GB2522511A

GB2522511A - Method

Info

Publication number: GB2522511A
Application number: GB1420652.8A
Authority: GB
Inventors: Richard Wastnage; Christopher Watt
Original assignee: QUALASEPT Ltd
Current assignee: QUALASEPT Ltd
Priority date: 2013-11-22
Filing date: 2014-11-20
Publication date: 2015-07-29
Also published as: GB201320660D0; GB201420652D0

Abstract

A method for increasing the shelf-life of a therapeutic protein such as an antibody (e.g. Trastuzumab) provides a closed system containing the protein freely suspended in a liquid, and a separate diluent. The protein and diluent are mixed prior to use without opening the closed system. Preferably the system is a double chamber bag having a drug chamber 1 and a diluent chamber 3 separated by a frangible or peelable seal 2. Resealable ports 4 allow drug and diluent to be added independently; administration port 5 is located in the diluent chamber so the undiluted drug cannot be delivered to a patient.

Description

Intellectual Property Office Application No. GB1420652.8 RTM Date:22 May 20t5 The following terms are registered trade marks and should be read as such wherever they occur in this document: Flerceptin (page 6,7, 8, 10 and 11) Dionex (page 7, 8) Zetasizer (page 8) NuPage (page 8) Versamax (page 9) Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo NI eth od

Field of invention

The present invention relates to methods for increasing the shelf-life of therapeutic proteins and in particular to storing therapeutic proteins in a form to increase the shelf-life stated as part of a marketing authorisation.

Background to the Invention

Most countries and territories around the world require specific documentation of medical products before they are authorised for marketing. This documentation typically provides a definitive description of the product in terms of chemical properties, clinical particulars, pharmacological properties and pharmaceutical particulars including shelf-life and storage requirements. In Europe this documentation is known at the Summary of Product Characteristics (SPC), Ready-to-use therapeutic proteins for intravenous infusion are supplicd in final diluted form and SPC's typically apply short shelf lives to these therapeutic proteins following reconstitution or dilution into their ready-to-use form. For example, the SPC for one therapeutic protein, Trastuzumab (a monoclonal antibody), indicates physical and chemical stability of reconstituted solutions for 48 hours when stored at 2-8°C in the original vial. However, only 24 hours stability has been demonstrated and assigned to the ready-to-use preparation of trastuzumab as an infusion solution when diluted in sodium chloride 0.9% in polyvinylchloride, polyethylene or polypropylene infusion bags at temperatures not exceeding 30°C. Limited data are available on the reasons for the application of these storage limits.

Dosage regimes for patients often require multiple doses of therapeutic proteins to be administered to a patient at intervals. The interval between each use is often longer than the ready-to-use therapeutic protein's shelf-life as set out in the SPC, particularly because therapeutic proteins are usually made up into their ready-to-use form by a pharmacist. The pharmacist is legally required to understand the SPC for the therapeutic protein and to take responsibility for the shelf-life of the product. The pharmacist prepares the ready-to-use form of the protein and transfers this to a medical professional for administration to a patient. The patient must then arrive at the appointed time and place in order for the therapentic protein to be administered.

Once the therapeutic protein has been made up into a ready-to-use form any therapeutic protein that is left after the initial dose has been administered therefore has to be discarded, as the shelf-life proscribed by the SPC will not allow the therapeutic protein to be stored until the next dose is needed. Additionally, the therapeutic protein may be made up by the pharmacist and delivered to the medical professional but the patient may miss their appointment. In this instance the whole dose of therapeutic protein may be wasted. Each dose of therapeutic protein can cost thousands of pounds and any wastage is therefore very expensive.

Aseptic manufacturing units are app'ying extended shelf lives on diluted ready-to-use infusion preparations of protein therapeutics. These ready-to-use infusion t5 preparations are stored at the final diluted concentrations and extensions of shelf-life from 7 days to 37 days have been applied, though no data to support these extensions is publicaily available.

