated at 30-35°C for 7 days.

At test days 0, 7 and 14 each cohort of inoculated TSB growth medium vials 102 were incubated at 20-25°C for 14 days.

At test days 0, 7 and 14 each cohort of inoculated FTM growth medium vials 102 was incubated at 30-35°C for 14 days.

The inoculated TSB and FTM growth medium vials 102 were examined for microbial growth daily on each of the first 7 days and at least twice during the 8^th to 14^th day of the incubation period.

TSB and FTM growth medium vial failures were recorded where turbidity was identified. The growth promoting quality of the TSB medium and the FTM medium in the presence of a 2.5 ml sample aliquot of Cisplatin was confirmed by dispensing 2.5 ml of drug into TSB and FTM vials, and then performing the Method Suitability Test as defined in USP <71> Sterility Tests.

Where growth was observed in either the TSB or FTM growth medium vials 102 following inoculation with Cisplatin, the growth medium was sub cultured to allow for identification of the contaminating species down to the species level up to a maximum of 5 contamination events.

The ability of TEVADAPTOR® systems to maintain a sterile barrier was determined based on the sterility testing of the TSB and FTM growth medium vials 102, absence of growth detected following two weeks incubation of the inoculated growth medium vials 102 and confirmation of the growth promoting quality of the growth medium in the presence of the 2.5 ml drug aliquot.

Results:

Interim Air sampling report:

Environmental Monitoring Assessment of the BSTL Pharmacy Unit - TEVA

Project

Tables 2 - 3 show the results of the Pre-Study environmental monitoring plate results for days 1-3, respectively.

TABLE 2

Key to Notes for Tables 2-5

lTSA Plates are for detecting bacterial organisms, but some fungal organisms may grow on them.

SDA plates are primarily for detection of fungal organisms, but some bacterial organisms may grow on them.

3NT- Not tested.

⁴FT- Failed test due to plate incorrectly positioned in air sampler unit. Sample Day Sample Date Sample Time Total CFU Detected/50mm surface area TSA Plates¹

Preliminary Study 27-Apr-2015 At Start 0

Day 1 At Start 0

Preliminary Study 28-Apr-2015 At Start 19

Day 2 At Start 10

Preliminary Study 29-Apr-2015 At Start 15

Day 3 At Start 17

Average ± Standard Deviations 10+8

TABLE 3

Tables 4 - 5 show the results of the Environmental Monitoring Plate results for study days 0, 7 and 14, respectively.

TABLE 4 Sample Sample Date Sample Time Total CFU

Day Detected/50mm

surface area TSA Plates¹

Study Day 0 28-Apr-2015 Before Start of Sampling 0

Before Start of Sampling 10

Before Start of Sampling 0

Before Start of Sampling 34

Before Start of Sampling ⁴FT

Study Day 7 05-May-2015 Before Start of Sampling 22

Before Start of Sampling 0

Before Start of Sampling 5

Before Start of Sampling 24

Before Start of Sampling 1

Before Start of Sampling 23

Study Day 14 12-May-2015 Before Start of Sampling 2

Before Start of Sampling 0

Before Start of Sampling 10

Before Start of Sampling 1

Before Start of Sampling 0

Before Start of Sampling 30

Average ± Standard Deviations 10+12

TABLE 5

14 Day Sterility Study Report

Study Vial Data Assessment: Cisplatin

Table 6 lists the sterility test results for the Cisplatin drug samples withdrawn from the 40 drug vials on days 0, 7 and 14. Table 7 summarizes the results for the Method Suitability Test required to establish the validity of sterility test method. Number of Vials

Sample Growth Number of Number of

Meeting the Sterility

Day Media Vials Tested Failed Vials

Test Requirement

TSB 40 40 0

Day 0

FTM 40 40 0

TSB 40 40 0

Day 7

FTM 40 40 0

TSB 40 40 0

Day 14

FTM 40 40 0

TABLE 6

TABLE 7 The above results show that sterility of 40 vials of Cisplatin was maintained throughout a 14 day period when drug samples were aseptically removed using the TEV ADAPTOR® closed system drug transfer device in a noncontrolled environment. Sampling from each vial was performed six times over the 14 day period, for a total of 240 samples removed from all of the vials. The Method Suitability Test results confirm the growth promoting capability of the growth medium in the presence of the drug aliquot, with a broad range of microbial strains, thereby confirming the validity of the sterility test results.

The foregoing results thus illustrate a method for handling a drug in liquid form including providing a drug in liquid form in a vial, the drug in liquid form being sterile, attaching a vial adaptor to the vial, attaching at least one syringe adaptor to the vial adaptor, at least one of the at least one syringe adaptor and the at least one vial adaptor being vented in a manner which prevents communication of micro-organisms and other particulates from the outside atmosphere to an interior of the vial, withdrawing a first quantity, but not all, of the drug in liquid form from the vial at a first point in time and thereafter withdrawing at least one second quantity of the drug in liquid form in the vial at at least one second point of time following the first point of time by at least 12 hours, the at least one second quantity of the drug in liquid form maintaining the sterility of the drug in liquid form.

Example II - Methotrexate

The following protocol was carried out:

General:

3. ICH Harmonized Guidelines for Internal Quality Control in Analytical Chemistry Laboratories, available on the ICH website. 4. Standard Protocol for Deriving the Chemical Stability of Aseptic Preparations, edition 2, 2011, National Health Service (NHS) Pharmaceutical Quality Assurance Committee.

7. Validation of the Environmental Bioburden was undertaken prior to conducting the main sterility study by active air sampling.

Aliquots were removed from 40 vials 100 containing Methotrexate and used to inoculate vials 102 containing microbial growth medium on a total of three test time points, at days 0, 7 and 14. On each test day, one sample aliquot was withdrawn for inoculation of Tryptic Soy Broth Growth Medium (TSB) and a second aliquot for the inoculation of Fluid Thioglycollate Growth Medium (FTM).

All growth media vials required for the study were prepared by Royal Derby Hospital in the UK in a single batch and batch released as part of their standard QC batch release process. All media were growth promotion tested (GPT) and quarantined for at least 2 weeks prior to use to demonstrate sterility of all media.

Sterility of the growth media vials was tested in accordance with the procedure set forth in Table 1 hereinabove.

Between samplings, the drug vials were stored according to the storage conditions listed on the drug label. All sampling took place in an "uncontrolled" preparation area which was tested for environmental bioburden prior to the sterility study.

All inoculated media vials were incubated according to the microbiological testing protocol described hereinbelow. Microbiological integrity testing of the TEVADAPTOR® Closed-

System Drug Transfer system in an "uncontrolled" environment when used with preservative free single dose Methotrexate vials Overview

Preparation of the 40 vials containing Methotrexate for sterility testing on days: 0, 7 and 14.

Materials

TEV ADAPTOR® vial adaptor 110, suitable for use with a 20mm vial cap.

TEV ADAPTOR® vial adaptor aseptic cap 120 as supplied with the TEVADAPTOR® vial adaptor 110, for use with a 20mm vial cap, for each TEV ADAPTOR® vial adaptor 110

TEVADAPTOR® syringe adaptor 130

5 ml Sterile luer lock syringes 140

Sterile 70% IMS prep pads Sterile wipes containing a mixture of 70% Isopropyl alcohol and 30% water made from 70 ml isopropyl alcohol mixed with 30 ml sterile water for infusion (WFI).

Equipment

Calibrated SAS active air sampling equipment. Background environmental microbiological assessment

The location did not contain the four levels of increasing protective measures of HEPA filtered air quality (ISO Class 7 or 8 Ante Area, ISO Class 7 Buffer Area, ISO Class 5 Primary Engineering Control Area and the ISO 5 laminar air flow hood in the Direct Compounding Area), air pressure cascades and gowning defined in USP Chapter <797> for sterile compounding.

On the day of the main study, lm³ samples with TSA and SDA were collected at the following time points: in the morning, the middle of the day and the afternoon. A total of six lm³ air samples were collected and subjected to testing. Surface viable counts on the work bench where sampling was performed was measured by lifting five Tryptic Soy Agar (TSA) contact plates prior to testing but after decontaminating the work bench surface using 70% isopropanol (IP A) spray and laboratory paper towel wipes. The five contact plates were incubated immediately after sampling.

Aseptic Transfer Procedure

The aseptic transfers each took place as illustrated generally in Figs. 1 A-

1K.

First, a previously unused and sterile TEVADAPTOR® vial adaptor 110, commercially available from Teva Medical Ltd. of Israel, and including a TEVADAPTOR® vial adaptor cap 120, was initially locked onto each vial 100, as seen at Fig. 1A. A preferred embodiment of the TEVADAPTOR® vial adaptor 110 is fully described in U.S. Patent 8,122,923, the disclosure of which is hereby incorporated by reference.

Thereafter, a previously unused and sterile TEVADAPTOR® syringe adaptor 130, commercially available from Teva Medical Ltd. of Israel, and including a TEVADAPTOR® syringe adaptor cap 135, was attached to a luer lock syringe 140, as seen in Fig. IB. A preferred embodiment of the TEVADAPTOR® Syringe Adaptor 130 is fully described in U.S. Patent 8,122,923, the disclosure of which is hereby incorporated by reference.

Thereafter, following removal of TEVADAPTOR® vial adaptor cap 120 and TEVADAPTOR® syringe adaptor cap 135, as seen in Fig. 1C, the previously unused and sterile TEVADAPTOR® syringe adaptor 130, having attached thereto the luer lock syringe 140, was operatively engaged with the TEVADAPTOR® vial adaptor 110, as seen in Fig. ID.

On each of days 0, 7 and 14, two aseptic transfers from each drug vial were performed using the TEVADAPTOR® syringe adaptors 130 and 5 ml luer lock syringes 140, in which only 2.5 ml of Methotrexate was drawn from each vial 100, as seen in Fig. IE.

Thereafter, the TEVADAPTOR® syringe adaptor 130, having syringe 140, containing 2.5 ml of Methotrexate, connected thereto, was disconnected from the TEVADAPTOR® vial adaptor 110, which remained locked onto vial 100 and was covered by TEVADAPTOR® vial adaptor cap 120, as seen in Fig IF.

An additional previously unused and sterile TEVADAPTOR® vial adaptor, designated by reference numeral 150, commercially available from Teva Medical Ltd. of Israel, and including a TEVADAPTOR® vial adaptor cap designated by reference numeral 160, was initially locked onto each media vial 102, as seen in Fig. 1G.

