GB2480276A

GB2480276A - Process for the preparation of an aqueous injectable enrofloxacin antimicrobial formulation

Info

Publication number: GB2480276A
Application number: GB1007857A
Authority: GB
Inventors: Michael Hilary Burke
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-05-11
Filing date: 2010-05-11
Publication date: 2011-11-16
Also published as: GB201007857D0

Abstract

A process for the preparation of an aqueous injectable enrofloxacin formulation comprises the steps of (i) preparing a solution of potassium hydroxide 50% w/w and cooling the solution to approximately 25 to 30°C; (ii) adding to a batch vessel, up to approximately 60 to 80% of the batch volume of purified water; (iii) bubbling nitrogen through the purified water (iv) adding n-butanol in the range of 1 to 5% w/v to the water and stirring until complete homogenisation; (v) adding enrofloxacin in the range of 1 to 15% w/v and stirring until complete dispersion of the enrofloxacin; (vi) adding stepwise, the solution of potassium hydroxide 50% w/w and testing the solution to ensure a pH of 11.0 to 11.5 has been obtained and that complete dispersion of the enrofloxacin has taken place; (vii) adding purified water up to 100% w/v; (viii) filtering the formulation at a maximum flow rate of less than 10ml per min through (a) a pre-filter of 0.6 microns; and (b) filtration through a sterilizing filter of 0.2 microns; followed by terminal sterilization; and (ix) collecting and packaging the resultant solution aseptically into separate vials.

Description

"A PROCESS FOR THE PREPARATION OF A STABLE INJECTABLE ANTIMICROBIAL FORMULATION"

Field of the Invention

The present invention is directed to a process for preparing a stable aqueous based injectable antimicrobial formulation comprising an effective amount of enrofloxacin.

Background to the Invention

Enrofloxacin is a synthetic, broad-spectrum antimicrobial substance, belonging to the fluoroquinolone group of antibiotics. It is bactericidal in action with activity against many Gram positive and Gram-negative bacteria and mycoplasmas. The mechanism of action of the quinolones is unique among antimicrobials, they act primarily to inhibit bacterial DNA gyrase, an enzyme responsible for controlling the supercoiling of bacterial DNA during replication. Resealing of the double stranded helix is inhibited resulting in irreversible degradation of the chromosomal DNA. The fluoroquinolones also possess activity against bacteria in the stationary phase by an alteration of the permeability of the outer membrane phospholipids cell wall but are inactive against strict anaerobes.

Specifically, Enrofloxacin is I -Cyclopropyl-6-fluoro-I,4-dihydro-4-oxo-7-(4-ethyl-I -piperacin-I-yl)quinoline-3-carboxylic Acid and has the following structural formula: N)ZX7

A

Enrofloxacin is commonly used as an antimicrobial in cattle and pigs. In cattle it is used to treat diseases of the respiratory and alimentary tract of bacterial or mycoplasmal origin (e.g. pasteurellosis, mycoplasmosis, coli-bacillosis, coli-septicaemia and salmonellosis) and secondary bacterial infections subsequent to viral infections (e.g., viral pneumonia), where clinical experience, supported where possible by sensitivity testing of the causal microorganism, indicates Enrofloxacin as the drug of choice.

In pigs, it is used to treat diseases of the respiratory and alimentary tract of bacterial or mycoplasm al origin (e.g. pasteurellosis, mycoplasmosis, coli-bacillosis, co/i-se pticaemia and salmonellosis) and multifactorial diseases such as atrophic rhinitis and enzootic pneumonia where clinical experience, supported where possible by sensitivity testing of the causal microorganism, indicates Enrofloxacin as the drug of choice.

Enrofloxacin is only slightly soluble in water. This can present problems in during the large scale manufacture of enrofloxacin formulations with water as the solvent. Thus, the solubility enhancement of enrofloxacin is of interest. Many different solutions for dealing with the low solubility of enrofloxacin have been proposed. For example, US 5,998,418 discloses enrofloxacin injection or infusion solutions comprising gluconic acid, glucoronic acid, glutamic acid, tartaric acid, plus a formulation aid (thickener, absorbents, antioxidants, and preservatives) and water. In this patent, the end formulation has a pH from approximately 3 to approximately 5 (see Examples I to 6).

