CN103965291B - Octreotide, the preparation method of octreotide acetate - Google Patents

Abstract

The invention discloses octreotide, the preparation method of octreotide acetate.The preparation method of this octreotide comprises the steps: to use high-efficient liquid phase reverse-phase chromatography that octreotide crude product solution carries out anti-phase purification, anti-phase desalination successively,；The filler of high-efficient liquid phase reverse-phase chromatography is SDVB (PS DVB) copolymer.Anti-phase purification and anti-phase desalination are combined by the present invention, design the more recent application of polymer filler SDVB, can be prepared on a large scale octreotide and octreotide acetate.

Description

Octreotide, the preparation method of octreotide acetate

Technical field

The present invention relates to field of biological pharmacy.More particularly it relates to octreotide, the preparation method of octreotide acetate.

Background technology

Angiotensin (or angiotensinamide) is for increasing Cardura, English name angiotensinamide or [Asn¹, Val5]-angiotensin II, chemical constitution is L-Asn-L-Arg-L-Val-L-Tyr-L-Val-L-His-L-Pro-L- Phe, the synthesis polypeptide being made up of eight amino acid residues, theoretical molecular 1031.18.Angiotensin is used for wound or hands Low blood pressure etc. caused when postoperative shock and general anesthesia or lumbar anesthesia, can prevent or treat peri-operation period hypotension, makees For first aid using medicine treatment shock stage hypotension it can also be used to treatment angiotensin converting enzyme inhibitor over administration and routine are controlled When treating invalid.

Pitressin Tannate, the synthesis polypeptide being made up of nine amino acid residues, its chemical constitution is L-Cys-L-Tyr- L-Phe-L-Gln-L-Asn-L-Cys-L-Pro-L-Arg-L-Gly (1 → 6)-disulfide bond tannate, the Theoretical molecular of vassopressin Amount 1084.24.Pitressin Tannate is vassopressin medicine, can promote that the heavily absorption of moisture is had by distal renal tubular and collecting tubule Having antidiuretic activity, its preparation is clinically used for treating diabetes insipidus.

Octreotide acetate (Octreotide acetate), the conjunction being made up of seven amino acid residue and Soviet Union's ammonia alcohol Become polypeptide, chemical constitution be D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl- N-[2R-hydroxyl-1R-(methylol) propyl group]-L-cysteinyl amine ring (2 → 7)-disulfide bond acetate, the theory of octreotide is divided Son amount is 1019.26, and in its crude drug lyophilized powder, acetic acid content is 5%～12%.Octreotide acetate can Developing restraint hormone, press down Gastrointestinal pancreas secrete polypeptides processed etc., its preparation is clinically used for needing the activeness acromegaly of long-term RUPTURED ESOPHAGEAL VARICES TREATED WITH OCTREOTIDE, such as art Rear growth hormone residual causes serum growth hormone level and increases, and not yet reaches the blood of abundant curative effect after hypophysis external-bean radiation therapy Clear level of growth hormone raises, and part is not suitable for operative treatment and the new preoperative therapy diagnosing growth hormone secreting adenoma patient.

Having listed the common purification process of polypeptide drugs mostly have employed preparative high performance liquid chromatography at present, and this method is to obtain The most efficient method of high-purity polypeptide target molecule, be also our solid phase synthesis angiotensin purification and main skill of desalination Art means.General polypeptide drugs purification preparation technology design is first mesolow chromatograph enrichment target polypeptides, then high pressure chromatographic refining, It is contemplated that our target polypeptides angiotensin molecular weight about 1kD, without suitable molecular sieve gel post (its applied sample amount is little, Flow velocity is low, and treating capacity is little, is relatively suitable for the molecular weight desalination more than 10kD albumen) or ultrafilter membrane selection.And mesolow chromatograph In conventional separation method have molecular sieve chromatography, ion exchange chromatography and hydrophobic interaction chromatography, these chromatograph sides The particle diameter of the filler used in method generally from tens microns to hundreds of micron, void size mostly is hundreds of nanometer, nothing Method obtains highly purified target polypeptides.If using high eluting salt, later stage process cannot find suitable desalination scheme, separates pure Changing effect the most limited, sample loss is very big, and the response rate calculates the most complicated, and preparation anti-phase for the later stage is the most unfavorable.The most scarce A kind of weary method effectively preparing polypeptide salt bulk drug, the most still in the urgent need to developing the purifying process of new polypeptide salt.