Thereafter, following removal of TEVADAPTOR® vial adaptor cap 160, as seen in Fig. 1H, the TEVADAPTOR® syringe adaptor 130, having syringe 140 connected thereto, was connected in one continuous movement to the designated growth media vial 102 (TSB or FTM) via TEVADAPTOR® vial adaptor 150, as seen in Fig II.

Thereafter, a 2.5 ml aliquot of Methotrexate from each drug vial was added to one TSB vial 102 and one FTM vial 102 from the syringe 140 via the TEVADAPTOR® syringe adaptor 130 and the TEVADAPTOR® vial adaptor 150, as seen in Fig 1J.

Thereafter, the TEVADAPTOR® syringe adaptor 130 was disconnected from the TEVADAPTOR® vial adaptor 150 of the growth media vial 102 and discarded after use. Aseptic TEVADAPTOR® vial adaptor cap 160 was fitted onto the TEVADAPTOR® vial adaptor 150, as seen in Fig IK.

All of the thus inoculated media vials 102 were sent for 14 day incubation.

The following sequence of actions took place

Day 0: Preparation of Test Methotrexate vials 100 for testing

The bench was wiped down using 70% IMS spray and sterile 70% IMS prep pads.

Each TEVADAPTOR® vial adaptor 110 was aseptically removed from its packaging and attached to a vial 100 leaving TEVADAPTOR® vial adaptor cap 120 in place. Day 0: Preparation of Growth Media vials 102 for testing

All aseptic work was carried out within the designated "uncontrolled" preparation area. The bench was wiped down using 70% IMS spray and sterile 70% IMS prep pads.

The cap was removed from a TSB vial 102 (TSB), the bung was swabbed with a sterile IP A 70% prep pad and left to dry for 2 minutes.

A TEVADAPTOR® vial adaptor 150 was aseptically removed from its packaging and attached to the TSB vial 102 leaving the TEVADAPTOR® vial adaptor cap 160 in place.

The TEVADAPTOR® syringe adaptor 130, having the syringe 140 attached thereto, was connected to the TEVADAPTOR® vial adaptor 110 of vial 100 and 2.5 ml of Methotrexate was withdrawn from the vial 100 by the syringe 140 via the TEVADAPTOR® syringe adaptor 130 and the TEVADAPTOR® vial adaptor 110.

The TEVADAPTOR® syringe adaptor 130, having the syringe 140, containing 2.5 ml of Methotrexate, attached thereto, was thereafter attached via TEVADAPTOR® vial adaptor 150 to the TSB growth medium vial 102.

The plunger of syringe 140 was depressed, thereby inoculating the TSB growth medium vial 102 with 2.5 ml of Methotrexate via the TEVADAPTOR® vial adaptor 150, while the TSB growth medium vial 102 was in an upright position and not inverted.

Following inoculation of the TSB media vial 102, the wings of the TEVADAPTOR® syringe adaptor 130 were depressed and the TEVADAPTOR® syringe adaptor 130 was disconnected from the TEVADAPTOR® vial adaptor 150 and from the TSB growth medium vial 102. The TEVADAPTOR® syringe adaptor 130 and syringe 140 were discarded.

A second aliquot of 2.5 ml of Methotrexate was then withdrawn from the same Methotrexate drug vial 100 for inoculating the FTM growth medium vial 102. The TEVADAPTOR® vial adaptor cap 120 was removed from the TEVADAPTOR® vial adaptor 110 and the septum of the TEVADAPTOR® vial adaptor 110 was swabbed with a sterile IP A 70% prep pad and left to dry for 2 minutes.

The TEVADAPTOR® syringe adaptor 130, having the syringe 140, containing 2.5 ml of Methotrexate, attached thereto, was thereafter attached via TEVADAPTOR® vial adaptor 150 to the FTM growth medium vial 102.

The plunger of syringe 140 was depressed thereby inoculating the FTM growth medium in the vial 102 with 2.5 ml of Methotrexate via the TEVADAPTOR® vial adaptor 150, while the FTM growth medium vial 102 was in an upright position and not inverted.

The above procedure was followed for each of the 40 vials of Methotrexate. A total of 80 samples were withdrawn from the vials 100 and used to inoculate 40 vials 102 of TSB and 40 vials 102 of FTM. All of the inoculated TSB growth medium vials 102 and FTM growth medium vials 102 were sent for incubation, as described in the microbiological procedure hereinbelow.

The surfaces of the bench were cleaned and all other materials were removed.

The vials 100 of Methotrexate were stored at room temperature, in accordance to the storage conditions listed on the drug label, within the "uncontrolled" preparation area.

Days 7 and 14

All Methotrexate vials 100 were sampled on each of days 7 and 14 according to the same sampling process as described above.

Microbiological Procedure

The TSA air sample plates were incubated for 5 days at 30-35°C.

The SDA air sample plates were incubated at 20-25°C for 7 days.

At test days 0, 7 and 14 each cohort of inoculated TSB growth medium vials 102 was incubated at 20-25 °C for 14 days.

At test days 0, 7 and 14 each cohort of inoculated FTM growth medium vials 102 was incubated at 30-35°C for 14 days. The inoculated TSB and FTM growth medium vials 102 were examined for microbial growth daily for each of the first 7 days and at least twice during the 8^th to 14^th day of the incubation period.

TSB and FTM growth medium vial failures were recorded where turbidity was identified.

The growth promoting quality of the TSB medium and the FTM medium in the presence of a 2.5 ml sample aliquot of Methotrexate was confirmed by dispensing 2.5 ml of drug into TSB and FTM vials, and then performing the Method Suitability Test as defined in USP <71> Sterility Tests.

Where growth was observed in either the TSB or FTM growth medium vials 102 following inoculation with Methotrexate, the growth medium was sub cultured to allow for identification of the contaminating species down to the species level up to a maximum of 5 contamination events.

Results:

Tables 2-3 hereinabove show the Interim air sampling and contact environmental monitoring data.

14 Day Sterility Study Report

Study Vial Data Assessment: Methotrexate

Table 8 lists the sterility test results for the Methotrexate drug samples withdraw from the 40 drug vials on days 0, 7 and 14. Table 9 summarizes the results for the Method Suitability Test required to establish the validity of sterility test method. Number of Vials

Sample Growth Number of Number of

Meeting the Sterility

Day Media Vials Tested Failed Vials

Test Requirement

TSB 40 40 0

Day 0

FTM 40 40 0

TSB 40 40 0

Day 7

FTM 40 40 0

TSB 40 40 0

Day 14

FTM 40 40 0

TABLE 8

TABLE 9 The above results show that sterility of 40 vials of Methotrexate was maintained throughout a 14 day period when drug samples were aseptically removed using the TEV ADAPTOR® closed system drug transfer device in a noncontrolled environment. Sampling from each vial was performed six times over the 14 day period, for a total of 240 samples removed from all of the vials. The Method Suitability Test results confirm the growth promoting capability of the growth medium in the presence of the drug aliquot, with a broad range of microbial strains, thereby confirming the validity of the sterility test results.

Example III - Trastuzumab

The following protocol was carried out:

General:

2. ICH Quality Guideline Q1E, Evaluation of Stability Data implemented as CPMP/ICH/420/02, available on the ICH website. 3. ICH Harmonized Guidelines for Internal Quality Control in Analytical Chemistry Laboratories, available on the ICH website.

4. ICH Q5C Guidelines, "Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products", available on the ICH website. This document, which provides guidance regarding the type of stability studies in support of Marketing Authorisation Applications (MAA) for biological medicinal products, was used as a framework. Tests relevant to this study were undertaken.

5. Santillo M, Barnes A, Douris G, Goddard G, Hiom S, Jackson M, Precious N, Simpson J and Weir P (2012) A standard protocol for deriving and assessment of stability. Part 2 - Aseptic Preparations (Biopharmaceuticals), Version 1, NHS Pharmaceutical Quality Assurance Committee. This document is commonly known as the "Yellow covered document."

6. United States Pharmacopoeia Chapter 71: Sterility Tests, available on the USP website.

7. All manipulations and inoculation conditions were in accordance with standard microbiological procedures, as seen in Collins, C. H.; Microbiological methods, 1964.

8. Validation of the Environmental Bioburden was undertaken prior to conducting the main sterility study by active air sampling.

Trastuzumab drug product was reconstituted using 7.2 ml water for injection (WFI) using a conventional needle and syringe to deliver the WFI to the control vials and using the TEV ADAPTOR® system to deliver the WFI to the test vials 202. Following reconstitution, 0.5 ml sample aliquots were withdrawn from both the control and test vials on days 0, 7, 14, 21 and 28 for physicochemical testing, comparing the stability of the control and test vials. On day 28, a 2.0 ml sample aliquot was withdrawn from each of two test vials for sterility testing in Tryptic Soy Broth (TSB) growth media. One (1) ml of the sample aliquot was dispensed into two TSB growth media vials and a further 1 ml aliquot was withdrawn from each of the two test vials for sterility testing in Fluid Thioglycollate Media (FTM) growth media.

All handling of the drug vials was performed in an ISO Class 5 laminar air flow hood situated in an uncontrolled environment. After sampling, the drug vials were stored at 2-8°C, in their original box, protected from light, in a non-controlled environment.

The samples of Trastuzumab for physicochemical testing were tested in duplicate and mean values reported. Testing was performed according to the USP proposed monograph for Trastuzumab and sterility testing was performed by direct inoculation of the 1.0 ml sample aliquot from each of two test vials into each of two Tryptic Soy Broth (TSB) growth medium vials and incubating one set of duplicate TSB samples for 14 days at 30-35°C and the second set of duplicate TSB growth media vials were incubated for 14 days at 20-25°C. An additional 1.0 ml sample aliquot of Trastuzumab was withdrawn from the two test vials, inoculated into each of two Fluid Thioglycollate Medium (FTM) growth medium vials and incubated for 14 days at 30- 35°C. The TSB and FTM vials were examined for microbial growth daily for the first 7 days and at least twice during the 8th to 14th day of the incubation period.

Microbiological and physicochemical integrity testing of the TEVADAPTOR® Closed System Drug Transfer Device in a controlled environment when used with preservative free, single dose Trastuzumab vials

Overview

Preparation of the two test vials containing Trastuzumab for physicochemical testing on days 0, 7, 14, 21 and 28 and sterility testing on Day 28 following drug reconstitution.

Materials

12 preservative free 150 mg vials of Trastuzumab,

75 ml of Tryptic soy broth in 100 mL Type 1 glass vial; tested for sterility and positive control tested to meet the USP/EP Growth Promotion test (n=2).

100 ml of Fluid Thioglycollate growth Media (FTM) in 100 ml Type 1 glass vial; tested for sterility and positive control tested to meet the USP/EP Growth Promotion test (n=2).