To date, enrofloxacin may be provided in an injectable solution with a low pH. At this pH a variety of stabilizers are required to improve solubility at or near physiological pH. For example, in U5 2010/0009979, the enrofloxacin formulation has a pH of 6.0.

Furthermore, at this pH many different stabilizers and co-solvents are required. U5 2009/0163484 discloses a number of different enrofloxacin formulations comprising various antioxidant sulphur compounds.

However, there remains a need for alternative formulations comprising enrofloxacin which are suitable for use as an injectable formulation. 5tability is necessary in order to allow for the formulation to be prepared well in advance of the intended use.

Additionally, there is a need for a process for preparing such a stable formulation.

Hence, the present invention is directed to an improved large-scale process for the production of an injectable enrofloxacin formulation.

Statement of the Invention

According to a general aspect of the invention, there is provided a process for the preparation of a stable aqueous based injectable enrofloxacin formulation with a pH of 11 to 11.5 comprising i) from I to 15% w/v of the active ingredient enrofloxacin, ii) from I to 5% w/v of the preservative n-butanol; and iii) to 100% w/v of purified water wherein the process takes place in a batch apparatus comprising a batch vessel having a capacity of at least 2,000 litres and comprises the following steps preparing a solution of potassium hydroxide 50% wlw and cooling the potassium hydroxide solution 50% w/w to approximately 25 to 3000; setting the batch volume of the formulation and adding up to approximately 60 to 80% of the batch volume of purified water for injection into the batch vessel; bubbling nitrogen through the purified water in the batch vessel and maintaining nitrogen bubbling throughout the process; adding n-butanol in the range of I to 5% w/v to the water and stirring until complete homogenization; adding the active ingredient enrofloxacin in the range I to 15% wlv and stirring until complete dispersion of the active ingredient enrofloxacin; testing and adjusting the pH of the solution by adding small amounts of potassium hydroxide solution 50% w/w to the solution in a stepwise manner and testing the solution to ensure a pH of 11.0 to 11.5 has been obtained and that complete dissolution of the active ingredient enrofloxacin has taken place; adding purified water up to 100% w/v; filtering the formulation at a maximum flow rate of less than lOmI per mm through (i) a pre-filter of 0.6 microns; and (ii) filtration through a sterilizing filter of 0.2 microns; followed by terminal sterilization; and collecting and packaging the resultant solution aseptically into separate vials.

Detailed Description of the Invention

In the specification the term "solution" refers to a single homogenous liquid, solid or gas phase that this mixture in which the components are uniformly distributed throughout the mixture. The formulation of the invention is an aqueous enrofloxacin solution In the specification the term "effective amount" refers to the amount of active agent necessary to have an anti-microbial effect.

In the specification the term "wlv" refers to the w/v of the final suspension formulation.

According to a first aspect of the invention, there is provided a large-scale process for the preparation of a stable aqueous based injectable enrofloxacin formulation with a pH of II to 11.5 comprising i) from Ito 15% w/v of the active ingredient enrofloxacin, ii) from I to 5% w/v of the preservative n-butanol; and iii) to 100% w/v of purified water wherein the process takes place in a batch apparatus comprising a batch vessel having a capacity of at least 2,000 litres, comprising the following steps preparing a solution of potassium hydroxide 50% wlw and cooling the potassium hydroxide solution 50% w/w to approximately 25 to 30°C; setting the batch size of the formulation and adding up to approximately 60 to 80% of the batch volume of purified water for injection into the batch vessel; bubbling nitrogen through the purified water in the batch vessel and maintaining nitrogen bubbling throughout the process; adding n-butanol in the range of I to 5% w/v to the water and stirring until complete homogenization; adding the active ingredient enrofloxacin in the range I to 15% wlv and stirring until complete dispersion of the active ingredient enrofloxacin; testing and adjusting the pH of the solution by adding small amounts of potassium hydroxide solution 50% w/w to the solution in a stepwise manner and testing the solution to ensure a pH of 11.0 to 11.5 has been obtained and that complete dissolution of the active ingredient enrofloxacin has taken place; adding purified water up to 100% w/v; filtering the formulation at a maximum flow rate of less than lOmI per mm through (i) a pre-filter of 0.6 microns; and (ii) filtration through a sterilizing filter of 0.2 microns; followed by terminal sterilization; and collecting and packaging the resultant solution aseptically into separate vials.

