This application claims priority to Indian patent application No. 2240/CHE/2009 filed on September 16, 2009, the contents of which are incorporated by reference in their entirety

Field of the Invention

The present invention relates to solid phase peptide synthesis of Enfuvirtide on TentaGel S RAM resin in a stepwise manner without isolating any intermediates.

Background of the Invention

Enfuvirtide (also referred to as “DP-1 78’; SEQ ID NO: I or Fuzeon or T-20), a novel anti-human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 envelope protein gp4l C- terminal heptad repeat (CHR).This drug is a natural interfacial sequence taken from the gp4l moiety of the HIV precursor protein gpl6O and represents the first case of a peptide drug derived from a protein fragment. Enfuvirtide is the first Fusion Inhibitor, as well as the first HIV entry inhibitor. Its mechanism of action is different from existing Anti HIV drugs in that the existing antiretrovirals work inside T-cells to stop virus replication, but Enfuvirtide works extracellularly where it blocks Fusion between the membrane of the host immune cell T-lymphocyte and the HIV-1 virus, a late stage of the entry process. This unique function gives fusion inhibitors the potential to treat multiple- class resistant stains of HIV. Enfuvirtide consisting of 36 amino acids and is very challenging to make on a large scale synthetically and thus represents a landmark in the industrial peptide chemistry. The sequence is as follows: CH3CO-Tyr-Thr-Ser-Leu-lle-His-Ser- Leu-Ile-Glu-Glu-Ser-Gln-Asn-Gln-Gln-G)U-LYS-Asfl-Glu-GIn-GIu-Leu-Leu-GIu-Leu-Asp-LyS-TrPAla-Ser-Leu-Trp-Asn-Trp-Phe-NH 2 and the following structural formula:

O. NH %. NH O.. c44 S Peptides are synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. The possibility of unintended reactions is obvious; therefore protecting groups are usually necessary.

Chemical peptide synthesis starts at the C- terminal end of the peptide and ends at the N-terminus. This is the opposite of protein biosynthesis, which starts at the N-terminal end.

The synthesis of peptides has been described by two general methods in the literature.

The first method is a solution phase procedure, based on fragment condensation. The process involves a time consuming, multi step synthesis, repeated purification between individual steps is required, which may result in pure product but with low yield. The second method for the synthesis of peptides utilizes the entire peptide chain using solid phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield, and resulted in a paradigm shift within the peptide synthesis community. As solid phase peptide synthesis techniques improved the rate at which a peptide could be synthesized, purification became the limiting factor in the production of high- quality peptides. Purification techniques improved with the introduction of reversed phase HPLC (high performance liquid chromatography). Further, SPPS of longer peptides were synthesized by hybrid approach using solid and solution phase synthesis. The individual fragments will be separately synthesized on the solid phase and then coupled in the solution phase to build the peptide product.

Enfuvirtide is first disclosed in US 5464933. This patent discloses that amidated peptides can be prepared using Rink resin (Advanced Chemtech) while peptides containing free carboxy termini were synthesized on Wang (p-alkoxy-benzyl-alcohol) resin (Bachem).

First residues were double coupled to the appropriate resin and subsequent residues were single coupled. Each coupling step was followed by acetic anhydride capping. Peptides were cleaved from the resin by treatment with trifluoroacetic acid (TFA) (10 ml), H20 (0.5 ml), thioanisole (0.5 ml), ethanedithiol (0.25 ml) and crystalline phenol (0.75 g). Purification was carried out by reverse phase HPLC.

Tarn, J. et al.(Dep. Microbiology Vanderbilt University; Organic Letters, 2002, Vol.4: 4167- 4170) describes inter alia, the preparation of DP-178 and related 30-mer peptides with the purpose of preparing three-helix bundles. Authors just mentioned that a general FMOC synthesis method is applied, giving the coupling reagents and resin cleavage conditions used. Nothing is said on the type of resin and/or resin- handle employed, as well as the excess of reagents used. No chromatogram is showed and no purification details or final purity is given. No indication of yields obtained is given and are entirely left subject to speculation. Since the work pertained only to labscale experimentation, no apparent need for devising an efficient synthetic process was given though.