TEVADAPTOR® vial adaptor 210, suitable for use with a 20mm vial cap. TEVADAPTOR® vial adaptor aseptic cap 220 as supplied with the TEVADAPTOR® vial adaptor for each TEVADAPTOR® vial adaptor 210.

TEVADAPTOR® syringe adaptor 230

5 ml Sterile luer lock syringes 240 from Terumo SS+T05ES1 Lot 12340/1752 expiry 11/2017 supplied by MediSave UK.

2 ml Sterile leur lock syringes 250 from Terumo SS+T02S1 Lot 13196/1595 expiry 06/2018 supplied by MediSave UK.

Sterile syringe needle 23 gauge length 1.25 inches code NN-2332R Lot 1301020 expiry 2017-12 supplied by MediSave UK.

Sterile 70% IMS solution prepared from 70 ml isopropyl alcohol and 30 ml sterile water for infusion (WFI).

USP water for infusion (WFI)

Equipment

Validated 20-25°C (+/- 1°C) and 30-35°C (+/- 1°C) incubators with continuously temperature monitored using a validated Comark temperature monitoring system.

Laminar Air Flow Hood Certified to EUGMP Class A according to BSEN14644; EUGMP; COSHH 2002; Reg 9

Background environmental microbiological assessment

Testing was performed in an ISO Class 5 certified laminar air flow hood, situated within a non-controlled laboratory environment.

Drug Reconstitution with TEVADAPTOR®

All aseptic manipulation work was carried out within the ISO Class 5 laminar air flow hood.

Drug reconstitution took place as illustrated generally in Figs. 2A-2S. The protective cap was removed from each of the control drug vials and each of test drug vials 202. Each of the control drug vials and each of the test drug vials 202, with the cap removed, was weighed accurately using a calibrated Sartorius Analytical balance model 120S to 4 decimal places. The weight was recorded for each of the Trastuzumab control drug vials and each of test drug vials 202.

The rubber stopper of each test drug vial 202 was swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

A previously unused and sterile TEVADAPTOR® vial adaptor 210, commercially available from Teva Medical Ltd. of Israel, and including a TEVADAPTOR® vial adaptor cap 220, was aseptically removed from its packaging and attached to each test drug vial 202 leaving the TEVADAPTOR® vial adaptor cap 220 in place, as seen in Fig. 2A.

The TEVADAPTOR® vial adaptor cap 220 was then removed from the TEVADAPTOR® vial adaptor 210 attached to each test drug vial 202. Each test drug vial 202, including the attached TEVADAPTOR® Vial Adaptor 210, was weighed accurately using a calibrated Sartorius Analytical balance model AC 120S to 4 decimal places and the weights were recorded.

The TEVADAPTOR® vial adaptor cap 220 from the TEVADAPTOR® vial adaptor 210 was then replaced on each of the TEVADAPTOR® vial adaptors 210 attached to each of the Trastuzumab test drug vials 202.

The protective cap was removed from a vial 225 of Water for Injection (WFI) and the bung, including the rubber stopper and aluminum band, was swabbed with a sterile IPA 70% preparation pad and left to dry for 2 minutes.

A TEVADAPTOR® vial adaptor 260, including a TEVADAPTOR® vial adaptor cap 270, was aseptically removed from its packaging and attached to WFI vial 225 leaving the TEVADAPTOR® vial adaptor cap 270 in place, as seen in Fig. 2B.

Thereafter, a previously unused and sterile TEVADAPTOR® syringe adaptor 230, including TEVADAPTOR® syringe adaptor cap 235, was operatively engaged with a sterile 5 ml syringe 240, as shown in Fig. 2C, and, following removal of TEVADAPTOR® vial adaptor cap 270 and TEVADAPTOR® syringe adaptor cap 235, as shown in Fig. 2D, with TEVADAPTOR® vial adaptor 260 attached to WFI vial 225, as shown in Fig. 2E. An aseptic transfer of approximately 5 ml of WFI from WFI vial 225 to syringe 240 was performed using the TEV ADAPTOR® syringe adaptor 230 and TEVADAPTOR® vial adaptor 260, as shown in Fig. 2F.

Thereafter, the TEVADAPTOR® syringe adaptor 230, together with the 5 ml syringe 240, was disconnected from the TEVADAPTOR® vial adaptor 260, which remained locked onto WFI vial 225, as seen in Fig 2G.

The TEVADAPTOR® vial adaptor cap 220 was then removed from TEVADAPTOR® vial adaptor 210 attached to test drug vial 202, as seen in Fig. 2H, and the TEVADAPTOR® syringe adaptor 230 with the 5 ml syringe 240, containing the WFI, was then operatively engaged with the TEVADAPTOR® vial adaptor 210 attached to test drug vial 202, as seen in Fig. 21.

5 ml of WFI from the 5 ml syringe 240 was aseptically transferred to the test drug vial 202 via the TEVADAPTOR® syringe adaptor 230 and TEVADAPTOR® vial adaptor 210, as seen in Fig. 2J.

Thereafter, the TEVADAPTOR® syringe adaptor 230 was disconnected from the TEVADAPTOR® vial adaptor 210, as seen in Fig. 2K.

Thereafter, a previously unused and sterile TEVADAPTOR® syringe adaptor, designated by reference numeral 280, including a TEVADAPTOR® syringe adaptor cap, designated by reference numeral 285, was operatively engaged with a sterile 2 ml syringe 250, as seen in Fig. 2L, and, following removal of TEVADAPTOR® syringe adaptor cap 285, as seen in Fig. 2M, with TEVADAPTOR® vial adaptor 260 attached to WFI vial 225, as seen in Fig. 2N.

An aseptic transfer of approximately 2.2 ml of WFI from WFI vial 225 to syringe 250 was performed using the TEVADAPTOR® syringe adaptor 280 and TEVADAPTOR® vial adaptor 260, as seen in Fig. 20.

Thereafter, the TEVADAPTOR® syringe adaptor 280, together with the 2 ml syringe 250, was disconnected from the TEVADAPTOR® vial adaptor 260, which remained locked onto vial WFI 225, as seen in Fig 2P.

The TEVADAPTOR® syringe adaptor 280 with the 2 ml syringe 250, containing the WFI, was then operatively engaged with the TEVADAPTOR® vial adaptor 210, as seen in Fig 2Q. 2.2 ml of WFI from the 2 ml syringe 250 was aseptically transferred to the test drug vial 202 via the TEVADAPTOR® syringe adaptor 280 and TEVADAPTOR® Vial Adaptor 210, as seen in Fig 2R.

Thereafter, the TEVADAPTOR® syringe adaptor 280 was disconnected from the TEVADAPTOR® vial adaptor 210, as seen in Fig 2S.

A total of 7.2 ml of WFI from WFI vial 255 was added to the test drug vial 202.

Thereafter, the test drug vial 202 was gently swirled until the entire drug contents were reconstituted in the WFI.

The procedure was repeated for each of the two test drug vials 202 of Trastuzumab, each vial being reconstituted with 7.2 ml of WFI.

Each Trastuzumab test drug vial 202 was then accurately weighed using a calibrated Sartorius Analytical weighing balance AC 120S to 4 decimal places and the weight of each test drug vial 202 recorded. This allowed an accurate determination of Trastuzumab concentration and confirmed the amount of diluent addition to each test and control Trastuzumab vial.

Drug Reconstitution with a Syringe and Needle

The protective cap was removed from a vial of WFI and the rubber stopper was swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

A 23 gauge sterile needle was aseptically attached to a 5 ml syringe.

The needle was inserted into the rubber stopper of the WFI vial and approximately 5 ml of WFI was withdrawn from the vial.

Air was removed from the syringe by holding the syringe upright to allow the air bubbles to migrate to the top of the syringe. The plunger rod was then depressed until all air was expelled from the syringe.

The protective cap was removed from a fresh vial of Trastuzumab to use as a control drug vial.

The Trastuzumab control drug vial was weighed accurately using a Sartorius Analytical balance model AC 120S. The weight was recorded.

The rubber stopper of the Trastuzumab control drug vial was swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes. The rubber stopper of the water for injection (WFI) vial was swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

A fresh 23 gauge needle and 5 ml syringe were removed from their packaging and the needle attached to the syringe and used to withdraw a 5 ml aliquot from the WFI vial.

The combined needle and syringe was then inserted into a vial of Trastuzumab and the 5 ml of WFI was transferred from the syringe into the vial.

The syringe was withdrawn from the vial and discarded.

The rubber stopper of the Trastuzumab control drug vial was swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

The rubber stopper of the water for injection (WFI) vial was swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

A fresh 23 gauge needle and a 2 ml syringe were removed from their packaging and the needle attached to the syringe and used to withdraw a 2.2 ml aliquot from the WFI vial.

The combined needle and syringe was then inserted into the control drug vial of Trastuzumab and the 2.2 ml of WFI was transferred from the syringe into the vial.

The syringe was withdrawn from the vial and discarded.

A total of 7.2 ml of WFI was added to the Trastuzumab control drug vial.

The drug vial was gently swirled until the entire drug contents were reconstituted in the WFI.

The procedure was repeated for each of the two Trastuzumab control vials on Day 0 of the study and performed in duplicate on each of the subsequent study test days 7, 14, 21 and 28, adding two Trastuzumab control drug vials per day, each drug vial being reconstituted with 7.2 ml of WFI. In total 10 Trastuzumab control drug vials were prepared during the study.

Aseptic Sampling Procedure with the TEVADAPTOR®

The TEVADAPTOR® vial adaptor cap 220 was removed from the

TEVADAPTOR® vial adaptor 210 attached to a reconstituted Trastuzumab test drug vial 202 and the elastomer was swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

A TEVADAPTOR® syringe adaptor was aseptically removed from its packaging and attached to a 2 ml syringe.

The TEVADAPTOR® syringe adaptor and syringe were connected to the TEVADAPTOR® vial adaptor 210 of the test drug vial 202 of Trastuzumab and 0.5 ml of the reconstituted drug was withdrawn from test drug vial 202.

The TEVADAPTOR® syringe adaptor was disconnected from the both the TEVADAPTOR® vial adaptor 210 and syringe and was discarded.

The 0.5 ml of drug sample was transferred to a sterile tube for physicochemical analysis.

The procedure was repeated for the remaining Trastuzumab test drug vial 202 with the TEVADAPTOR® vial adaptor 210 attached thereto.