It will be understood that the process is a large scale manufacturing process, thus, the batch size or volume of the formulation may be chosen as for example, 2000 litres or any other amount suitable for large scale manufacture.

The formulation must be readily and efficiently prepared, stable and effective in use. This formulation has a pH of 11.0 to 11.5. This pH range has been found optimal to achieve a stable product using a simple and efficient large-scale manufacturing process.

Additionally, we have recognized that that in order to achieve a complete solution of enrofloxacin in water the pH adjusting agent potassium hydroxide should be used.

Furthermore, the manufacturing process uses a limited number of excipients thereby increasing efficiency.

In general terms, the process involves introducing the bulk of the solvent (water) into the batch vessel and dissolving the individual components therein. After making up the desired volume with the remainder of the solvent, the formulation is then sterile filtered through suitable bacteria-retaining filters and/or heat-sterilised. However, the process is dictated by the excipients used, which have been chosen to result in a stable homogenous formulation of enrofloxacin.

One of the major challenges in this large-scale manufacture of an injectable solution of enrofloxacin is to formulate a homogenous solution of the active agent.

Ideally, the injectable Enrofloxacin solution of the invention comprises: Preferred Quantity Names of substances Function and/or Percentage Active substance Enrofloxacin From 50 mg -100.0 mg Active Excipients n-Butanol 30.0 mg Preservative Potassium Hydroxide q.s. up to 11 to 11.5 pH adjustment Water for injection q.s. up to 1.0 ml Solvent The present invention is directed to a large-scale process for the manufacture of a formulation containing enrofloxacin according to the above proportions According to one embodiment of the invention, a solution (50% w/w) of potassium hydroxide is used. The use of a potassium solution ensures that the potassium hydroxide is easier to handle and the pH adjustment can be controlled more accurately.

However, a strong exothermic reaction takes place when making an aqueous solution of potassium hydroxide. Thus, the direct addition of potassium hydroxide can produce an unnecessary and potentially deleterious heating of the solution and is to be avoided. For this reason, the potassium hydroxide solution is prepared and cooled to 25 to 30°C prior to use.

The optimal pH range of the end formulation is 11.0 to 11.5. This pH is required to ensure that the active Enrofloxacin is completely dissolved when diluted in water. At a pH lower than 10, the mix of Enrofloxacin in water becomes a suspension because it is not completely dissolved. By adding Potassium Hydroxide 50% w/w solution, the pH increases and Enrofloxacin is completely dissolved at this pH. We found the pH of 11.0 to 11.5 to be particularly effective.

Furthermore, the use of a preservative is also desirable to provide an antimicrobial activity. However, many commonly used preservatives are inactive at a pH of 10.5 to 12.0. Thus, butanol was chosen to act as a preservative.

According to another embodiment of the invention, butanol is present at a level of 30 mg/mI (3%), where it was found to be most effective against the microogranims S. aureus, P. aeruginosa, C. albicans and A. niger. This concentration was found to be the optimum concentration to obtain an adequate preservation of the end formulation.

Ideally, purified water is used.

According to one embodiment of the invention, the process further comprises testing the solution after the addition of potassium hydroxide solution 50% w/w to ensure the density of the solution is from approximately 1.025 to 1.035 g/ml and that a clear yellow solution is obtained. If these values are not obtained further potassium hydroxide solution 50% is added until they are obtained.

Ideally, n-butanol is added at a level of approximately 3% w/v.

Ideally, the active ingredient enrofloxacin is added in the range of 5 to 10% wlv.

Optionally, the nitrogen is applied throughout the process at a pressure below 4kg/cm2.