However, methods used to apply extended shelf lives to other therapeutic proteins cannot be directly applied to antibodies because antibodies do not only degrade through physio-chemical mechanisms but also suffer from a functional loss of activity over time, All of these mechanisms of degradation must be analysed in order to fully characterise the integrity of an antibody upon storage and this must be carried out using the final container for the antibody. It is not possible to predict how the antibody will degrade, or at what rate. Conventional stability testing for therapeutic proteins has focussed on physio-chemical properties of the therapeutic proteins rather than looking at the effect of storage on the functional activity of the therapeutic proteins. Additionally, it is not sufficient to measure the frmnctional activity of the therapeutic proteins at a single time point, instead the activity needs to be measured over multiple days, providing data that can be compared over different time points, It is an object of the present invention to reduce wastage of therapeutic proteins. n j

Description

The present invention provides a method for increasing the shelf-life of a therapeutic protein comprising providing a therapeutic protein and a diluent in a dosed system wherein the therapeutic protein and diluent are physically separate within the system and can be brought into contact with each other prior to use of the therapeutic protein without opening the closed system.

Methods of the present invention allow therapeutic proteins to be made ready-to-use in the vicinity of a patient rather than in a pharmacy. Additionally, therapeutic proteins stored according to the method of the present invention can have an extended shelf-life compared a market authorisation, with physio-chemical properties and functional activity being maintained within acceptable limits.

The closed system used in methods of the invention can be provided by a container having at least two compartments. In embodiments of the invention the container may have three or more compartments or four or more compartments. In alternative embodiments of the invention the closed system may be provided by two or more conjoined containers, each container having at least one compartment, The container is typically sealed from the environment and in preferred embodiments of the invention the container is gas and liquid impermeable. Compartments within the container may be capped with air or an inert gas atmosphere such as a nitrogen atmosphere. The container can comprise or be made of plastics material, especially polypropylene or polyethylene, low or high density, polyvinylchloride or other polymer used in manufacture of containers or in the medical industry, e.g. polyethylene terephthalate. In preferred embodiments of the invention the container is made of polyolefin, In preferred embodiments of the invention compartments of the container are separated from each other by a frangible seal or a peelable seal. The frangible or peelable seal prevents the therapeutic protein and diluent from coming into contact with each other until such time as the seal is broken. Preferably the therapeutic protein and diluent are physically and chemically separate until the seal is breached.

ConsequeniJy, the seal is preferably gas and liquid impermeable and can comprise or be made of plastics material. In embodiments of the invention the seal is formed by an inner layer of the container material, for example, by the controlled application of heat and pressure.

Once the therapeutic protein and the diluent have been brought into contact with each other they form a ready-to-use solution, which is suitable for administration to a patient. In order to make the therapeutic protein ready-to-use the therapeutic protein and the diluent can be brought into contact with each other by breaking the frangible seal or by opening the peelable seal. Breaking the seal will generally be done by the user physically manipulating the outside of the container until the seal is broken. It is especially preferred that the seal can be broken or otherwise opened without opening the closed system to the environment. The therapeutic protein can therefore be made into a ready-to-use form by a user immediately prior to administration to a patient rather than having to be made up by a pharmacist. For example, this means that a therapeutic protein could be made ready-to-use only once a patient has arrived for their appointment, if the patient were to miss their appointment the protein would not be made ready-to-use and consequently would not be wasted.

In embodiments of the invention the container is a sterile, flexible container such as a bag, preferably an infusion bag.

In preferred embodiments of the invention the compartment containing the therapeutic protein does not have a port for administration to a patient. Consequently, the therapeutic protein can only be administered to a patient after it has mixed with the diluent, thereby avoiding the risk that undiluted therapeutic protein is administered to a patient.

The therapeutic protein is stored in the container in a liquid form, for example, in a solution. The liquid form is also known as a ready-to-mix form and can comprise pharmaceutically acceptable carriers and excipients. Suitable carriers and excipients include, but are not limited to, polysorbates, trehalose, sorbitol, sucrose and polyethyleneglycols, Suitable concentrations of the ready-to-mix therapeutic protein include, but are not limited to, typical pre-dilution concentrations as described in the SPC for the specific protein, generally in the range of 0.1 mg/mL to 150 mg/mt.

preferably 20 mg/mL to 125 mg/mL. More preferably, the storage concentration of the read-to-mix therapeutic protein is higher than the SPC recommended administration concentration. The concentration of the ready-to-mix therapeutic protein is preferably at least 5% higher than the concentration specified in the Summary of Product Characteristics (SPC) The ready-to-mix therapeutic protein is stored in a liquid form prior to contact with the diluent. Preferably the therapeutic protein is freely suspended in a pharmaceutically acceptable liquid, such as a buffer solution. In other words, the ready-to-mix therapeutic protein is not attached to a solid support or otherwise immobilised. Preferably the ready-to-mix therapeutic protein is not in a lyophilised form.