The sampling procedure was performed on days 0, 7, 14, 21 and 28 using the opened Trastuzumab test drug vials 202 stored with the TEVADAPTOR® system. When not in use the Trastuzumab test drug vials 202 fitted with TEVADAPTOR® vial adaptors 210 were stored according to the manufacturer's instructions for use at 2-8°C, in a pharmacy refrigerator, protected from light in the original drug vial packaging with a black oversleeve protecting the drug contents from light.

Aseptic Sampling Procedure with Syringe and Needle

A 23 gauge sterile needle was aseptically attached to a 2 ml syringe.

The needle was inserted into the rubber stopper of the Trastuzumab control drug vial and 0.5 ml of drug was withdrawn from the vial.

The syringe was withdrawn from the vial stopper and the syringe contents were transferred to a sterile tube for physicochemical analysis.

The syringe was discarded.

The sampling procedure was performed on days 0, 7, 14, 21 and 28 following reconstitution of each of the two fresh unopened Trastuzumab control drug vials for that sampling day. When not in use, the fresh Trastuzumab control drug vials were stored according to the manufacturer's instructions for use at 2-8°C, in a pharmacy refrigerator, protected from light in the original drug vial packaging with a black oversleeve protecting the drug contents from light. Aseptic Sampling Procedure for Sterility Testing with TSB growth media

On day 28, the TEVADAPTOR® vial adaptor caps 220 were removed from TEVADAPTOR® vial adaptors 210 attached to two reconstituted Trastuzumab test drug vials 202, the elastomers were swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

Two TEVADAPTOR® syringe adaptors were aseptically removed from their packaging and each was attached to a 2 ml syringe.

One TEVADAPTOR® syringe adaptor and syringe was connected to the TEVADAPTOR® vial adaptor attached to each test drug vial 202 of Trastuzumab and 2.0 ml drug was withdrawn from the test drug vial 202.

The protective cap was removed from four vials of Tryptic Soy Broth growth medium (TSB), the rubber stoppers were swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

A TEVADAPTOR® vial adaptor was aseptically removed from its packaging and attached to each of the four TSB vials leaving the TEVADAPTOR® vial adaptor cap in place.

One TEVADAPTOR® syringe adaptor and syringe containing the 2.0 ml drug sample aliquot was attached to the TEVADAPTOR® vial adaptor on the first of the two duplicate TSB vials for the first Trastuzumab drug sampled from test vial 1. One ml (1.0ml) of the contents of the syringe was transferred into the first TSB vial. The TEVADAPTOR® syringe adaptor, with the syringe attached, was disconnected from the TEVADAPTOR® vial adaptor. The same TEVADAPTOR® syringe adaptor and syringe containing the remaining 1.0 ml drug sample aliquot within the syringe was then re-attached to the TEVADAPTOR® vial adaptor on the second of the two duplicate TSB vials for the first Trastuzumab drug sampled from test vial 1. The contents of the syringe (1.0 ml) were transferred into the second TSB vial. The TEVADAPTOR® syringe adaptor, with the syringe attached, was disconnected from the TEVADAPTOR® vial adaptor attached to the TSB vial and discarded.

The above procedure was then repeated for the second Trastuzumab test drug vial.

Aseptic Sampling Procedure for Sterility Testing with FTM growth media

On day 28, the TEVADAPTOR® vial adaptor caps 220 were removed from TEVADAPTOR® vial adaptors 210 attached to the two reconstituted Trastuzumab test drug vials 202, the elastomers were swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

One TEVADAPTOR® syringe adaptor and 2 ml syringe was connected to the TEVADAPTOR® vial adaptor attached to each test drug vial 202 of Trastuzumab and 1.0 ml of the drug was withdrawn from the test drug vial 202.

The protective cap was removed from two vials of Fluid Thioglycollate growth medium (FTM), the rubber stoppers were swabbed with sterile IPA 70% preparation pad and left to dry for 2 minutes.

A TEVADAPTOR® vial adaptor was aseptically removed from its packaging and attached to each of the FTM vials leaving the TEVADAPTOR® vial adaptor cap in place.

One TEVADAPTOR® syringe adaptor and syringe containing the 1.0 ml drug sample aliquot was attached to the TEVADAPTOR® vial adaptor on each of the FTM vials.

The contents of each syringe were transferred into the FTM vials.

The TEVADAPTOR® syringe adaptor, with the syringe attached, was disconnected from the TEVADAPTOR® vial adaptor attached to the FTM vial and discarded. Incubation of the Sterility Test Samples

One set of duplicate TSB vials inoculated with 1.0 ml Trastuzumab were incubated for 14 days at 20-25 °C. The other set of duplicate TSB vials inoculated with 1.0 ml of Trastuzumab were incubated for 14 days at 30-35°C.

The FTM vials inoculated with 1.0 ml Trastuzumab were incubated for 14 days at 30-35°C.

The vials were inspected on each day for the first 7 days and then on the 8^th and 14^th day for signs of microbial growth.

Physicochemical Testing

The sample aliquots withdrawn using a syringe and needle or using the TEVADAPTOR® system on Days 0, 7, 14, 21 and 28 were subjected to the physicochemical analyses shown in Table 10.

TABLE 10 (continuation)

TABLE 10 (continuation)

TABLE 10

Report Method for Impurities Analysis:

Measurement of related impurities with a molecular mass higher than that of Trastuzumab by size exclusion chromatography (SEC) in order to assesses the amount of high molecular weight impurities within the Trastuzumab presentations at each time point as a percentage of the main Trastuzumab active drug substance peak.

Methodology:

Trastuzumab samples were analyzed according to the protocol provided in the USP summary validation report July 25, 2013 without variation: related impurities with molecular mass higher than that of Trastuzumab: size exclusion chromatography (SEC). An assessment was made, using this analysis technique, as to the amount of dimer impurity for Trastuzumab that was present on each of the sampling test days. This was reported as a percentage of the main Trastuzumab peak as well as the loss of Trastuzumab in each of the Test samples prepared using TEV ADAPTOR® systems as compared with the control reference freshly prepared on the day of the test using a standard needle and syringe approach.

Stability Indicating Test of Method:

The method was taken directly from the USP method and reproduced without variation. In addition, the method was assessed for its ability to separate excipients from Trastuzumab drug substance (DS) including both the monomeric active pharmaceutical ingredient (API) and the high molecular weight impurity dimer. The method was demonstrated to be capable of separating all species and to be able to separate the Trastuzumab monomer from dimer with the required resolution according to the USP criteria of not less than (NLT) 2.0 minutes..

The resolution between the dimer and the intact peak representing Trastuzumab monomer was found to be 2.37 minutes which was within the acceptance criteria of not less than (NLT) 2.0 minutes. Analyses were performed at two wavelengths for detection, 214 nm and 280 nm. The data presented in Table 11 below is based on detection at 214 nm. Similar results were obtained with detection at 280nm with no deleterious effect on resolution of method.

Forced Degradation Studies for Trastuzumab Resolution solution at lOmg/ml were performed to generate dimer as a control. To this end, the 10 mg/ml solution was exposure to UV light (354nm) for 2 hours. Following exposure to UV light the percentage of high molecular weight impurity dimer increased from 0.2-0.3% to 1%. No further attempt to degrade the Trastuzumab drug substance was made. Results are show below in Table 11. Table 12 demonstrates system resolution between the dimer and Trastuzumab monomer peaks.

Table 11 includes the data showing the level of high molecular weight impurity (dimer) present in the lOmg/ml Trastuzumab resolution solution sample following exposure to UV light at 365nm for 2 hours. Data presented was obtained at 214nm detection.

TABLE 11

Table 12 includes the data showing the resolution between the high molecular weight impurity (dimer) present within the lOmg/ml Trastuzumab resolution solution sample and the main intact Trastuzumab peak. Data is following exposure to high powered UV light at 365nm for 2 hours.

TABLE 12

Inter and Intra assay Precision Analysis:

Analysis of Trastuzumab resolution solutions was performed in replicate (N = 6) for Trastuzumab resolution solution at three concentrations: 75 micogram/ml; 750 microgram/ml and 7500 microgram/ml.

Intra-assay precision was evaluated from the data for each of the three different Trastuzumab concentrations. The obtained intra-assay precision (%CV < 3.7) for analysis of the Ί5μ§/Ώή, 750μ§/ιη1 and 7500 μ§/ι 1 control samples complies with the acceptance criterion (%CV < 5.0). The obtained accuracy 96-104% and 97-104%, respectively, for analysis of the 750 μ /ηι1 and 7500 μ§/ηύ control samples complies with the set criterion for accuracy, 90-110%.

Linearity of instrument response:

A number of replicate (N=9) linear instrument calibrations were performed for Trastuzumab resolution solutions over a four day period preceding the study.

The obtained data for the correlation coefficient is 0.9949 - 0.9994 (214 nm) and 0.9973-1.0000 (280 nm) which is in accordance with the set criterion for linearity (>0.9900).

A typical instrument response curve for Trastuzumab resolution solution with detection at 214nm is shown in Fig. 3.

A typical instrument response curve for Trastuzumab resolution solution with detection at 280nm is shown in Fig. 4.

Limits of Detection (LQD) and Limits of Quantitation (LQQ):

Limit of detection and limit of quantification were determined from the average standard deviation and slope calculated from repeated analysis of the lowest calibration standard prepared from Trastuzumab resolution solution (CAL1) which had a concentration of 10 μ§/ΐΏΐ.

LOD = 3.3 x Std dev. response/Slope = 3.3 x 0.4241/0.0623 = 22.5 μ^πύ

LOQ = 10 x Std dev. response/Slope = 10 x 0.4241/0.0623 = 68.1 μ^πύ

Note that the size of the peaks obtained for Trastuzumab monomer at 22.5 microgram/ml indicate that a lower detection limit could be possible, but further investigation was not necessary for the purpose of this study.

A typical chromatogram for resolution solution Trastuzumab at detection wavelength of 280nm is shown in Fig. 5. A typical chromatogram for the formulation buffer for the Trastuzumab resolution solution under identical chromatographic separation conditions at detection wavelength of 214nm is shown in Fig. 6.

The only peak detected was the excipient with a retention time of 23.8 minutes which is outside of the window for detection of Trastuzumab monomer or high molecular weight impurity dimer which elute at 16.5 minutes and 14.1 minutes respectively.

Table 13 shows the percentage recovery of Trastuzumab drug substance in TEV ADAPTOR® system (Test TZM) when stored for up to 28 days versus Trastuzumab freshly prepared on the day of test in a reference control glass vial. Data is from analysis by high performance size exclusion liquid chromatography (HPLC-SEC) with detection at a wavelength of 214nm. Note: The data was obtained from duplicate test and control devices with duplicate sampling and triplicate injections.