After the preparation of the formulation, it is then filtered and packaged ideally into vials but optionally into any other suitable containers.

It will be understood that the container into which the formulation is filled can be made of glass or plastic. It is also possible for the container material to offer protection from light or oxygen.

The resultant product is intended to be administered to cattle and pigs by subcutaneous or intramuscular route, where each ml contains from approximately 50 to 100 mg of Enrofloxacin (Enrofloxacin 5% or 10% Injection)

Figures and Example

The invention will now be described by reference to the following non-limiting examples and figures.

Figure 1 shows a flowchart for the manufacturing process of the injectable Enrofloxacin solution of the invention.

In step 1, approximately 60 to 80%, preferably 75%, of the final batch volume of water for injection is added into a stainless steel mixing vessel.

In step 2, nitrogen is bubbled through the water. This prevents the oxidation of the ingredients during manufacture. Nitrogen is kept bubbling until the end of the manufacturing process. Ideally, the nitrogen is applied at a pressure below 4kg/cm° throughout the process until after filtration.

In step 3, the preservative n-butanol was added into the water. N-butanol prevent the solution from microbiological contamination, thus, is added at the beginning of the manufacturing process.

In step 4, the active ingredient enrofloxacin is added to the mixing vessel.

It is important to note that the sequence of addition of ingredients in the process is very important.

In step 5, the pH of the solution is adjusted to a pH of approximately 11.0 to 11.5 using a previously prepared and cooled potassium hydroxide 50% wlw solution. The potassium hydroxide solution is added to ensure the active ingredient enrofloxacin dissolves completely, and to ensure the required pH is achieved. The potassium hydroxide is added to the solution in the mixing vessel in a sequential and stepwise manner. Thus, small amounts of the potassium hydroxide solution are added and the solution is tested each time to ensure that a pH of 11 to 11.5 is obtained.

The potassium hydroxide 50% w/w is prepared prior to commencing the process by weighing the required amount of water, adding the potassium hydroxide and stirring until complete dissolution. As this is an exothermic process, there is an increase of the temperature of the solution, hence, the solution is cooled to 25-30°C prior to use.

In step 6, the density of the solution is measured to ensure it adheres to the desired

product specification.

In step 7, depending on the amount of potassium hydroxide 50% w/w solution added, the final volume of the formulation was adjusted with water to 100% of the final batch volume.

In step 8, the appearance, pH and density of the bulk were checked to ensure they complied with the desired product specification (see below). If all the parameters were correct, the product was then filtered through two filters. If the parameters were not correct further pH adjusting agent or purified water may be added.

Quality control checks are carried out during the manufacturing process to ensure the final product has the correct specification. This involved checking: -pH adjustment -Appearance of the final solution -pH of the final solution -Density of the final solution -Final volume of the bulk -Integrity test of the sterilization filter -Differential pressure during filtration -Volume check during the filling Carrying out these controls during the manufacturing process ensures that end product is within specifications and advantageously no intermediate analysis is required. The desired end specification for the formulation is as follows: Appearance The finished product must be a clear yellow solution, free from particle matter.

Density The density of the finished formulation should be approximately 1.025 -1.035 g I ml, preferably approximately 1.030 gI ml.

The pH of the finished formulation should be approximately 10.5 to 12.0 to ensure that the active ingredient Enrofloxacin is completely dissolved.

Enrofloxacin assay The acceptable limit for the assay of the active principle in the finished product is 95.0 - 105.0 % (95.0 -105.0 mg Iml Enrofloxacin).

Related substances I lmrurities The limits for impurities are: -Impurity A (Fluorquinolonic Acid): «= 0.2% -Impurity B (Ciprofloxacin): «= 0.5% -Any other single impurity: «= 1.0% -Total impurities: «= 1.0% Butanol Assay The acceptable limit for the assay of the preservative in the finished product is 90.0 - 110.0 % (27.0-33.0 mg /ml Butanol).

Sterility The finished formulation must comply with current Eur. Ph. criteria for injectable forms.

Once the finished formulation has been verified to meet the desired product

specification, it is then further processed.