In preferred embodiments of the invention the shelf-life as stated as part of a market authorisation for the therapeutic protein is increased, preferably by at least 30 days, more preferably by at least 56 days. In embodiments of the invention the shelf-life may be increased by up to 60, 70, 80 or 90 days.

The therapeutic protein may be an antibody or an antibody fragment and is preferably a monoclonal antibody.

Suitable monoclonal antibodies for use in methods of the invention include Rituximab, Infliximab, Trastuzumab, Bevacizumab, Natalizumab, Cetuximab, Tocilizumab, Muromonab-CD3, Abciximab, Basiliximab, Palivizumab, Gemtuzumab, Alemtuzumab, Tositumomab, ibritumomab tiuxetan, Omalizumab, Panitumumab, Eculizumab, Canakinumab, Catumaxomab, Ofatumnmab, Belimumab, Ipilimnmab, Brentuximab and Pertuzumab,

Brief description of the drawings

The invention is now described in specific embodiments with reference to accompanying drawings in which: Figure 1 shows a suitable storage device which comprises a double chamber bag according to one embodiment of the invention. The bag has a drug chamber I for containing the therapeutic protein, which is separated from a diluent chamber 3 by a frangible or peelable seal 2. Resealable activated ports 4 allow drug and diluent to be added independently to their respective chambers, while the administration port 5 is necessarily located in the diluent chamber so that a patient can only receive the therapeutic protein after it has been combined with the diluent. If the contents of the bag were administered to a patient without the frangible or peelable seal being broken the patient would receive a dose of diluent only, thereby avoiding the risk that the patient could inadvertently receive an undiluted dose of the therapeutic protein.

Figure 2 shows SDS-PAGE analysis of Trastuzumab (Herceptin) stored in a single chamber bag and analysed under non-reducing conditions on storage days 0, 1 and 28.

From left to right; protein ladder marker, blank, sample 1, sample 2, sample 3, sample 4, sample 5, sample 6, sample 7, sample 8. Primary band at -lSOkDa represents Trastuzumab monomer.

Figure 3 shows SDS-PAGE analysis of Trastuzumab (Herceptin) stored in a double chamber bag under non-reducing conditions on storage days 0, 2 and 28. From left to right; protein ladder marker, blank, sample 1, sample 2, sample 3, sample 4, sample 5, sample 6, sample 7, sample 8. Primary band at 150kDa represents Trastuzumab monomer, Faint band relating to aggregate observed above monomer, Bands also present at] OOkDa and 26kDa.

Figure 4 shows mean results of particle counting of trastuzumab stored in a single chamber bag at a diluted concentration of 0.21 mg/mt +1-SD (particles). Data gives the numbers of particles >10 micron and >25 micron in size, Figure 5 shows biological activity of Trastuzumab (Herceptin) stored in a single chamber bag at a concentration of 0.63mg/mL. Biological activity shown in the graph is the percentage inhibition of BT474 cell growth compared to untreated cells.

Trastuzumab stored in a single chamber bag inhibited BT474 cell growth by 21.1% on Day 0 when tested at a final concentration of 3 tg/ml. At this concentration, inhibition of growth had fallen to 5,1% by Day 35.

Figure 6 shows biological activity of Trastuzumab (Herceptin) stored in a double chamber bag. Biological activity shown in the graph is the percentage inhibition of BT474 cell growth compared to untreated cells. Trastuzumab stored in a double chamber bag inhibited BT474 cell growth by 20% on Day 0 when tested at a final concentration of 3 j.tg/ml. At this concentration, inhibition of growth remained constant up to and including, Day 35.

Figure 7 shows a comparison of results from single chamber and double chamber bags which have been indexed to demonstrate clearly an extension of frmnctional activity in the double chamber bag.

Examples

NI etho ds Visual inspect/or Each of the samples were checked by the unaided eye under normal laboratory fluorescent light for evidence of particulates, precipitation, colour change and turbidity at the indicated storage time.

Size-exclusion HPLC analysis Degradation/aggregation was analysed using a stability indicating FIPLC method using a Dionex Ultimate 3000 system iii conjunction with a MAbPac SEC-i (5 p.m.

300A, 4.0 x 300 mm) column maintained at a constant 25°C. Mobile phase consisted of 50 mIvI sodium phosphate and 300 nilVi sodium chloride at pH 7 and was pumped at a flow rate of 0.2 mL/min. The duration of each isocratic run was 30 minutes and UV absorbance was detected at 280 nm. A control blank of 20 p.L mobile phase was run between each sample in order to ensure complete elution and to assess carry over.