TABLE 13

Table 14 shows percentage of Trastuzumab dimer (high molecular weight impurity) within Trastuzumab samples prepared in TEVADAPTOR® system (% Test) when stored for up to 28 days versus Trastuzumab freshly prepared on the day of test in reference control glass vial (% Control). Data is from analysis by high performance size exclusion liquid chromatography (HPLC-SEC) with detection at a wavelength of 280nm. Data obtained at 214nm shows similar results (data not shown).

TABLE 14

Percentages of dimer were based on peak area calculation for the two resolved peaks corresponding to the Trastuzumab monomer and Trastuzumab dimer at retention times of 16.5 minutes and 14.1 minutes respectively. No account was made for differences in extinction coefficients for the two products.

Acceptance criteria for the amount of high molecular weight impurity present was set at 1% and according to the UK National Healthservice (NHS) Quality Assurance Committee 2012 "A Standard protocol for deriving and assessment of stability: Part 2 - Aseptic Preparations of Biopharmaceuticals" the increase in high molecular weight impurities should not exceed 2% relative to the main active pharmaceutical ingredient peak. No criteria are set within the USP validation report for Trastuzumab, only to determine the amount of % dimer in each solution.

All test and control Trastuzumab solutions were within the acceptance criteria (<1%).

Conclusion:

Analysis by size exclusion chromatography demonstrates that Trastuzumab solutions for injection remain stable for up to 28 days when prepared using TEVADAPTOR® systems. No significant difference was observed between Test Trastuzumab solutions prepared in TEV ADAPTOR® systems compared with reference control Trastuzumab freshly prepared in reference type one glass vials using the standard needle and syringe approach on the day of test. Reference Procedure Peptide Mapping of Trastuzumab following digestion

Procedure was performed according to the published Trastuzumab Validation Report Jul 25, 2013 on the USP website with the deviation that a Thermo Scientific (UK) Acclaim™ RSLC C18 column, 12θΑ, 100x2.1 mm, 2.2 μιη column was used as a replacement for the Waters BEH C18 column stated in the USP procedure. This is a direct replacement. The Thermo Acclaim column has the same packing material as the Waters BEH column, with no deviations. Also quenching of the protein digest was performed using 2 microliters of 50% Trifluoroacetic acid (TFA) instead of 1 microliter of concentrated TFA to assist with the sample manipulations.

The purpose of this test procedure was to identify any change or loss to the separate Trastuzumab peptide chains by performing enzymatic digest following by a reverse phase chromatographic separation of all breakdown peptide fragments. Any changes to the Trastuzumab molecule including: fragment and amino acid removal or loss can be easily identified and semi-quantified using this approach. Results:

Matrix chromatogram did not show any significant peak response within the integration window.

The chromatographic profile obtained from both the reference control solutions of Trastuzumab and test solutions contained within the TEVADAPTOR® system showed all CDR regions 1, 2 and 3 for both light and heavy chains fragments following digestion.

Chromatographic profiles from a negative control material showed a significant difference in peak profile to that obtained with Trastuzumab - data not shown.

The Chromatographic profile seen in Fig. 7 shows a typical profile for

Trastuzumab reference solution following protein digest and peptide mapping using Reverse Phase high performance liquid chromatographic separation according to the USP summary validation procedure.

The relative retention times of each peptide fragment from CDR regions 1, 2 and 3 originating from light and heavy chains were assigned on the basis of data presented in the USP summary validation report.

Table 15 shows data from peptide mapping profiles performed at Day 0 for both Trastuzumab within TEV ADAPTOR® system, referred to as Test (n=2) and Trastuzumab freshly reconstituted in a glass vial on the day of analysis, referred to as Control (n=2). All devices were sampled in duplicate.

TABLE 15

Table 16 shows data from peptide mapping profiles performed at Day 28 for both Trastuzumab within TEVADAPTOR® system, referred to as Test (n=2) and Trastuzumab freshly reconstituted in a glass vial on day of analysis, referred to as Control (n=2). All devices sampled in duplicate.

TABLE 16

Conclusion for Peptide Mapping:

After 28 days storage in TEVADAPTOR® systems reconstituted Trastuzumab solution for injection was not distinguishable from reference control solutions of Trastuzumab prepared aseptically on the day of test (Day 28) in glass vials. On day 0 and day 28 Trastuzumab reconstituted for use in TEVADAPTOR® systems showed all three CDR regions 1, 2 and 3 for both light and heavy chains in accordance with reference control solutions of Trastuzumab.

Reference:

USP Medicines Compendium Summary Validation Report July 25, 2013. Visual Appearance

Table 17 shows the results of visual appearance testing, including visible particulates, for Trastuzumab test drug vials control drug vials on Days: 0, 7, 14, 21 and 28.

TAB E 17

Key to Table 17:

Clarity: turbid (T) / Clear (C)

Color: Colorless (CLS) /Yellow tinge (YT) / Strongly Colored (SC)

Content: Presence of visible particulates; Yes (Y) / No (N)

All test drug vials were within the accepted criteria of no visible particulates detected when viewed against white and black backgrounds. Duplicate control drug vials tested at each time point. Test performed according to USP 37: <788> Injections - Foreign and Particulate Matter and EP 2.9.20 Particulate Contamination: Visible Particles. 12ϋ

Table 18 shows variation in pH measurements for Trastuzumab test drug vials versus control drug vials on Days: 0, 7, 14, 21 and 28.

TABLE 18

All Trastuzumab Test drug vials were within the accepted criteria, of +0.5 pH units, of the Trastuzumab Control drug vials on the test day. Report Method for Limit of Charged Variants Analysis (After carboxypeptidase treatment): Cation Exchange Chromatography (CEX)

Methodology:

Trastuzumab samples were analyzed according to the protocol provided in the USP summary validation report July 25, 2013 without variation: Limit of Charged Variants Analysis (After carboxypeptidase treatment): Cation Exchange Chromatography (CEX).

The system suitability performance for the analysis of Trastuzumab samples obtained during this study meets the USP requirements of: there being no less than (NLT) 1.3 minutes resolution between the acid variant Trastuzumab pre-peak Glul and the main Trastuzumab (K0) peak corresponding to the Trastuzumab with all four of the terminal L-lysine cleaved following treatment with carboxypeptidase. Furthermore, the system suitability for Trastuzumab analysis by cation exchange chromatography (CEC) also met the requirement of not more than (NMT) 1% variance in the retention times for the main Trastuzumab peak (K0) during analysis of both test and reference control samples.

The methodology was followed as given in the proposed USP summary validation document without variation.

Stability Indicating Test of Method:

The method was taken directly from the USP method and reproduced without variation. The method demonstrated resolution between acidic and basic charged variants and allowed quantification of the main intact Trastuzumab peak (K0) in the presence of both acidic and basic variants. The method employed the treatment of Trastuzumab samples with carboypeptidase which results in the removal of up to four terminal L-lysine residues from the Trastuzumab molecule, depending on the charge variant state at prior to treatment. It was not possible during this analysis to test the ability of the method to separate different basic charged variants prior to treatment with carboypeptidase because the batch and lot of Trastuzumab used in this study did not contain any basic charge variants. This reflects the fact that all four terminal L-Lysine residues are cleaved during manufacture of the product as tested.

As with other chromatographic analyses reported for Trastuzumab stability testing, all analyses were performed at two wavelengths for detection, 214 nm and 280 nm, the data presented in Table 19 was from detection at 214nm. The data for 280nm, which is not included, shows identical results. No deleterious effects or loss of resolution within the data was observed when using the less specific detection wavelength of 214nm.

Treatment of Trastuzumab resolution solution (RS) with carobxypeptidase according to the methods used should cleave any terminal L-lysine residues from the polypeptide chains (four in total) of Trastuzumab producing the main Trastuzumab (K0) peak in the chromatogram. As noted above, in the batch of Trastuzumab used in the study, all four potential L-lysine residues from each peptide chain had already been removed by exogenous carboxypeptidases from the manufacturing process of the drug substance. As such, the analysis of Trastuzumab resolution solution (RS) pre-treatment with caboxypeptide and following treatment with this enzyme showed no difference in the chromatographic profile obtained. The peak corresponding to Trastuzumab K0 was always the largest peak and there was no significant presence of any basic charge variant species relating to Kl, K2, K3 and K4 (where K represents an L-Lysine amino acid residue) with retention times greater than the Trastuzumab K0 species as separated under the conditions used.

Results:

The result from the analysis of Trastuzumab presentations prepared using TEVADAPTOR® system and stored for up to 28 days and Trastuzumab freshly prepared under aseptic conditions using the standard needle and syringe approach are presented below. All test and control samples were analyzed in duplicate from duplicate containers and analyzed in triplicate.

Table 19 shows the resolution and system suitability for the Cation exchange (CEX) chromatographic separation method, specifically the resolution between the Trastuzumab K0 peak and the acid variant pre -peak Glul. Detection was performed at 214nm. Results for 280nm are identical in terms of system performance.

TABLE 19

A typical chromatogram for resolution solution Trastuzumab without carboxypeptide treatment on day 28 at a detection wavelength of 280nm is shown in Fig. 8.

As seen in Fig. 8, the main intact Trastuzmab peak (K0) appearing at a retention time of 23.51 minutes and to the shorter retention side of this peak are the acid variants starting with Glul herein described as the Trastuzumab pre -peak, due to its shorter retention time of 21.25 minutes. To the right of the main Trastuzumab peak (K0) is the window where basic variants with between one and four L-Lysine residues would elute. The window between 27 minutes and 42 minutes does not show any presence of another basic charge variant Trastuzumab species. This chromatographic separation was performed using a resolution reference sample of Trastuzumab without pre-treatment with carboxypeptidase to show the number of potential charge variants. Due to modifications as a result of manufacture the Trastuzumab used in this study, there is no charge in variant profile.

A typical chromatogram for the treatment of Trastuzumab formulation buffer with caroxypeptidase under identical CEX chromatographic separation conditions as used for Trastuzumab samples, performed at a detection wavelength of 280nm is shown in Fig. 9.