In step 9, the finished formulation is filtered through pre-filter and a sterilizing filter.

Ideally, a 0.6 microns pre-filter is used and a 0.2 microns sterilizing filter is used. The first filter is designed to clarify and pre-filter the formulation. Ideally, it is made of polypropylene which is designed not to contaminate the formulation. Ideally the second filter is a hydrophilic modified polyvinylidenfluoride (PVDF) membrane. Again, this material is chosen so as no contamination of the formulation ensues. Ideally, the maximum flow rate through the filters is less than 10 mI/mm, optionally I mI/mm. After filtration, the material is subjected to terminal sterilization.

In step 10, the filtered finished formulation is dispensed into lOOmI and 250m1 vials.

Ideally, 100 ml and 250 ml amber glass Type I vials are used. This ensures the formulation is protected from the light and the vial can store alkaline solutions. Ideally, the stoppers used for closing the vial were made of grey teflonised chlorobutyl, because this material is adequate for contacting with an alkaline solution. The fluorocarbon film, made of a modified ethylene-tetrafuoroethylene copolymer (ETFE) reduces absorption of the drug product, by acting as an effective barrier against organic and inorganic extractables, minimizing interaction between the drug and the closure. This film is also resistant to aggressive preparations that could not be compatible with uncoated rubber materials.

In step 11, the injectable formulation vials are packaged for storage and/or transport.

EXAMPLES

Two formulations were made (i) enrofloxacin 5% injection formulation and (ii) enrofloxacin 10% injection formulation as follows: Enrofloxacin 10% iniection Quantity and/or Names of substances Function Percentage Active substance Enrofloxacin 100.0 mg Active substance Excipients n-Butanol 30.0 mg Preservative Potassium Hydroxide q.s. up to pH 10.5 -12.0 pH adjustment Water for injection q.s. up to 1.0 ml Solvent Industrial-scale batch size 10% Formulation for 2,000 L Enrofloxacin 200 Kg n-butanol 60 kg Potassium hydroxide q.s. up to pH 11.0-11.5 Water for injections q.s. up to. 2,000 L Enrofloxacin 5% injection Quantity and/or Names of substances Function Percentage Active substance Enrofloxacin 50.0 mg Active substance Excipients n-Butanol 30.0 mg Preservative Potassium Hydroxide q.s. up to pH 10.5 -12.0 pH adjustment Water for injection q.s. up to 1.0 ml Solvent Industrial-scale batch size 5% Formulation for 2,000 L Enrofloxacin 100 Kg n-butanol 60 kg Potassium hydroxide q.s. up to pH 11.0-11.5 Water for injections q.s. up to. 2,000 L

MANUFACTURING METHOD

Equipment: Stainless steel vessel, with stirrer and nitrogen bubbling Auxiliary stainless steel vessel Weighing scales under laminar flow Pre-filters and sterilizing filters Filling line located in an area with controlled air Labelling machine The process of the invention is ideally performed using a batch apparatus comprising a batch vessel having a capacity of at least 2000 L. The vessel has a top liquid inlet and a bottom liquid outlet to a delivery line. An additive line is also connected to the bottom liquid outlet and leads to a homogenizer including a pump for delivery of liquid from the additive line to the vessel inlet. An additive hopper may be connected to the additive line for delivery of excipients into this additive line. Live cells are provided from measuring the weight of the contents of the vessel.

In the process of the invention, a predetermined quantity, such as 1500 litres of water (60 to 80% of the final batch volume) is first delivered into the batch vessel. The formulation excipients to be added are introduced separately below by loading into the additive hopper, homogenising with water from the vessel and mixing by pumping around the circuit from the top inlet to the bottom outlet of the vessel.

After a batch is complete, the additive line, hopper, and associated homogeniser and pump may be easily disconnected and cleaned in situ. If required, the unit may be readily moved to another batch vessel while the first vessel is being emptied and cleaned.

The method of the invention involves the following steps: Step A -Preparation of a potassium hydroxide 50% w/w solution: 1. Weigh the required amount of water for injection.