W020061 08594 application discloses the process of Enfuvirtide using PEG resin. According to this process said peptide which may have protected and/or unprotected amino acid side chains is prepared bound to a solid phase which is an amphophilic PEG resin and wherein the peptide comprises a prolin-free, helix-forming segment consisting of a run of six contiguous amino acid residues comprising at least four protected or unprotected helix-forming amino acids residues.

Enfuvirtide synthesis typically utilizes both solid and liquid phase procedures to synthesize and combine groups of specific peptide fragments to yield the Enfuvirtide product (Bray, B. L., Nature Rev., 2:587-593 (2003)). Further, US 6281331 disclosed an efficient large scale production achieved by a combination of partial synthesis of segments of T-20 on solid phase followed by segment condensation typically in the liquid phase.

Enfuvirtide was prepared by linear SPPS on rink amide resin. However the poor crude purities ranging 30-40% and low overall yields (6-8%) from this route were unacceptable for further development. Later an analog of Enfuvirtide T-999 a H(V Fusion peptide was synthesized on stepwise manner using Sieber amide resin. Efficient full-length synthesis on solid-phase has not been demonstrated so far, such approach typically failing to produce more proper product than useless side products, purification of impurities posing further problems and again diminishing final yields. An improved route was needed for the synthesis of Enfuvirtide. Our initial attempts to use rink amide resin (100-200 mesh, 1.lmmol/g) for the synthesis of Enfuvirtide were unsuccessful. Alternatively, the use of Fmoc-rink amide MBHA resin was led to poor yields and purity.

The present invention relates to an improved process for the preparation of Enfuvirtide using TentaGel SRAM resin. It involves one pot synthesis of Enfuvirtide without isolating any intermediate. After the initial successful synthesis, the linear synthesis on TentaGel S RAM was repeated at a scale of 50g resin for further evaluation. This built behaved similarly to the small scale trial with crude purity of 68%.
Obiect of the Invention

The main object of the present invention is to provide an improved process for the preparation of Enfuvirtide.

Another object of the present invention is to provide an improved process for synthesis of Enfuvirtide or pharmaceutically acceptable salts using TentaGel S RAM.

Another object of the present invention is to provide an easy coupling of amino acids by using DIC/HOBt Yet another object of the present invention is to provide a process to get directly C-terminal amide on cleavage of the peptide from the resin.

Summary of the Invention

The main aspect of the invention is to provide an improved process for the preparation of Enfuvirtide or its pharmaceutically acceptable salts, which comprises coupling of amino acids optionally having protecting groups on TentaGel S RAM in peptide sequence to give a compound of formula (IV) which is cleaved from the resin with simultaneous deprotection of protecting groups to give a crude compound of formula V, which is purified to give an Enfuvirtide and if required it may converted to its pharmaceutically acceptable salt.

Another aspect of the present invention is to provide a coupling procedure which is faster, efficient, racemization free and does not require addition of any base during coupling. The formed byproduct can be removed by simply washing with solvent.

Another aspect of the present invention is to provide an improved process by precipitation or crystallization of the peptide after cleavage to get more pure Enfuvirtide.
Yet another aspect of the present invention is to provide the process to get directly C-terminal amide on cleavage of the peptide from the resin which is a required functional group for Enfuvirtide.

Detailed Description of the Invention

For the purpose of clarity and as an aid in the understanding of the invention, as disclosed and claimed herein, the following terms and abbreviations are defined below AcOH acetic acid

Soc tert-butyloxycarbonyl
tBu tert-butyl
DCM Dichloromethane
N, N’-diisopropyfcarbodjjmjde
DMF N, N’-Dimethylformamide
Fmoc 9-fluorenylmethoxycarbonyl
HOBt N-hydroxybenzotriazole
MTBE Methyl tert-butyl ether
SPPS solid phase peptide synthesis
TFA trifluoroacetic acid
TIS trilsopropylsilane
Trt trityl

As used herein, the term ‘Capping’ relates to a process of protecting free functional groups on the polymeric resin as acetyl or ester form.

The present invention relates to an improved process for large scale production of Enfuvirtide or its pharmaceutically acceptable salts with increased yield and purity on

TentaGel S RAM resin without isolating any intermediate.

The synthesis of the present invention is preferably carried out in the presence of appropriate protecting groups unless otherwise noted.