Summary of the invention

The technical problem to be solved is to overcome the complicated process of preparation of polypeptide salt, receipts in prior art The defect that rate is low, and provide a peptide species, the preparation method of polypeptide salt.The preparation method of the polypeptide salt of the present invention with polypeptide is Initiation material, through high pressure Reverse phase chromatography and desalting steps, prepares highly purified polypeptide salt, particularly angiotensin vinegar The method of hydrochlorate, Pitressin Tannate and octreotide acetate.

Inventor finds through research repeatedly, and the filler of current high pressure reverse-phase chromatography classics purification is silica matrix, Its post is imitated, and resolution is high, but chromatographic condition pH2-7 tolerance is narrower；And Agilent polymer filler PLRP-S is styrene-two Vinyl benzene (PS-DVB) copolymer, can tolerate broader pH scope (pH scope is up to 1-14), and available 1M NaOH is molten Liquid regenerates, it is also possible to be precisely controlled pore size and aperture form so that the accessible diffusion of solute molecule, relative to silica gel base Matter, which increases effective surface area, can keep good post effect and resolution, and improve the volume containing the sample of filler.

The present invention is to solve above-mentioned technical problem by the following technical programs:

The invention provides the preparation method of a peptide species, it comprises the steps: to use high-efficient liquid phase reverse-phase chromatography Polypeptide crude product solution is carried out successively anti-phase purification, anti-phase desalination,；The filler of high-efficient liquid phase reverse-phase chromatography is benzene second Alkene-divinylbenzene (PS-DVB) copolymer.

Wherein, described polypeptide crude product can be each peptide species crude product of this area, preferably basic polypeptide crude product, more preferably Ground is angiotensin crude product, vassopressin crude product or octreotide crude product.Described angiotensin crude product is preferably patent Shen Please the disclosed angiotensin crude product using Solid phase synthesis of CN201210131066.5, HPLC purity is 80～90%；Institute The vassopressin that the vassopressin crude product stated is preferably patent application CN201210131067.X disclosed employing Solid phase synthesis is thick Product, HPLC purity is 80～90%；Described octreotide crude product preferably uses the octreotide crude product that Solid phase synthesis prepares, HPLC purity is 80～90%.

Described polypeptide crude product solution preferably polypeptide crude product is dissolved in the acetonitrile solution that concentration of volume percent is 5% The 10g/L solution formed.

Wherein, the condition of described anti-phase purification is preferably as follows: mobile phase A be percent by volume be the trifluoro second of 0.1% Aqueous acid, Mobile phase B be percent by volume be the trifluoroacetic acid acetonitrile solution of 0.1%, flowing phase C be described polypeptide crude product Solution, flow velocity is 180～220mL/min (preferably 200mL/min), and detection wavelength is 220nm；

Carrying out online loading, eluting according to the condition of following table, percentage ratio is percent by volume；

Elution step	Elution time	Eluent
			1	0～2min	95%A+5%B
2	2～22min	100%C
			3	22～40min	95%A+5%B
4	40～100min	95%A+5%B → 65%A+35%B
			5	100～110min	40%A+60%B
6	110～120min	95%A+5%B

Collect the eluent that retention time is 45～95min and obtain polypeptide-trifluoroacetic acid solution.

Wherein, the condition of described anti-phase desalination is preferably as follows: mobile phase A is water, and Mobile phase B is acetonitrile, and flowing phase C is 0.1mol/L NaOH solution, flow velocity is 180～220mL/min (preferably 200mL/min), and detection wavelength is 220nm；

Carrying out online loading, eluting according to the condition of following table, percentage ratio is percent by volume；

Elution step	Elution time	Eluent
			1	0～5min	95%A+5%B
2	5～45min	75%A+25% polypeptide-trifluoroacetic acid solution
			3	45～55min	95%A+5%B
4	55～75min	75%A+5%B+20%C
			5	75～95min	95%A+5%B
6	95～155min	95%A+5%B → 65%A+35%B
			7	155～175min	40%A+60%B
8	175～180min	95%A+5%B

Collect the eluent that retention time is 100～155min and i.e. can get polypeptide solution.