Data acquisition and integration was obtained using Dionex Chromeleon software version 6.80. Results are expressed as a percentage concentration relative to Day 0.

Dynamic light scattering analysis (DLS) For DLS analysis, aH samples were ana'ysed on a Mavem Instruments Zetasizer Nano-S utilising a red laser at a wavelength of 633 nm and a I-leflma Quartz-Suprasil cuvette (Type 1O5251OO5-QS, Light path 3x3 mm, Centre 965 mm), Approximately 1OOiL (3 drops sufficient to cover the cell ndow) of sample solution were transferred directly into the analysis cuvette. Analysis of each sample was performed in triplicate at +19°C using Malvern Instruments Zetasizer software version 6.20 and a cumulants fit analysis.

Microflow Imaging (MFI) A Fluid Imagingtm' FIowCAM VS-I was used with a Fluid ImagingTM FC8OFV flow cell to assess particles sized 10 tm-1O0 tm. Flow rate was 0.1 mi/mm, imaging rate was 20 frames per second, and efficiency was 34.5 %. A 0.9% NaC1 blank was run at start up and the system calibrated with 10 m beads. Data were analysed using Visual SpreadsheetTM software. Samples were each tested three times.

SDS gel electrophoresis 2 ML of each test sample was diluted with 20 tL of 2x Laemmili reducing SDS loading buffer. 5!IL of each sample was then loaded on to a 15 well Invitrogen NuPage 10% BIS-TPJS Gel along with ijiL of unstained protein reference ladder (Invitrogen BenchMark). Each gel was run at 200V for 50-60 minutes and then visualized by treating with coomassie blue stain for 1 hour before dc-staining.

Functional aceivily assay for Trasmuzurnab (Herceptin,) Functional activity testing of reconstituted Trastuzumab was performed using a growth assay method, BT474 cells were maintained in McCoy's 5A medium supp'emented with O% heat inactivated foetal bovine serum (hi-FBS), CeHs were harvested from culture, washed and seeded in 96 well plates at 1x104 cells in 100 jiL per well, in quintuplicate, and cultured overnight at 37°C. Cells were then incubated at the specified storage times in the absence (control cells) or presence of trastuzumab diluted to a final concentration of 3 j.tg/mL for a further 48 hours. Cell supernatants were then removed and replaced with RPM11640 medium without phenol red, supplemented with 10% hi-FBS and 1.2 mM MTT. Cells were then cultured for 4 hours at 37°C, culture medium removed, and cells dissolved in 100 ML DMSO.

Absorbance was recorded at 540 nm (A540) using a Molecular Devices Versamax plate reader. A540 values from cells in the absence of trastuzumab were set to 100%, and degree of inhibition of growth by trastuzumab samples determined by comparison to untreated samples.

Stability study of diluted Trastuzumab stored in a single-chambered polyolefin infusion bag The purpose of this study was to detennine the physical/chemical stability and functional activity of aseptically prepared trastuzumab solutions in a single-chamber infusion device over extended storage periods of 35 days. The trastuzumab lyophilised powder was reconstituted with water for injection to an initial concentration of 21 mg/mL according to the manufacturer's instructions, The reconstituted trastuzumab concentrate was further diluted with 0.9% sodium chloride solution to a therapeutically relevant final concentration of either 0.21 mg/mL or 0.63 mg/nil in the respective infusion bag for storage. Three replicate bags were prepared at each concentration, and the diluted trastuzumab infusion bags stored under refrigeration at 2-8°C. Samples of test solutions were withdrawn from the infusion bags under aseptic conditions after specified days of storage for analysis of physical/chemical stability and functional activity.