In one embodiment of the present invention, the process for the preparation of Enfuvirtide comprises the steps of:

a) swelling of the TentaGel S RAM resin in a solvent;

b) anchoring first protected terminal amino acid to a resin;

c) capping the resin obtained in step b);

d) selective deprotection of the amino group;

e) coupling carboxyl terminus of the next N-protected amino acid to the amine group;

f) repeating step c, and d to form a peptide sequence;

g) cleaving the peptide from the resin as well as side chain protections using a cocktail mixture;

h) enriching the crude Enfuvirtide purity by precipitation or recrystallisation; and

i) purifying the enriched Enfuvirtide to give pure Enfuvirtide acetate.

In another embodiment, the specifications of the resin used for the SPPS is as below

According to the present invention this TentaGel S RAM resin utilized in the process for the preparation of Enfuvirtide acts as a support material. The selection of polymeric support and attached linker is very critical for overall outcome of the solid phase peptide synthesis. The TentaGel S RAM resin is very effective for the preparation of Enfuvirtide and is comprising of grafted copolymers consisting of a low cross linked polystyrene on which the polyethylene glycol is grafted. This resin found to improve efficiency of difficult peptide coupling steps and unexpectedly enhancing the purity and yield. It is found that the synthesis is very fast and efficient. The yields and purity are good; there is no aggregate or difficult couplings observed during synthesis. There is no major truncated peptide observed and all the couplings went smoothly with single addition! coupling.

In another embodiment, the resin used for synthesis of peptide undergoes swelling in presence of solvent selected from methylene chloride, tetrahydrofuran, N,N-dimethylformamide, N-methyl pyrrrolidone or mixture thereof. The resin is then treated with N-terminus protected amino acid in presence of coupling agent for a desired period of time to affect the coupling.

In yet another embodiment, deprotection of the protected amino acid anchored to the resin is done selectively in the presence of nucleophilic base such as 20% piperidine in dimethylformamide, methylene chloride or N-methyl pyrrolidine.

In yet another embodiment, before proceeding to next step, the unreacted linkers on the resin (polymer) is protected to avoid the undesired peptide chain formation. Preferably the free groups on resin are protected with acetylatirig reagents such as acetic anhydride in a solvent such as dichloromethane in the presence of a base such as pyridine. This process of capping is performed after anchoring the first protected amino acid to resin.

In yet another embodiment, the coupling agents used for the coupling are selected from DIC and HOBt or HOBt, HBTU and DIEA. The amount of protected amino acid used in the present invention is selected from 1 M to 3M with respect to resin loading capacity.

Resin Particle size

(pm) Matrix Loading

TentaGel SRAM 90 Poly(oxyethylene)-RAM

Polymer bound 0.24 mmol/g

In yet another embodiment, the coupling reaction is carried out in the presence of solvents selected from dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrolidone or mixtures thereof.

In yet another embodiment, the resin after completion of the reaction is optionally washed with solvents such as DMF and 0CM to remove residual reagents and byproducts.

In yet another embodiment, adding a solution of next Fmoc amino acid that has been preactivated with DIC/HOBt. Coupling efficiency after each coupling step should be controlled during the synthesis by means of Ninhydrin test and individual couplings showing unexpected low coupling efficiency should be repeated prior to continuing with further cycle of deprotection and coupling. The process is repeated till the completion of all the amino acids in stepwise manner and the reactions were monitored using Kaiser
Test.

The functional group present on the amino acids used in the process of the present invention may be appropriately protected to avoid any undesired by products. Suitable protecting groups are described in the literature (See for example, P Wuts,and T.W. Greene, Protective Groups in Organic Synthesis John Wiley & Sons, 4th Edition 2007). The protecting group may vary depending upon the particular amino acid which may include but are not limited to tBu or Trt or BOC.

In yet another embodiment, the N-terminus is acetylated on resin with acetylating agent such as acetic anhydride in presence of a base such as pyridine before cleavage of peptide from the resin.

In yet another embodiment, cleavage of the peptide from the resin and deprotecting the orthogonal functional protecting groups on amino acid groups is carried out using TFA and a cocktail of carbocation scavengers to provide crude Enfuvirtide with an HPLC purity of approximately 70%. Cleaving the peptide from the resin involves treating the protected peptide anchored to the resin with an acid having at least one scavenger.