2. In a separate vessel, add Potassium Hydroxide to part of the water for injection and stir until complete dissolution.

3. As it is an exothermic process, there is an increase of the temperature of the solution, thus, the solution is cooled to approximately 25 to 30°C.

4. The potassium hydroxide 50% w/w solution is stored until further use.

Step B -Preparation of the enrofloxacin formulation: 1. Add part of the remaining water for injection, for example from 60 to 80%, preferably 75% for injection to the vessel; 2. Start Nitrogen bubbling through the water and maintain it until the end of the process; 3. Add the pre-determined amount of n-butanol directly to the mixing vessel and stir until complete homogenization; 4. Add the pre-determined amount of Enrofloxacin in small stepwise portions and stir until complete dispersion; 5. Adjust the pH of to approximately 11.0 to 11.5 by adding small stepwise portions (aliquots) of the Potassium Hydroxide solution 50% wlw previously prepared in a stepwise fashion. pH is measured after each addition of the potassium hydroxide solution. After addition of the potassium hydroxide the formulation is stirred intensely to ensure the enrofloxacin is completely dissolved and a clear yellow liquid is obtained; 6. Check the formulation against desired product specification table to ensure the formulation has the desired appearance, density and pH.

7. If necessary adjust final volume with further purified water.

The following specifications are set for the product:

TESTS CHARACTERISTICS SPECIFICATIONS TEST METHOD

Yellow clear solution free from

Description Visual inspection

particular matter pH 10.5-12.0 Density 1.025 -1.035 g/ml Format 100 ml: 100.0-105.0 ml Extractable volume _______________________________ ___________________ Format 250 ml: 250.0 -260.0 ml Assay Enrofloxacin 95.0 -105.0 mg/mI HPLC Related substances -Impurity B (Ciprofloxacin) «= 0.5% HPLC -Any other individual impurity «= 1.0% HPLC -Total impurities «= 1.0% HPLC -Impurity A (Fluorquinolonic Acid) «= 0.2% TLC Preservative Butanol 27 -33 mg/mI GO Sterility Sterile Eur. Ph.

Stability studies carried out at 25°C and 40°C Samples of the formulation were prepared and stored at different conditions of temperature (25°C and 40°C) to check any incompatibility, degradation and/or physical changes in the proposed formulation.

Bioburden 1. Before filtration a 200m1 sample is taken from the formulation and tested to ensure that the maximum acceptable bioburden of 10 CFU/ 100 ml using standard testing procedures Step C-Filtration 1. Filter the formulation from step B,through a polypropylene pre-filter (0.6 pm) and then through a sterilizing filter (0.2 pm) made of a hydrophilic modified polyvinylidenfluoride (PVDF) membrane. The maximum flow rate through the filters is set at less than 10 mI/mm.

2. Followed by terminal sterilization in an autoclave.

Step D-Filling labelling and packaging 1. Fill the vials in the filling line with the formulation from Step C; 2. Take samples of the product for Quality Control analysis.

3. Label and pack the vials

RESULTS

ENROFLOXACIN 10% INJECTION PACKAGING SIZE: 100 mI/STORAGE CONDITIONS: 40°C ± 2°C /75 ± 5°C % R.H.

Initial 3 6

Parameters Specifications

analysis months months Yellow, clear solution free

1.-Description Complies Complies Complies

from particular matter 2.-pH 10.5-12.0 11.2 11.3 11.1 3.-Density 1.025-1.035g/ml 1.032 1.032 1.031 4.-AssayEnrofloxacin 95.0-105.0mg/mI 100.0 100.4 100.3 5-Impurity A (Fluorquinolonic ac.) «= 0.2% N.D. N.D. N.D.

6.-Impurity B (Ciprofloxacin) «= 0.5% 0.02 0.06 0.06 7.-Any other individual impurity(*) «= 1.0% 0.02 0.03 0.11 8.-Total impurities «= 1.0% 0.02 0.10 0.20 9.-Butanol 27-33 mg/mI 31 29 31 10.-Sterility test According current Eur.Ph. Complies N.P Complies NP.: Not performed in this case

N.D: Not detectable.