Wherein, the filling condition of described high-efficient liquid phase reverse-phase chromatography is preferably as follows: filler is Agilent PLRP-S benzene Ethylene-divinylbenzene (PS-DVB) copolymer, aperture 10nm, particle diameter 10 μm, fill column density 0.33g/mL, post pressure is 650psi。

Present invention also offers the preparation method of polypeptide salt, it comprises the steps: to obtain many by above-mentioned preparation method Peptide solution mixes with acid,.

Wherein, described acid is preferably glacial acetic acid or tannic acid.

In the present invention, after described mixing terminates, the most also carry out concentrating under reduced pressure, lyophilization, i.e. can get polypeptide Salt lyophilizing powder body.The pressure of described concentrating under reduced pressure preferably-0.15～-0.05MPa, the temperature of described concentrating under reduced pressure is relatively It is 25～35 DEG C goodly.

Wherein, described polypeptide is preferably angiotensin, vassopressin or octreotide.

Wherein, described polypeptide salt is preferably angiotensin acetate, vassopressin tannate or octreotide acetate.

Angiotensin, vassopressin and octreotide are all a kind of peptide materials, unstable in the basic conditions, degradable, Especially under strong alkali environment, concentration that integrated survey of the present invention alkali cleaning is de-and time, to ensure to reduce sample in desalination processes Destruction and loss.

Innovative point of the present invention is to be combined anti-phase purification and anti-phase desalination, containing an alkalescence essence ammonia in angiotensin Acid residue (its guanidine radicals side chain pKa value is 12.48) and an alkaline histidine residues, containing an alkaline arginine in vassopressin Residue, then contains the residue of a basic lysine in octreotide, this polypeptide combines TFA (trifluoro second under strongly acidic conditions Acid) very capable, and may have multiple alkaline residue site can be in conjunction with, need highly basic could TFA remove with displacement, for It is prepared on a large scale and desalinating process, designs the more recent application of polymer filler PLRP-S.Novelty of the present invention has been used instead Phase absorption method desalination, uses on-line dilution loading to solve problem associated with anti-phase purification and anti-phase desalination.

On the basis of meeting common sense in the field, above-mentioned each optimum condition, can combination in any, obtain the preferred embodiments of the invention.

Agents useful for same of the present invention and raw material are the most commercially.

The most progressive effect of the present invention is:

(1) method that present invention utilizes acid-base neutralization, first with in highly basic and strong acid, utilizes dredging of polypeptide and reverse phase filler Water combines, the strong acid root that neutral eluting is free on a column, and the strong acid root using in Alkaline Elution and combining, preparation deacidification Polypeptide solution.

(2) present invention uses online loading, and without dilute sample solution, directly in liquid phase systems pipeline, biased sample is molten Liquid and mobile phase A, Mobile phase B, be loaded in chromatographic column, it is to avoid after dilution, sample volume is excessive, what concentration was diluted asks Topic, is suitable for continuous print and produces.

(3) present invention has used reverse phase absorption method desalination innovatively, uses on-line dilution loading to solve anti-phase purification With anti-phase desalination associated with problem, optimize production technology, applicable industrialization produces continuously.

Detailed description of the invention

Further illustrate the present invention below by the mode of embodiment, but the most therefore limit the present invention to described reality Execute among example scope.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or according to business Product description selects.

Embodiment 1 (HPLC method detection angiotensin crude material and purification midbody solution purity and quantitative)

Instrument: Waters2695/2489 high performance liquid chromatograph

Detached dowel: Waters XBridge-C18,4.6 × 150mm, 5 μm

Flowing phase: A is 20mM NaH₂PO₄-H₃PO₄(pH3.0), B be percent by volume be the acetonitrile solution of 50%

Flow velocity is 1.0mL/min, and detection wavelength is 220nm, and room temperature detects,

Gradient see table shown in 1, and percentage ratio is percent by volume.