Table 20 shows percentage recovery of Trastuzumab (KO) charge variant drug substance in TEVADAPTOR® system (Test Trastuzumab KO) when stored for up to 28 days versus Trastuzumab (KO) charge variant drug substance from freshly prepared reference control samples prepared using a standard needle and syringe approach on the day of test. Data is from analysis by high performance cation exchange (CEX) liquid chromatography (HPLC-CEX) with detection at a wavelength of 280nm. The data was obtained from duplicate test and control devices with duplicate sampling and triplicate injections

TABLE 20

Acceptance Criteria:

The acceptance criteria used was based on the USP criteria for system suitability, of not less than a resolution of 1.3 minutes between the acidic variant pre- peak Glul and the main intact Trastuzumab (K0) peak, which this test method passed for all study time points:. The USP also requires not more than (NMT) 1.0% system variability in the Trastuzumab (K0) main peak retention time, which was also met in the reported test method. In addition to the USP, which does not state an acceptable limit for the amount of Trastuzumab (K0) following treatment with caboxypeptidase in test samples compared with control samples; thus, the criteria defined in the UK National Healthservice (NHS) Quality Assurance Committee 2012 "A Standard protocol for deriving and assessment of stability: Part 2 - Aseptic Preparations of Biopharmaceuticals" was adopted. All test Trastuzumab solutions prepared using TEVADAPTOR® system should be within 95-105% (for the presence of Trastuzumab (K0)) of control samples freshly prepared using standard needle and syringe approach on all test days.

Conclusion:

Analysis by cation exchange chromatography (CEX) demonstrates that Trastuzumab solutions for injection remain stable for up to 28 days when prepared using TEVADAPTOR® systems. No significant difference in the amount of Trastuzumab charge variant K0 following treatment with carboxypeptidase was observed between Trastuzumab solutions prepared in TEVADAPTOR® systems compared with Trastuzumab freshly prepared in reference type one glass vials on the day of test. All test solutions remained within 95-105% of the starting control values as determined using reference standards of Trastuzumab freshly prepared on the day of test.

ASSESSMENT OF TRASTUZUMAB AND DETECTION OF LOW MOLECULAR WEIGHT IMPURITIES

As defined in the relevant section of the proposed USP Summary Validation Report, two low molecular weight impurity species are assessed in this test approach:

Limit of Non-Glycosylated Heavy Chain Impurities: CE-SDS (Under Reducing Conditions) &

Limit of Low Molecular Weight Impurities: CE-SDS (Under non- reducing conditions) The capillary electrophoresis (CE) separation method for determining the amount of both Non-Glycosylated Heavy Chain (NGHC) fragment and low molecular weight impurity (Heavy Heavy Light, HHL) species within Trastuzumab presentations was performed in accordance with the page 7 of the USP Summary Validation Report July 25, 2013.

The only deviation to the protocol published in the USP Summary validation report was that data presented in this report was produced using a different instrument. All data presented here was obtained using an Agilent 2100 BioAnalyzer Lab on a Chip CE instrument and Agilent's proprietary 230 Protein Assay using Fluorescence detection of the separated molecular species on chip. This instrumentation provides superior performance to the standard Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) approach enabling higher sensitivity and complete separation of intact Trastuzumab (HHLL) from its low molecular weight impurity product HHL which represents loss of a single peptide light chain. The achieved resolution of the HHLL from HHL is in accordance with the separation capability required as per the USP summary validation report guidelines. This was not possible using gradient gel electrophoretic separation methods (data not included). It was also not possible to achieve the desired level of sensitivity and resolution for detection of the low molecular weight Trastuzumab impurity (Non-Glycosylated Heavy Chain, NGHC, 53.5 kDa) peptide fragment from the Glycosylated Heavy Chain form (HC, molecular size 58 kDa) using standard polyacrylamide gel electrophoresis. With the Agilent BioAnalyzer system, both the level of detection sensitivity and system resolution were met, as reported in the USP Summary Validation report (down to the 1% limit for the NGHC species).

Fig. 10 shows a typical electropherogram from the separation of intact

Trastuzumab (HHLL) under denaturing, non-reducing conditions performed as part of the validation for the Capillary Electrophoresis separation method for testing of loss of a light chain peptide fragment (low molecular weight impurity test).

As seen in Fig. 10, the largest peak represents intact Trastuzumab (HHLL). This species contains all four polypeptide chains comprising two identical heavy (HC) and two identical light chains (LC), appearing at the highest molecular weight of 157 kDa. The small low molecular weight impurity peak (HHL), at a molecular size of 141 kDa, represent the loss of a single peptide light chain (HHLL-L = HHL). All other peaks are either system peaks or molecular weight reference ladder samples i.e. the high and low molecular weight markers at 250 kDa and 10 kDa, respectively. No other peaks in the detection window for Trastuzumab related peaks are observed in the electropherogram.

Fig. 11 shows a typical electropherogram from the separation of the non- glycosylated form of Trastuzumab peptide heavy chain (NGHC) and the glycosylated form of the Trastuzumab peptide heavy chain (HC). Separation was performed under denaturing, reducing conditions, as part of the validation for the Capillary Electrophoresis separation method for: Limit of NGHC Impurities: CE-SDS.

As seen in Fig. 11, the largest peak represents the glycosylated form of the Trastuzumab peptide heavy chain (HC). This fragment appears at the highest molecular weight of -58 kDa. This is separated from the non-glycosylated form of the same Trastuzumab peptide heavy chain designated (NGHC), which appears at the lower molecular size of 53.5 kDa. The smaller Trastuzumab peptide light chain (LC) fragment peak is also reported for completeness and appears at a molecular size of -28 kDa. All other significant peaks are either system peaks or molecular weight reference ladder peaks and are not due to Trastuzumab related species. These peaks appear outside of the separation window of interest for Trastuzumab related peaks.

Table 21 shows Capillary Electrophoresis analysis under denaturing

Sodium Dodecyl Sulphate (SDS) non-reducing conditions for Trastuzumab Test drug vials prepared with TEVADAPTOR® systems and Trastuzumab Control drug vials prepared using standard needle and syringe reconstituted on the day of test: Measurements were performed for TEVADAPTOR® system vials of Trastuzumab on the following test days: Day 0, 7, 14, 21 and 28. This test is for the limit of loss of a single peptide light chain fragment from intact Trastuzumab (low molecular weight impurity test). Measurements were performed in duplicate with duplicate sampling on each day of test. Assigned Trastuzumab species ID & Size of

Test Day (Percentages of total protein) Molecular Species kDa

0 7 14 21 28

Trastuzumab HHL 141 kDa (Av Test) 5.1 5.8 5.8 4.9 5.8

Trastuzumab HHL 141 kDa (Av Control) 5.1 5.6 6.1 4.8 6.1

Difference IControl-Testl (Percentage, %) 0.0 0.1 -0.4 0.1 -0.3

Trastuzumab HHLL intact 157 kDa (Av Test)

85.6 88.6 88.9 90.5 88.5

Trastuzumab HHLL intact 157 kDa (Av Control) 88.9 87.5 87.8 90.4 89.0

Difference IControl-Testl (Percentage, %) -3.3* 1.0 1.0 0.0 -0.5

TABLE 21

Key to Table 21

Test = with TEVADAPTOR® system.

Control = aseptically prepared Trastuzumab using standard needle & syringe approach. HHLL = Heavy Heavy Light Light = intact Trastuzumab

HHL = Heavy Heavy Light Chain low molecular weight impurity.

* anomalous result on Day 0 for Test. Acceptance Criteria:

The USP validation guidelines indicate acceptance criteria for system suitability only and do not indicate acceptable limits for impurities including low molecular weight impurities as identified using this test approach. As such the UK National Healthservice (NHS) Quality Assurance Committee 2012 "A Standard protocol for deriving and assessment of stability: Part 2 - Aseptic Preparations of Biopharmaceuticals" criteria was applied to the data. All test Trastuzumab solutions prepared using TEVADAPTOR® system should be within 95-105% (for the presence of intact Trastuzumab (HHLL) species) as well as Glycosylated Heavy Chain following treatment under denaturing and reducing conditions with DTT when compared with reference control preparations of Trastuzumab freshly prepared on the day of test using a standard needle and syringe approach. The results show that All Trastuzumab Test drug vials prepared using TEVADAPTOR® systems are within the accepted criteria (95.0-105%) of the Trastuzumab Control drug vials that were freshly prepared on the day of test for both the intact Trastuzumab species (HHLL) and for the low molecular weight impurity peak due to loss of a single light chain designated HHL appearing at a molecular size of 141 kDa. For all Test Trastuzumab samples the percentage change (loss) of intact HHLL over the 28 day period is not greater than 10% when compared with control samples. All Trastuzumab test samples prepared using TEVADAPTOR® systems therefore meet the acceptance criteria as it is described in this document.

Table 22 shows Capillary Electrophoresis (CE) analysis under denaturing Sodium Dodecyl Sulphate (SDS) Reducing conditions for Trastuzumab Test drug vials prepared with TEVADAPTOR® systems versus Trastuzumab Control drug vials freshly prepared using standard needle and syringe reconstituted on the day of test: limit of Non-Glycosylated Heavy Chain (NGHC) impurity. Measurements were performed for Test (TEVADAPTOR® system vials) and control samples of Trastuzumab in duplicate with duplicate sampling on the following test days: Day 0, 7, 14, 21 and 28.

Test Day (Percentages of total protein)

Assignment of Trastuzumab Peptide

0 7 14 21 28 Fragments: LC; NGHC & HC kDa

Fragment Light Chain 26.4 kDa

31.6 31.7 32.3 31.7 31.6 (Av Test LC)

Fragment Light Chain 26.4 kDa

31.6 31.6 31.8 31.5 31.5 (Av Control LC)

Difference IControl-Testl (Percentage, %) 0.0 0.2 0.5 0.3 0.2

Non-Glycosylated Heavy Chain 53.5 kDa

0.6 0.8 0.7 0.8 0.7 (Av Test NGHC)

Non-Glycosylated Heavy Chain 53.5 kDa

0.5 0.9 0.6 0.6 0.7 (Av Control NGHC)

Difference IControl-Testl (Percentage, %) 0.1 -0.1 0.1 0.1 0.0

Glycosylated Heavy Chain 58.1 kDa

67.5 67.7 66.4 67.4 67.1 (Av Test HC)

Glycosylated Heavy Chain 58.1 kDa

67.8 66.8 67.5 67.1 66.9 (Av Control HC)

Difference IControl-Testl (Percentage, %) -0.3 0.9 -1.1 0.3 0.2

TABLE 22

All Trastuzumab Test drug vials were within the accepted criteria (95.0- 105%) of the Trastuzumab Control drug vials on the day of test for the amount of Non- Glycosylated Heavy Chain (NGHC) impurity present within the Test presentations of Trastuzumab compared with control samples. The limit of Non-Glycosylated Heavy Chain impurity tests performed show that the there is no significant increase in the NGHC low molecular weight impurity peak within the Test samples prepared with TEVADAPTOR® systems when compared with Control Trastuzumab presentations. All samples were within the acceptable limits as defined in the USP document for system suitability and as defined in this document based on the National Health Service (NHS) Pharmaceutical Quality Assurance Committee protocols for assessment of stability of biopharmaceuticals - 2012. Conclusions:

Under both denaturing and reducing as well as denaturing and non reducing conditions Trastuzumab prepared and stored in TEV ADAPTOR® systems for up to 28 days showed no significant difference to Trastuzumab freshly prepared under aseptic conditions on the day of test (control solutions). All test samples prepared using the TEV ADAPTOR® system were within the acceptance criteria of (95-105%) of control reference samples and showed no significant increase in the amount of either low molecular weight impurity: Non-Glycosylated Heavy Chain Trastuzumab fragment or Heavy Heavy Light Chain Trastuzumab fragment on all test days over a 28 day period.