PRODUCT: ENROFLOXACIN 5% INJECTION PACKAGING SIZE: 100 ml STORAGE CONDITIONS: 40°C ± 2°C /75 ± 5°C % R.H.

Parameters Specifications Initial 3 months 6 months _____________________________ ______________________ analysis _________ ___________ Yellow clear solution free 1.-Description from particular matter Complies Complies Complies 2.-pH 10.5-12.0 11.0 11.1 10.9 3.-Density 1.010-1.020g/ml 1.013 1.013 1.012 4.-Assay Enrofloxacin 47.5-52.5 mg/mI 49.9 50.3 49.7 5-lmpurityA(Fluorquinolonicac.) «=0.2% N.D. N.D. N.D.

6.-Impurity B (Ciprofloxacin) «= 0.5% 0.02 0.06 0.07 7.-Any other individual impurity(*) «= 1.0% N.D. 0.03 0.04 8.-Total impurities «= 1.0% 0.02 0.10 0.10 9.-Butanol 27-33 mg/mI 30 30 30 10.-Sterility test According current Eur.Ph. Complies NP. Complies N.P.: Not performed in this case.

N.D: Not detectable.

Laboratory samples stored at 25°C and 40°C were analysed at initial and 2 months storage. No significant changes were detected between the two set points. Additionally, samples from the reference product (stored at room temperature) were also analysed and the results were very similar to those obtained in the laboratory samples. Samples stored at 40°C were analysed again after 6 months of storage and results were within

specifications.

In conclusion, the final formulation did not show any incompatibility among the ingredients of the product during the development pharmaceutics. No significant changes in the samples were observed in any of the storage conditions studied during the formulation development. This compatibility should be confirmed with stability studies according to VICH Guidelines.

The invention is not limited to the embodiments described above but may be varied within the scope of the claims.

Claims

CLAIMSI. A process for the preparation of a stable aqueous based injectable enrofloxacin formulation with a pH of II to 11.5 comprising i) from Ito 15% w/v of the active ingredient enrofloxacin, ii) from I to 5% wlv of the preservative n-butanol; iii) to 100% wlv of purified water; wherein the process takes place in a batch apparatus comprising a batch vessel having a capacity of at least 2,000 litres and comprises the following steps preparing a solution of potassium hydroxide 50% wlw and cooling the potassium hydroxide solution 50% w/w to approximately 25 to 3000; setting the batch volume of the formulation and adding up to approximately 60 to 80% of the batch volume of purified water into the batch vessel; bubbling nitrogen through the purified water in the batch vessel and maintaining nitrogen bubbling throughout the process; adding n-butanol in the range of I to 5% w/v to the water and stirring until complete homogenization; adding the active ingredient enrofloxacin in the range I to 15% wlv and stirring until complete dispersion of the active ingredient enrofloxacin; testing and adjusting the pH of the solution by adding potassium hydroxide solution 50% w/w to the solution in a stepwise manner and testing the solution to ensure a pH of II.0 to II.5 has been obtained and that complete dissolution of the active ingredient enrofloxacin has taken place; adding purified water up to 100% w/v; filtering the formulation at a maximum flow rate of less than lOmI per mm through (i) a pre-filter of 0.6 microns; and (ii) filtration through a sterilizing filter of 0.2 microns; followed by terminal sterilization; and collecting and packaging the resultant solution aseptically into separate vials.
2. The process according to claim I wherein the batch volume is 2000 litres.
3. The process according to claim I or claim 2 further comprising testing the solution after the addition of potassium hydroxide solution 50% w/w to ensure the density of the solution is from approximately 1.025 to 1.035 g/ml and that a clear yellow solution is obtained.
4. The process according to any of claims I to 3 wherein n-butanol is added at a level of approximately 3% w/v.
5. The process according to any of claims I to 4 wherein the active ingredient enrofloxacin is added in the range of 5 to 10% w/v.
6. The process according to any of the preceding claims wherein the nitrogen is applied throughout the process at a pressure below 4kg/cm2.
7. A process for preparing an aqueous based injectable enrofloxacin formulation substantially as described herein with reference to the examples and figures.