Table 1 flows phase gradient

Elution step	Elution time	Eluent
			1	0～7min	90%A+10%B → 60%A+40%B
2	7～13min	60%A+40%B → 50%A+50%B
			3	13～35min	50%A+50%B → 100%B
4	35～40min	100%B
			5	40～45min	90%A+10%B

Embodiment 2 (75mm internal diameter L&L4003 prepares post filling)

Using Load&Lock dynamic axial compression and static locking technology, filler is styrene-divinylbenzene copolymer (reverse phase filler Agilent PLRP-S), aperture 10nm, particle diameter 10 μm, fill column density 0.33g/mL, be filled to post bed pressure 650psi, uses Varian chromatograph loading system, 370g dry powder filler, after the stirring homogenate of 2L methanol, pours 75mm internal diameter L& into L4003 prepares post, and compression ratio is 3:1, and carrier gas is N₂, regulation nebulizer gas pressure makes oil gauge pressure be 2000psi, dynamic axial It is compressed to post bed height 26cm, as preparing post used by anti-phase purification and anti-phase desalination scheme.

Embodiment 3

(1) the anti-phase purification of angiotensin crude material

Instrument: Varian SD-1 high-pressure liquid phase preparation system

Chromatographic column: embodiment 2 self-chambering prepare post Load&Lock400375 × 260mm, PLRP-S10 μm 10nm

Flowing phase: A be percent by volume be the trifluoroacetic acid aqueous solution of 0.1%, B be percent by volume be 0.1% trifluoro Acetic acid acetonitrile solution, angiotensin crude product (the disclosed blood using Solid phase synthesis of patent application CN201210131066.5 Angiotensin crude product) it is that solvent dissolves and is configured to 10g/L solution through the acetonitrile solution of percent by volume 5%, filter clarification Rear sample liquid is flowing phase C, and measuring its HPLC purity as described in Example 1 is 84.98%, retention time is 10.34min, HPLC data are shown in Table 2.

The peak result of table 2 angiotensin crude product HPLC detection

Peak is numbered	Retention time (min)	Area (microvolt * second)	Highly (microvolt)	% area
					1	5.723	114660	9972	0.68
2	6.470	13557	1592	0.08
					3	7.598	9823	899	0.06
4	8.133	76178	7881	0.45
					5	8.738	67006	7818	0.40
6	9.088	8971	1327	0.05
					7	9.631	46650	4709	0.28
8	10.335	14357014	1242791	84.98
					9	10.988	276934	44721	1.64
10	11.138	1118879	118916	6.62
					11	11.817	131299	8221	0.78
12	12.251	21447	2115	0.13
					13	12.541	30951	3707	0.18

The anti-phase purification condition of the present embodiment is as follows: flow velocity 200mL/min, 220nm detect, and purification gradient see table Shown in 3, it is that 56.5-62min target main peak is collected as angiotensin-trifluoroacetic acid solution to retention time, by implementing It is 98.89% that the method for example 1 measures its HPLC purity, and retention time is that 10.51min, HPLC data see table 4.

The gradient (percentage ratio is percent by volume) of the anti-phase purification of table 3

Elution step	Elution time	Eluent
			1	0～2min	95%A+5%B
2	2～22min	100%C
			3	22～40min	95%A+5%B
4	40～100min	95%A+5%B → 65%A+35%B
			5	100～110min	40%A+60%B
6	110～120min	95%A+5%B

The peak result of table 4 angiotensin-trifluoroacetic acid solution HPLC detection

Peak is numbered	Retention time (min)	Area (microvolt * second)	Highly (microvolt)	% area
					1	5.706	3447	360	0.02
2	8.863	7995	949	0.06
					3	9.808	14220	1760	0.10
4	10.514	13765923	1133552	98.89
					5	11.191	129102	13699	0.93

(2) the anti-phase desalination of angiotensin-trifluoroacetic acid solution

Instrument: Varian SD-1 high-pressure liquid phase preparation system

Chromatographic column: embodiment 2 self-chambering prepare post Load&Lock400375 × 260mm, PLRP-S10 μm 10nm

Flowing phase: mobile phase A is pure water, and Mobile phase B is acetonitrile, flowing phase C is 0.1M NaOH solution, flow velocity 200mL/ Min, 220nm detect, and the gradient of anti-phase desalination is shown in Table 5, and is that 120-132min target main peak is carried out to retention time Being collected as angiotensin and remove TFA solution, measuring its HPLC purity by method in embodiment 1 is 99.51%, and retention time is 10.39min, HPLC data are shown in Table 6.