According to the present invention, the peptide cleavage and global deprotection reagents used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents. Scavengers are selected from EDT, DDM, TIPS, TES, Phenol, thioanisole or mixture thereof. The cocktail mixture used for the reaction is selected from group comprising of TFAITIS IThioanisole/DCMIEDT and water in the preferred volumes of 85%12. 5%12. 5%12. 5%12.5%15% respectively.

In yet another embodiment, after the completion of the reaction, the reaction mixture may optionally be filtered and washed with acid or an organic solvent. The crude Enfuvirtide is isolated by combining the reaction mass with an organic solvent; the organic solvent is selected form diethyl ether, isopropyl ether, tert-butyl methyl ether, tert-butyl ethyl ether, isopropyl ether, ethyl acetate or mixture thereof.

In yet another embodiment, purification of the crude Enfuvirtide is carried out by preparative RPHPLC to produce pure Enfuvirtide or its pharmaceutically acceptable salts.

According to the present invention, crude Enfuvirtide is dissolved in methanol and loaded on C18 column (50 X 250 mm, 100 A) using a linear gradient of aqueous TFA (0.035%) and acetonitrile (0.045% TFA) from 40% to 60% over 45 mm. Pure product is isolated either in the form of Enfuvirtide by evaporation of acetonitrile followed by lyophilization or in the form of Enfuvirtide acetate by evaporation of the acetonitrile, passing the aqueous layer through ion exchange column using 10% acetic acid in methanol, evaporation of the methanol followed by precipitation from organic solvent . The organic solvent is selected from diethyl ether, isopropyl ether, tert-butyl methyl ether, tert-butyl ethyl ether, isopropyl ether, preferably isopropyl ether.

In yet another embodiment, the Enfuvirtide fractions were analyzed by reverse phase HPLC by preparative HPLC method using 30-70% solvent B (0.036% TFA in Acetonitrile) in solvent A (0.045% TFA in water) over 50 mm at 1.Oml/min using a diasopak SP 5micron 4.6x 250mm column and UV Detector at 220nm.

According to the present invention, the purity of the Enfuvirtide is not less than 98.0%, preferably not less than 99.0%.

MALDI-TOF and LC-MS analysis of peptide samples were performed in Agilent technologies accurate mass Q-TOF LCMS InstrUment.

In still another embodiment, preparation of the pharmaceutical composition comprises a
therapeutically effective amount of Enfuvirtide, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier for treatment of AIDS or HIV infection.

“Therapeutically effective” means the amount of agent required to provide a meaningful patient benefit as understood by practitioners in the field of AIDS and HIV infection. In general, the goals of treatment are suppression of viral load, restoration and preservation of immunologic function, improved quality of life, and reduction of HIV-related morbidity and mortality.

The present invention schematically shown below

The process for the preparation of enfuvirtide is summarized in synthetic scheme-I depicted below:

TentaGel SRAM
Fmoc-NH-Linker 1
20% Piperidine in DMF
2. Fmoc-Phe-OI-l
3. DIC/HOBt
Fmoc-Phe-NH- (II)
1. 20% Piperidine in DMF
2. Fmoc-Trp(Boc)-OH
3. DIC/HOBt
Fmoc-Trp(Boc)-Phe-NH- (Ill)
Repeat Steps 1&2

Ac-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-IIe-His(Trt)-Ser(tBu)-Leu-IleGlu(OtBu)-Glu(OtBu)-Ser(tBu)-GIn(Trt)-Asn(Trt)-GIn(Trt)-Gln(Trt)GIu(OtBu)-Lys(Boc)-Asn(Trt)-GI u(OtBu)-Gl n(Trt)-Glu(OtBu)-LeuLeu-GIu(OtBu)-Leu-Asp(OtBu)-Lys(B
-Trp(Boc)-Ala-Ser(tBu)-Leu Trp(Boc)-Asn(Trt)-Trp(Boc)-Phe-N (IV)