Physical/chemical stability of the diluted trastuzumab was assessed afler 0, 2, 13, 29 and 35 days of storage for 0.21 mg/mL using MFI particle counting and 0, and 28 days of storage for 0.63 mg/mL using a stability indicating 1-1.PLC assay utilizing UV absorbance at 280nm, dynamic light scaftering, visual inspection and SDS-PAGE, according to the methods described above, The functional activity of the diluted trastuzumab at 0,63 mg/mL was assessed after 0, 2 and 35 days of storage using an MTT growth assay according to the method described above, Stability study of reconstituted Trastuzumab stored in a multi-chamber polyolefm infusion bag The purpose of this study was to determine the physical/chemical stability and functional activity of asepticaHy prepared trastuzumab solutions in a double-chamber infusion device over extended storage periods of 35 days. The trastuzumab lyophilised powder was reconstituted with water for injection to an initial concentration of 2] mg/mL according to the manufacturer's instructions. Four replicate bags were prepared by filling 10 mL (210 mg) of trastuzumab solution into the small drug chamber and the bags stored under refrigeration at 2-8°C. Tests were performed on infusion solutions after preparation in a Qualasept Ltd 250m1 5 layer polyolefin multi-chamber inifision bag with a pealable seal (PB02752603T13CPP070). All tests were performed using the commercially available Herceptin (Roche) powder formulation for concentrate for infusion. 250m1 tS multi-chamber infusion bags were aseptically prepared by Qualasept Ltd in a grade A clean room environment (Class 11 Biological safety cabinets) using a suitably validated process and operator. Samples of test solutions were withdrawn from the infusion bags under aseptic conditions after specified days of storage for analysis of physical/chemical stability and functional activity.

Physical/chemical stability of the diluted trastuzumab was assessed after 0, 2, 28 and 56 days of storage using a stability indicating HPLC assay utilizing UV absorbance at 280nm, dynamic light scattering, visual inspection and SDS-PAGE, according to the methods described above. The ifinctional activity of the diluted trastuzumab was assessed after 0, 14, 35 and 56 days of storage using an MTT growth assay according to the method described above.

Results Physical and chemical stability testing of reconstituted Trastuzumab Trastuzumab (1-lerceptin) inftision solution, aseptically prepared in accordance with the SPC at a concentration of 2]mg/ml infrision concentrate, stored at 2-8°C in a polyolefin multi-chamber infusion device was found to be physio-chemically stable for a minimum of 56 days as indicated by the methods outlined.

Trastuzumab abundance as determined by SE-HPLC (see tables 1 and 2) did not show an overall reduction for the reconstituted concentrate when analysed in relation to the Day 0 sample for either the single chamber or the double chamber storage device.

% Trastuzumab Remaining Concentration DayO Dayl +SD Day28 +SD (mg/mi) 0.63 00 100.62 1.2 98.87 1.2 Table 1: HPLC analysis of Herceptin stored in single chamber bag % Trastuzumab Remaining Concentration -DayO Day2 +SD Day28 +SD DaySo +SD (mg/mi) 21.0 100 94.49 8.3 105.44 10.2 98.54 8.4 Table 2: HPLC analysis of Herceptin stored in double chamber bag Comparison of chromatographic profiles across the study period showed the infusion concentrate (2 Img/ml) was extremely robust, demonstrating almost no signs of both aggregate or fragment peaks. The diluted 0.63mg/mi solution seemed equally robust up to day 28 after which the emergence of peaks due to degradation became increasingly evident.

The SDS-PAGE results of trastuzumab test solutions were compared across all sample time points with no significant differences detected (see figures 2 and 3). A single major band was observed in the region of the I 5OkDa marker under non-reducing conditions, This band relates to intact trastuzumab of known molecular weight 145kDa, A faint band was also evident above the major band indicating the low level presence of aggregated antibody and supports the results obtained by SE-HPLC.

Upon visual inspection, all test solutions remained clear for the duration of the study with no evidence of precipitates or particulate matter. No change in colour or turbidity was observed across the entire study period.

Analysis by DLS demonstrated that only very low levels of high molecular weight aggregates could be detected in the diluted 0.63 mg/mL preparation across the period of the study (see table 3). However, in terms of relative abundance by volume this accounted for <0, 9°/b for the diluted samples. The average hydrodynamic radius of secondary peak aggregates was 1.Ôi.tm. No high molecular weight aggregates were observed in the concentrated 21 mg/mL preparation across the study (see table 4).

Flydrodynaniic Radius (nm) Concentration Da! 0 ±811 Da I ±SD Day 21 ±811 (mg/mi) Mononier/Dimer 0.63 7.2 0,8 6.7 0.9 6,0 0.2 HMW Aggregate 0.63 2208 585 2450 561 3140 0 "A UMW by: Intensity 063 6.14 3,16 0,03 Aggregate Volume 0.04 0.01 0.00 Table 3: Mean results ofDLS analysis for a single chamber bag. Results expressed as mean monomer peak contribution +1-%RSD. Each sample assayed in triplicate, 3 IS replicates of four samples at each concentration, n=]2. Day

Concentration 0 15 35 56 21mg/mI 100.00 + 0.00 100.00 + 0.00 100.00 + 0.00 100.00 + 0.00 Table 4: Mean results of DLS analysis for double chamber bag. Results expressed as mean monomer peak contribution +1-%RSD. Each sample assayed in triplicate, 3 replicates of four samples at each concentration, n=12.