Gradient (percentage ratio is percent by volume) used by the anti-phase desalination of table 5

Elution step	Elution time	Eluent
			1	0～5min	95%A+5%B
2	5～45min	75%A+25% angiotensin-trifluoroacetic acid solution
			3	45～55min	95%A+5%B
4	55～75min	75%A+5%B+20%C
			5	75～95min	95%A+5%B
6	95～155min	95%A+5%B → 65%A+35%B
			7	155～175min	40%A+60%B
8	175～180min	95%A+5%B

Table 6 angiotensin is except the peak result of TFA Solution H PLC detection

Peak is numbered	Retention time (min)	Area (microvolt * second)	Highly (microvolt)	% area
					1	8.799	11184	1521	0.05
2	9.730	3823	557	0.02
					3	10.387	22413365	1987946	99.51
4	11.098	36099	4959	0.16
					5	11.326	53415	7451	0.24
6	11.750	6245	657	0.03

Embodiment 4 (angiotensin becomes salt, concentration and lyophilizing)

Angiotensin embodiment 3 obtained removes TFA solution 1L, adds 2mL glacial acetic acid, obtains angiotensin acetic acid Saline solution, removes the partial acetonitrile in solution at-0.1MPa, the spin concentration that reduces pressure under the conditions of 30 DEG C, is distributed into rustless steel pallet In, liquid surface height controlling is 0.5～1.0cm, and (Shanghai Dong Fulong science and technology share is limited to cover gauze feeding vacuum freeze drier Company LYO-1) carry out lyophilization according to pre-designed freeze-drying curve, freeze-dried obtain angiotensin acetate Lyophilized powder solid 13.3g, by feeding intake, 40g angiotensin crude product calculated yield is 33.2%, measures it as described in Example 1 HPLC purity is 99.18%, and retention time is that 10.53min, HPLC data are shown in Table 7.

The peak result of the HPLC detection of table 7 angiotensin acetate lyophilized powder

Peak is numbered	Retention time (min)	Area (microvolt * second)	Highly (microvolt)	% area
					1	8.864	7464	921	0.06
2	10.529	11398620	1121376	99.18
					3	11.176	23762	2733	0.21
4	11.406	51069	5939	0.44
					5	11.797	11452	642	0.10

Embodiment 5 (angiotensin acetate Mass Spectrometer Method)

Waters micromass ZQ substance level Four bar Electrospray Mass Spectrometry (ESI-MS) is used to measure angiotensin acetic acid Salt molecular weight analyte, result display molecular mass peak [M+1]⁺Measured value 1031.93, leading ion fragment peak [M+2]²⁺Measured value 516.65, all coincidence theory values 1031.18.

Embodiment 6 (acetic acid content and TFA residual in ion-chromatographic determination angiotensin acetate sample)

Instrument: U.S.'s Dionex500 ion chromatograph

Detached dowel: IonPacAS₁₄-AG₁₄

Detection mode: conductance

Leacheate: 1.0mM NaHCO₃+3.0mM Na₂CO₃

Take after sample 100mg adds the dissolving of 10mL leacheate and cross DionexOnGuard II RP pre-treatment pre-column, enter ion color Spectrometer detection level.Testing result shows, acetic acid content is 9.35% (mass percent), and TFA content is not for detecting (detection limit 5μg/g)。

Embodiment 7 (HPLC method detection vassopressin crude material and purification midbody solution purity)

Instrument: Waters2695/2489 high performance liquid chromatograph

Detached dowel: Agilent XDB-C184.6 × 250mm, 5 μm

Flowing phase: A be percent by volume be 0.1%TFA aqueous solution, B be percent by volume be the second of 0.1%TFA-50% Nitrile aqueous solution

Flow velocity is 1.0mL/min, and detection wavelength is 214nm, and room temperature detects,

Gradient see table shown in 8, and percentage ratio is percent by volume.