Report Method for determination of Trastuzumab Potency using a Biological binding assay (Enzyme Linked Immunosorbent Assay, ELISA)

Methodology:

Trastuzumab samples were analyzed according to the protocol supplied by AbD Serotec who manufactured and supplied the HCA-166 Human Anti- Trastuzumab (Fab antibody, V5 and StrepX-StrepX-tagged) as well as the HCA168 Human anti-Trastuzumab (Fab FLAG and HIS-6-tag) antibodies used in this test method. A LYNX rapid Horse Radish peroxidase (HRP) conjugation kit supplied by AbD Serotec was used as supplied to conjugate HRP to the detection antibody HCA168. HCA166 (0.5mg/ml) was used as supplied following dilution down to a working concentration of 1 microgram/ml by dilution in phosphate buffered saline (PBS).

The methodology in the USP summary validation document does not contain any reference data to the method of ELISA for determining biological potency, only referring to a method involving the use of an anti-proliferation assay using a HER2 positive human cancer cell line BT474.

Due to the inherent variability that exists with biological assays as compared with physicochemical methods the acceptance criteria has been relaxed as discussed and proposed within the National Health Service (NHS) Pharmaceutical Quality Assurance protocol for assessing stability in Biopharmaceutical products - 2012. System Suitability and Repeatability of Method:

Results:

The binding curves obtained with Trastuzumab resolution solutions from validation runs performed on several validation runs and throughout the 28 day study period showed excellent agreement and inter repeatability with the method used. Two example of binding curves for Trastuzumab resolution solution are shown is Figs. 12 and 13, for Days 0 and Day 28 of the study, respectively. Both exhibit good binding characteristics across the range of Trastuzumab concentrations tested from 0 to 500ng/ml and show a linear instrument response at Trastuzumab concentrations of between 100 to 220 ng/ml. For the data collection, a Biotek Epoch plate reader was used at a wavelength of 450nm and all analysis was performed using the proprietary Gen5 software supplied with the instrument. The analysis software used a 4 parameter fit algorithm to fit all of the experimental data to the curve. This was assessed as the best fit for the data and the recommended standard fitting algorithm for interpretation of ELISA binding data.

Fig. 12 shows a typical binding curve for resolution solution Trastuzumab prepared at concentrations between 0 and 500ng/ml on day 0 at a detection wavelength of 450nm.

Fig. 13 shows a typical binding curve for resolution solution Trastuzumab prepared at concentrations between 0 and 500ng/ml on day 28 at a detection wavelength of 450nm.

Figs. 12 and 13 show the reproducibility of the ELISA method for Trastuzumab. The binding assay provides almost identical response curves for Trastuzumab across the range of drug concentrations within the assay (0 to 500ng/ml) which covers around 75% of the dynamic response of the instrument at 450nm detection wavelength.

Table 23 shows the percentage recovery of intact efficacious Trastuzumab drug substance in TEV ADAPTOR® system (Test Trastuzumab) when stored for up to 28 days versus Trastuzumab resolution solution drug substance obtained from freshly prepared reference control samples prepared using a standard needle and syringe approach on the day of test. Data is presented as % recovery based on absorbance readings at 450nm for the Horse Radish Peroxidase substrate Ultra-TMB. The data of Table 23 was obtained from duplicate test and control devices with duplicate sampling and triplicate injections.

TABLE 23

Acceptance Criteria:

As the USP does not state an acceptable limit for the amount of Trastuzumab as determined using a Biological potency binding assay (ELISA), we used the UK National Healthservice (NHS) Quality Assurance Committee 2012 "A Standard protocol for deriving and assessment of stability: Part 2 - Aseptic Preparations of Biopharmaceuticals" criteria. All test Trastuzumab solutions prepared using TEVADAPTOR® system should be within 90-110% (for the amount of potent intact Trastuzumab) of control samples freshly prepared using standard needle and syringe approach on all test days.

Conclusion:

Analysis by a Bespoke Trastuzumab ELISA binding assay was used to quantify the amount of potent Trasuzumab within the presentations prepared using TEVADAPTOR® systems and these presentations compared with freshly prepared Trastuzumab solutions in control glass vials reconstituted using the standard needle and syringe approach.

Trastuzumab solutions prepared in TEVADAPTOR® systems compared with Trastuzumab freshly prepared in reference type one glass vials on the day of test showed no significant difference over a 28 day extended period. All test solutions remained within 90-110% of the starting control values as determined using reference standards of Trastuzumab freshly prepared on the day of test. The data for Day 0 in TEVADAPTOR® systems shows a wider variance than usual but within the variability of the data set the test solutions prepared using TEVADAPTOR® systems is not significantly different to those prepared using the standard syringe and needle approach on Day 0 and all other test days. Biological Activity- Cellular Proliferation Assay

Following the recommended guidelines in the proposed USP summary validation document for the assessment of stability of Trastuzumab a cell based assay was designed to measure Trastuzumab potency through an anti-proliferation assay. The approach uses an immortal human breast cancer cell line that over expresses the surface cell receptor HER2 which is the target for therapy using Trastuzumab. Trastuzumab is able to selectively bind to the HER2 receptor and prevent both homodimerisation and heterodimerisation processes, which inhibits a downstream cellular cascade pathway resulting in loss of cell activity including ability to proliferate. The main deviation in the test method employed to that published in the USP Summary validation report concerned the standard manual tissue culture practices and procedures that were followed, including cell counting (counts obtained using an inverted Olympus phase contrast microscope and a Heamacytometer) and manual cell seeding into 96 multiwell plates. The repeatability of cell seeding is a particular challenge for this type of assay as it affects the ratio between drug dose and cell count within a well, which will have an impact on cell response. The work published in the USP was performed using automated cell counting equipment and automated liquid cell suspension handling systems, with high repeatability in dispensing and seeding cells into multiwell plates. All other details including cell type (ATCC no. HTB-20, obtained from Lab of the Government Chemist in the UK as distributor for ATCC) were followed exactly to the method supplied in the USP document. In the work reported here the form of Resazurin fluorescent dye used was that of a commercially available reagent (ALAMAR BLUE, Sigma Aldrich, UK), which was used as supplied. Cells were subcultured on receipt from LGC UK in T75 flasks (Nuncleon) and incubated in a Sanyo incubator with 5% C0₂ and with high humidity (RH) at 37°C. Cell suspensions were harvested from the T75 flasks at around 70% confluence and used at a concentration of 0.9-1.0 x 10⁵ cells per ml in medium B. All 96 microwell plates were prepared using a set of Trastuzumab resolution solution (RS) standards in triplicate wells along with test and control wells (triplicate), negative controls (no cells) and positive controls. Control blanks were also prepared for each 96 microwell plate using media only without addition of Resazurin dye (ALAMAR BLUE). Fluorescence was measured using a Thermo Scientific ASCENT Fluoroskan plate reader operating Thermo Scientific ASCENT software and using the 530nm, 590nm excitation and emission filter set. Dose response curves were prepared using Trastuzumab RS for all plates measured, with additional triplicate wells for all Trastuzumab test solutions from samples prepared using TEVADAPTOR® systems, control wells using standard syringe and needle approach and system controls (Negative, positive). ALAMAR BLUE was aliquoted out on each day of test and autoclaved at 120°C according to the manufacturer's instructions for use to form a fully reduced product for comparison (100% fluorescence value).

The potency assay described for Trastuzumab compliments the Trastuzumab biological binding assay in terms of assessment of biological function providing additional information as to the functionality of the Trastuzumab drug product following preparation and storage. The challenge with all cellular assays is the repeatability of the data and variance which can make it difficult to assess accurately a small-medium change in drug potency (circa 10-20%) which is more easily accomplished using an ELISA, the latter being a more robust assay. Fig. 14 below shows a typical drug dose response curve for Trastuzumab resolution solution (RS) as performed using the above cell assay.

Results:

The results from the biological potency assay using the cell line HTB-20 (BT474) indicate that Trastuzumab preparations when made with TEVADAPTOR® systems maintain their integrity for up to 28 days as indicated by the biological anti- proliferation assay seen below in Table 24. The drug dose response curve obtained was highly repeatable and matched that observed in the guidance USP document for potency assessment for Trastuzumab. Although apparent high fluorescent values were obtained by the authors of the USP study data this can be simply a product of high amplifier gain on the photomultiplier tube detection sensor. Consistently low recorded fluorescence units were obtained using the Ascent plate reader with the gain set to a minimum value as to reduce noise in the signal response (dark current). Negative controls were performed without Trastuzumab and these wells showed no reduction in cellular response (placebo) nor did wells treated with another monoclonal antibody which lacks a binding domain for HER2 (Rituximab) - data not shown. Due to the difficulty in reproducibly seeding all wells with the same number of viable cells a significant amount of validation and optimization was performed for this assay prior to undertaking the study.

As can be seen from the typical drug dose response curve shown in Fig. 14, the response curve does not flatten off at low Trastuzumab concentrations indicating that higher dilutions of the Trastuzumab drug are required to observe a non drug dose response characteristic. This may impact on the number of concentration points available over the concentration range where there is a drug dose relationship. We ensured that a number of concentration points fell on the linear response part of the drug dose response curve. Due to practical constraints in terms of numbers of wells only 9 Trastuzumab resolution solution concentrations were used for standards within the plate and these were always present within the plate alongside test wells (triplicate) to provide an internal reference. At high Trastuzumab concentrations the drug dose response flattens off showing very low to no remaining cellular activity at concentrations greater than 0.3 micrograms/ml with respect to Trastuzumab. This is consistent with the USP data. This indicates that at this drug dose level all HTB-20 cells are no longer capable of proliferating to form colonies. The Trastuzumab concentration range for a drug dose response is consistent with those values reported in the USP summary validation document. Table 24 shows data from Trastuzumab samples taken from TEV ADAPTOR® prepared test devices and Trastuzumab controls freshly prepared on the day of test using the standard needle and syringe method for Days 0 and 28 only.