Particle counting of trastuzumab stored in a single chamber bag at a diluted concentration of 021 mg/mt indicated a significant increase in particle number over the study period, and consistently increasing at each time point (see table 5 and figure 4), A diluted concentration of 0.21 mg/mL is within the SPC defined concentrations for clinical use, indicating it is not possible to store trastuzumab at clinically relevant concentrations (as would be the case if stored in a single chamber bag) for extended periods of time, whilst retaining necessary product quality (as set out by regulatory bodies).

A >tOtm ± SD 10 to 25im >25tm ± SD DayO 303 136 284 20 15 Day2 377 252 348 29 33 Day 13 487 202 452 35 34 Day29 1612 Sb 1479 33 H7 Day 35 2338 932 2173 165 105 Table 5: Mean results of particle counting of trastuzumab stored in a single chamber bag at a diluted concentration of 0.21 mg/mL +/-SD (particles). Data clearly shows that numbers of particles >10 micron in size and >25 micron in size consistently and dramatically increase over the study period, indicating this product would not meet quality criteria for clinical use.

I'm ictional actn'ity testing of reconstituted J)astnznrnab Biological activity was assessed by the ability of the antibody to inhibit the growth of BT474 cells, All samples were tested at a final concentration of 3 g/ml (see figures 5, 6 and 7). The activity shown in the graphs is percentage inhibition of growth compared to untreated cells, Data shown are mean of four replicates with standard deviation, Trastuzumab stored in a double chambered bag inhibited BT474 cell growth by 4% on Day 0, and 13% on Day 56. Trastuzumab stored in the single chamber bag inhibited cell growth by 21.1% on Day 0 which had to fallen to 5.1% on Day 35 (trastuzumab tested at 3 g/mL).

Claims

Claims 1. A method for increasing the shelf-life of a therapeutic protein comprising providing a therapeutic protein and a diluent in a closed system wherein the therapeutic protein and diluent are physically separate within the system and can be brought into contact with each other prior to use of the therapeutic protein without opening the closed system, wherein the therapeutic protein is freely suspended in a liquid, prior to being brought into contact with the diluent.
2. The method of claim 1, wherein the concentration of the therapeutic protein suspended in liquid prior to contact with the diluent is at least 5% higher than the concentration specified in the Summary of Product Characteristics (SPC).
3. The method of claim 1 or claim 2, wherein the therapeutic protein is an antibody or antibody fragment.
4. The method of claim 3, wherein the therapeutic protein is a monoclonal antibody.
5. The method of claim 3 or 4, wherein the antibody is selected from Rituximab, infliximab, Trastuzumab, Bevacizumab, Natalizumab, Cetuximab, Tocilizumab, Muromonab-CD3, Abciximab, B asiliximab, Palivizumab, Gemtuzumab, Alemtuzumab, Tositumomab, Ibritumomab tiuxetan, Omalizumab, Panitumumab, Eculizumab, Canakinumab, Catumaxomab, Ofatumumab, Belimumab, Ipilimumab, Brentuximab, Pertuzumab,
6. The method of any of claims ito 5, wherein the closed system is provided by a container having at least two compartments.
7. The method of claim 6, wherein the compartments are separated by a frangible seal.
8. The method of claim 7, wherein the therapeutic protein and the diluent are brought into contact with each other by breaking the frangible seal.
9. The method of any of claims 6 to 8, wherein the compartment containing the therapeutic protein does not have a port for administration to a patient.
10. The method of any of claims 6 to 9, wherein the container is a bag.
11. The method of any of claims 1 to 10, wherein the liquid comprises excipients.
12. The method of any of claims Ito, wherein the shelf-life as stated as part of a market authorisation for the therapeutic protein is increased by at least 30 days.
13. The method of claim 12, wherein the shelf-life as stated as part of a market authorisation for the therapeutic protein is increased by at least 56 days.
14. The method of any of claims ito 13, wherein the therapeutic protein freely suspended in liquid and the diluent are brought into contact with each other to form a solution suitable for administration to a patient.
15. A method for increasing the shelf-life of a therapeutic protein as hereinbefore described with reference to the examples.