Cleavage Cocktail

95%TFA)TIS/ThioanisoleED

T/Water

Recrystallization

Ethanol/Ethyl Acetate

Ac-Tyr-Thr-Ser-Leu-lle-His-Ser-Leu-lle-Glu-GIu-Ser-Gln-Asn-GlnGln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-AlaSer-Leu-Trp-Asn-Trp-Phe-NH2
(V)

Purification using Prep Salt exchange using ion exchange resin

Enfuvitride Acetate

Examples

Preparation of Enfuvirtide (N-Acetyl-L-tyrosyl-L-threonyl-L-seryl-L-leucyl-L-isoleucyl-L-histadyl-Lseryl-L-leucyl-L-isoleucyi-L-glutaminyl-L-glutamyl-L-seryl-L-glutaminyl-L-asparaginyl-L-glutaminylL-glutaminal-L-giutamyi-L-lysyl-L-asparaginyl-L-giutamyl-L-glutaminyl-L-glutamyl-L-leucylL-leucyl-L-glutamyl-L-leucyl-L-aspartyl-L-lysyl-L-tryptophyl-L-alanyl-L-seryl-L-ieucylL-tryptophyl-L-asparginyl-L-tryptophyl-L-phenyialaninamide)

Manual synthesis

TentaGel S RAM Resin (5g, 1.2 mmol loading 0.24 mmol/g) was swelled in dichloromethane (30 mL) for about 2h, followed by DMF (30 mL) for 2 h. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF. The Fmoc-Phe(0.93 g 2.4 mmol) was added to initiate the first coupling step. The coupling was carried out using HOBt (0.33 g, 2.4 mmol), DIC (0.37mL, 2.4 mmol) in DMF (40 mL). The completion of the coupling was confirmed by Ninhydrin test. After washing of the resin, the Fmoc protecting group was removed with 20% piperidine in DMF. The resin was repeatedly washed with DMF and DCM before the addition of the next amino acid. These steps were repeated each time with successive amino acid according to the peptide sequence. After the complete synthesis of the linear peptide the N-terminal Fmoc group is removed and acetylated using acetic anhydride/pyridine. All amino acids used were Fmoc protected. Trifunctional amino acids side chain were protected as follows: Lys (Boc) Asn (Trt), Gin (trt), Tyr (t-Bu), His (Trt), Ser (t-Bu), Glu (Ot-Bu), Asp (Ot-Bu), Trp (Boc) and Thr (t-Bu).

The cleavage of the peptide from the resin with simultaneous deprotection of the protecting groups was done by treating with cleavage mixture (TFA (85%); TIS (2.5%); thioanisole (2.5%); DCM (2.5%), EDT (2.5%) and water (5%)) at room temperature for 2 h. The cleavage mixture was collected by filtration. The resin was washed with TFA and Dichioromethane. The product was precipitated by the addition of 10 volumes of MTBE. The precipitate was filtered and washed several times with MTBE, ethyl acetate and dried, HPLC purity-70%. The identity of the peptide was confirmed by LC-MS (m/z [M+2H] 2+ 2246.0, [M+3H] 2+ 1498.0 and [M+4H] 2+ 1123.0) MALDI (4492.0) and RP-HPLC (RT 31.5 was 70 % purity).

Synthesis using SPP Synthesizer

General Procedure

The assembly of the peptide chain was carried out in the following manner. The resin was transferred to the RV of the peptide synthesizer (Advance chemtech Model 90) and the linear peptide assembled on it using 1.5 times mole excess amino acid derivatives, on the peptide synthesizer. The first amino acid, Fmoc-Phe-OH, was coupled to the resin by deprotecting the Fmoc group on the resin, followed by activating the Fmoc-Phe-OH by DICIHOBt. For coupling of next amino acid, Fmoc-Trp (Boc)-OH, the Fmoc group of first amino acid was deprotected followed by activating the Fmoc-Trp (Boc) by DIC/HOBt. This process is repeated with all the amino acids till the entire linear peptide chain is assembled on the solid support. Each coupling was carried out for 90 mm. After couplings are complete, the resin was washed with organic solvent preferably DMF followed by DCM, and then dried under vacuum.