Table 24 shows biological potency of Trastuzumab as determined using a cellular anti-proliferation assay (HTB-20, ATCC) for samples prepared using TEVADAPTOR® systems and stored for 28 days and freshly prepared control Trastuzumab preparations using a standard needle and syringe approach on the day of test. Sampling Date

Sample ID

Day 0 Day 28 Acceptance Criteria

Trastuzumab potency (%) Test

91.8 + 5.4 96.6 + 7.6

samples (n=2) (90-110%)

Trastuzumab potency (%) for

100.0 ± 4.7 100.0 + 9.1

Control samples (n=2) (90-110%)

Difference

8.2 + 5.4 3.4 + 9.1

Control - Test 0-20%

TABLE 24.

Acceptance Criteria:

The USP validation guidelines indicate acceptance criteria for system suitability based on repeatability, a dose response curve with a fold anti-proliferation of >1.3, whereas placebo should show no dose response according to the guidance document. In the study data reported, repeatability was demonstrated by performing the assay with Trastuzumab resolution solutions (RS) prepared fresh on each day of test, however further investigations in to the effects of exposure of Trastuzumab RS to elevated temperature and loss of potency were not performed in this study. In the absence of a defined acceptance criteria for the level of potency that should be retained by Trastuzumab test solutions we have adopted the criteria as set out in the National Health Service (NHS) Pharmaceutical Quality Assurance "yellow cover" document for a standard protocol for deriving assessment of the stability of aseptic preparations - part 2 Biopharmaceuticals - 2012. This provides a wider acceptance limit than that applied to small molecule stability, especially for biological assays such as the anti-proliferation assay reported here. The measured experimental data presented for Trastuzumab preparations made using TEVADAPTOR® systems shows good agreement with freshly prepared control reference solutions made on the day of test using a standard needle and syringe approach. All Test samples were within 20% of control sample potency values and are therefore deemed to have passed. Conclusions;

Trastuzumab prepared using TEVADAPTOR® systems is demonstrated to be as efficacious as freshly prepared Trastuzumab from control preparations made using a standard syringe and needle approach on the day of test. Trastuzumab when prepared using TEVADAPTOR® systems can be safely stored for up to 28 days in the TEVADAPTOR® enclosure without deleterious loss of potency as determined by a specific anti-proliferation assay using a human immportal cell line that over expresses the target cell surface receptor HER2 (HER2+). No drug dose dependence was observed for a non specific monoclonal drug substance (Rituximab) that does not bind selectively to the HER2 receptor.

Study Vial Sterility Test

Table 25 lists the sterility test results for the Trastuzumab drug samples withdrawn on day 28 at the end of the Trastuzumab stability study.

Table 26 below summarizes the results for the Method Suitability Test required to establish the validity of sterility test method.

TABLE 25 Growth Promotion Method

Suitability Test Result

Growth (Growth observed within 5 Medium Microbial Strain days following inoculation with <100 Colony Forming

Units)

TSB Staphylococcus aureus NCTC 10788 Meets Requirements

TSB Bacillus subtilis NCTC 10400 Meets Requirements

TSB Pseudomonas aeruginosa NCTC 12924 Meets Requirements

TSB Candida albicans NCPF 3179 Meets Requirements

TSB Aspergillus brasiliensis NCPF 2275 Meets Requirements

FTM Staphylococcus aureus NCTC 10788 Meets Requirements

FTM Bacillus subtilis NCTC 10400 Meets Requirements

FTM Pseudomonas aeruginosa NCTC 12924 Meets Requirements

FTM Candida albicans NCPF 3179 Meets Requirements

FTM Aspergillus brasiliensis NCPF 2275 Meets Requirements

FTM Clostridium sporogenes ATCC 11437 Meets Requirements

TABLE 26

Testing established that sterility was maintained in the single dose Trastuzumab drug vials when prepared using TEVADAPTOR® systems and used over a 28 day period (accessed 5 times over 28 days for physicochemical sampling and an additional 3 times for sterility testing) as can be seen from the data presented above. This demonstrates the ability of TEVADAPTOR® to maintain a sterile barrier to the drug substance Trastuzumab following first puncture of the drug vial and this barrier is maintained up to 28 days when accessed on multiple days over the 28 day in use period.

It will be appreciated by persons skilled in the art that the present invention is not limited to what has been specifically shown and described hereinabove. Rather, the scope of the present invention includes combinations and subcombinations of the features described hereinabove as well as modifications thereof which would occur to a person skilled in the art upon reading the foregoing description.

Claims

1. A method for handling a drug in liquid form comprising:

providing a drug in liquid form in a vial, said drug in liquid form being sterile;

attaching a vial adaptor to said vial;

attaching at least one syringe adaptor to said vial adaptor, at least one of said at least one syringe adaptor and said at least one vial adaptor being vented in a manner which prevents communication of micro-organisms and other particulates from the outside atmosphere to an interior of said vial;

withdrawing a first quantity, but not all, of said drug in liquid form from said vial at a first point in time; and thereafter

withdrawing at least one second quantity of said drug in liquid form in said vial at at least one second point of time following said first point of time by at least 12 hours, said at least one second quantity of said drug in liquid form maintaining said sterility of said drug in liquid form.

2. A method for handling a drug in liquid form according to claim 1 and wherein said at least one second point of time follows said first point of time by at least

24 hours.

3. A method for handling a drug in liquid form according to claim 1 or claim 2 and wherein said at least one second point of time follows said first point of time by at least 2 days.

4. A method for handling a drug in liquid form according to any of claims 1-3 and wherein said at least one second point of time follows said first point of time by at least 7 days.

5. A method for handling a drug in liquid form according to any of claims

1-4 and wherein said at least one second point of time follows said first point of time by at least 14 days.

6. A method for handling a drug in liquid form according to any of claims

1-5 and wherein said at least one second point of time follows said first point of time by at least 28 days.

7. A method for handling a drug in liquid form according to any of claims 1-6 and wherein said sterility comprises sterility as measured by test performed under

USP <71> sterility test.

8. A method for handling a drug in liquid form according to any of claims 1-7 and wherein said second quantity of drug in liquid form also maintains physical and chemical properties of said drug in liquid form.

9. A method for handling a drug in liquid form according to any of claims 1-8 and wherein said at least one of said at least one syringe adaptor and said at least one vial adaptor is vented to the atmosphere in a manner which prevents communication of micro-organisms and other particulates from the outside atmosphere to an interior of said vial.

10. A method for handling a drug in liquid form according to any of claims 1-9 and wherein said drug in a liquid form is used in the preparation of a high risk level compound sterile preparation (CSP).

11. A method for handling a drug in liquid form according to any of claims 1-10 and wherein said method is performed within a Class 5 laminar air flow hood (LAF).

12. A method for handling a drug in liquid form according to claim 11 and wherein said method does not require a cleanroom environment as defined in USP <797>.

13. A method for handling a drug in liquid form according to any of claims

1-12 and wherein said withdrawing at least one second quantity of said drug in liquid form in said vial comprises withdrawing multiple quantities at multiple respective points of time following said first point of time by at least 12 hours.

14. A method for handling a drug in liquid form according to any of claims

1-13 and wherein said providing a drug in liquid form in a vial comprises:

providing a non-liquid drug in a first vial;

attaching a first vial adaptor to said first vial;

providing a liquid for reconstituting said non-liquid drug in a second vial; attaching a second vial adaptor to said second vial;

attaching at least one syringe to at least one syringe adaptor; attaching said at least one syringe adaptor, with said syringe attached, to said second vial adaptor;

transferring at least a quantity of said liquid for reconstituting said non- liquid drug from said second vial into said at least one syringe via said at least one syringe adaptor and said second vial adaptor;

thereafter, transferring said at least a quantity of said liquid for reconstituting said non-liquid drug from said at least one syringe into said first vial via said at least one syringe adaptor and said first vial adaptor.

15. A method for handling a drug in liquid form according to claim 14 and also comprising reconstituting said non-liquid drug by moving said first vial.

16. A method for handling a drug in liquid form according to claim 14 and wherein said non-liquid drug comprises Trastuzumab.

17. A method for handling a drug in liquid form according to claim 1 and wherein said drug in liquid form is Cisplatin.

18. A method for handling a drug in liquid form according to claim 1 and wherein said drug in liquid form is Methotrexate.

19. A method for handling a drug in liquid form according to claim 1 and wherein said drug in liquid form comprises Trastuzumab that has been reconstituted.

20. A method for handling a drug in liquid form according to claim 1 and wherein said drug in liquid form in a vial comprises a non-liquid drug in a vial that has been reconstituted.

21. A method for handling a drug in liquid form according to any of claims 1-13 and wherein said vial adaptor comprises:

a spike adapted for penetrating said vial;

a mechanical lock for locking said vial adaptor to said vial once said spike penetrates said vial; and

an element operative to vent the interior of said vial in a manner which prevents communication of micro-organisms from the outside atmosphere to an interior of said vial.

22. A method for handling a drug in liquid form according to claim 21 and wherein said vial adaptor also includes a septum equipped syringe port.

23. A method for handling a drug in liquid form according to claim 21 or claim 22 and wherein said mechanical lock includes at least one locking element, operative to irreversibly lock said vial adaptor to said vial.

24. A method for handling a drug in liquid form according to claim 23 and wherein said at least one locking element includes at least one radially extending portion and at least one transversely extending portion.

25. A method for handling a drug in liquid form according to any of claims 1-13 and wherein said vial adaptor includes at least one locking element, operative to irreversibly lock said vial adaptor to said vial.

26. A method for handling a drug in liquid form according to claim 25 and wherein said at least one locking element includes at least one radially extending portion and at least one transversely extending portion.

27. A method for handling a drug in liquid form according to any of claims

1-13 and wherein said at least one syringe adaptor comprises:

a septa housing;

at least two septa enclosed in said septa housing defining a space therebetween; and

a needle, including a tip located in said space when said syringe adaptor is not connected to said vial adaptor.

28. A method for handling a drug in liquid form according to claim 27 and wherein said septa housing is movable relative to said needle, thereby to expose said tip.

29. A method for handling a drug in liquid form according to claim 28 and wherein at least a portion of said needle is protected by a needle protector.

30. A method for handling a drug in liquid form according to claim 29 and wherein said needle protector comprises an elastomeric tubing element.

31. A method for handling a drug in liquid form according to any of claims 1-30 and wherein said vial adaptor is a TEVADAPTOR® vial adaptor.

32. A method for handling a drug in liquid form according to any of claims

1-31 and wherein said at least one syringe adaptor is a TEVADAPTOR® syringe adaptor.