Cleavage of the peptide from the resin to yield peptide amide

The assembled peptide resin (15 g) was treated with 200 mL of cleavage cocktail consisting of TFA (85%); TIS (2.5%); thioanisole (2.5%); DCM (2.5%) EDT (2.5%) and water (5%) for 2 h. at room temperature. The reaction mixture was filtered and precipitation of the peptide was carried out by the addition of 500 mL of cold MTBE with constant stirring. The crude peptide was isolated by filtering through sintered funnel followed by washing with cold MTBE (100 mL x 3) to remove the scavengers used in the cleavage cocktail. The crude peptide precipitate is recrystallized using Methanol or Ethanol-IPE, dried under vacuum, yield - 5 g and HPLC purity - 65%. Further purification of the peptide can be achieved by treating with acetonitrile at 50°C for one hour (slurry). The identity of the peptide was done by HPLC and confirmed by LCMS.
Preparative HPLC Purification

The crude Enfuvirtide was dissolved in methanol loaded on to preparative C18 column (50X250mm, 100 A°). The peptide was purified using a linear gradient of aqueous TFA (0.035%) and acetonitrile (0.045% TFA) from 40% to 60% over 45 mm.. The pure fraction containing the Enfuvirtide was pooled. The Acetonitrile was evaporated and the aqueous layer was lyophilized to give the Enfuvirtide as white solid. Alternatively after evaporation of the acetonitrile the aqueous layer was passed through ion exchange column for desalting using 10% acetic in methanol .The Methanol was evaporated and the peptide precipitated from IPE. The peptide analyzed by RPHPLC and confirmed by MALDI or LC-MS.

Purity by HPLC -> 99%

Claims

A process for the preparation of Enfuvirtide comprising the steps of:

a) swelling of the TentaGel S RAM resin in a solvent;

b) anchoring first protected terminal amino acid to a resin;

c) capping the resin obtained in step b);

d) selective deprotection of the amino group;

e) coupling carboxyl terminus of the next N-protected amino acid to the amine group;

f) repeating step c, and d to form a peptide sequence;

g) cleaving the peptide from the resin as well as side chain protections using a cocktail mixture;

h) enriching the crude Enfuvirtide purity by precipitation or recrystallisation; and i) purifying the enriched Enfuvirtide to give pure Enfuvirtide acetate.

2. The process according to claim 1, wherein said resin specifications are as below

3. The process according to claim 1, wherein said solvent used in step a) for swelling of resin is selected from methylene chloride, tetrahydrofuran, N, N-dimethylformamide, Nmethylpyrrrolidone or mixture thereof.

4. The process according to claim 1, wherein said capping in step c) is carried out with acetylating reagents such as acetic anhydride in a solvent such as dichloromethane in the presence of a base such as pyridine.

5. The process according to claim 1, wherein said reagent used for the deprotection in step d) is selected from nucleophilic base such as 20% piperidine.

6. The process according to claim 1, wherein said solvent in step d) is selected for the deprotection is selected from dimethylformamide, methylene chloride or N-methyl pyrrolidine.

7. The process according to claim 1, wherein said coupling agent used in step e) is selected from DIC and HOBt or HOBt, HBTU and DIEA.

8. The process according to claim 1, wherein said solvent in step e) is selected from dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrolidone or mixtures thereof.

9. The process according to claim 1, wherein next Fmoc amino acid in step f) is pre-activated with DIC/HOBt during peptide sequence formation.

10. The process according to claim 1, wherein said cocktail mixture used for cleavage of the reaction in step g) is selected from group comprising of TFAITIS /Thioanisole/DCM/EDT and water in the preferred volumes of 85%12.5%/2.5%/2.5%12.5%/5% respectively.

11. The process according to claim 1, wherein said solvent used for the precipitation or recrystallisation in step h) is selected from diethyl ether, isopropyl ether, tert-butyl methyl ether, tert-butyl ethyl ether, isopropyl ether, ethyl acetate or mixture thereof.

12. Purification of Enfuvirtide is carried out by preparative RP-HPLC using 30-70% solvent B (0.036% TFA in Acetonitrile) in solvent A (0.045% TFA in water) over 50 mm at 1 .Oml/min using a diasopak SP 5micron 4.6x 250mm column and UV Detector at 220nm, to produce pure Enfuvirtide or its pharmaceutically acceptable salts.

13. Enfuvirtide with purity more than 98.0%, preferably not less than 99.0%.