Table 8 flows phase gradient

Elution step	Elution time	Eluent
			1	0～5min	95%A+5%B
2	5～25min	95%A+5%B → 50%A+50%B
			3	25～30min	100%B
4	30～35min	95%A+5%B

Embodiment 8

(1) the anti-phase purification of vassopressin crude material

Instrument: Varian SD-1 high-pressure liquid phase preparation system

Chromatographic column: embodiment 2 self-chambering prepare post Load&Lock400375 × 260mm, PLRP-S10 μm 10nm

Flowing phase: A be percent by volume be the trifluoroacetic acid aqueous solution of 0.1%, B be percent by volume be 0.1% trifluoro Acetic acid acetonitrile solution, vassopressin crude product (the disclosed vassopressin using Solid phase synthesis of patent application CN201210131067.X Crude product) it is that solvent dissolves and is configured to 10g/L solution through the acetonitrile solution of percent by volume 5%, after filtering clarification, sample liquid is Flowing phase C, measuring its HPLC purity as described in Example 7 is 88.13%, and retention time is 14.40min.

The anti-phase purification condition of the present embodiment is as follows: flow velocity 200mL/min, 220nm detect, and purification gradient see table Shown in 9, it is that 48-54min target main peak is collected as vassopressin-trifluoroacetic acid solution, by the side of embodiment 7 to retention time It is 99.19% that method measures its HPLC purity, and retention time is 14.60min.

The gradient (percentage ratio is percent by volume) of the anti-phase purification of table 9

Elution step	Elution time	Eluent
			1	0～2min	95%A+5%B
2	2～22min	100%C
			3	22～40min	95%A+5%B
4	40～100min	95%A+5%B → 65%A+35%B

5	100～110min	40%A+60%B
			6	110～120min	95%A+5%B

(2) the anti-phase desalination of vassopressin-trifluoroacetic acid solution

Instrument: Varian SD-1 high-pressure liquid phase preparation system

Chromatographic column: embodiment 2 self-chambering prepare post Load&Lock400375 × 260mm, PLRP-S10 μm 10nm

Flowing phase: mobile phase A is pure water, and Mobile phase B is acetonitrile, flowing phase C is 0.1M NaOH solution, flow velocity 200mL/ Min, 220nm detect, and the gradient of anti-phase desalination see table shown in 10, is that 102-112min target main peak enters to retention time Row is collected as vassopressin and removes TFA solution, and measuring its HPLC purity by method in embodiment 7 is 99.81%, and retention time is 14.51min。

Gradient (percentage ratio is percent by volume) used by the anti-phase desalination of table 10

Elution step	Elution time	Eluent
			1	0～5min	95%A+5%B
2	5～45min	75%A+25% vassopressin-trifluoroacetic acid solution
			3	45～55min	95%A+5%B
4	55～75min	75%A+5%B+20%C
			5	75～95min	95%A+5%B
6	95～155min	95%A+5%B → 65%A+35%B
			7	155～175min	40%A+60%B
8	175～180min	95%A+5%B

Waters micromass ZQ substance level Four bar Electrospray Mass Spectrometry (ESI-MS) is used to measure its molecular mass peak [M +1]⁺Measured value 1084.40, leading ion fragment peak [M+2]²⁺Measured value 542.75, all coincidence theory value 1084.24.By implementing Example 6 records vassopressin and removes TFA content in TFA solution is not detect (detection limit 5 μ g/g).

Embodiment 9 (vassopressin becomes salt, concentration and lyophilizing)

Vassopressin embodiment 8 obtained removes TFA solution 1L, removes at-0.1MPa, the spin concentration that reduces pressure under the conditions of 30 DEG C Partial acetonitrile in solution, is slowly added to the tannic acid aqueous solution that mass volume ratio is 20%, and tannic acid consumption is added by each ten thousand units Pressure element adds one gram of tannic acid meter, while add stirring, obtains vassopressin tannate solution, is distributed in rustless steel pallet, liquid level control System, 0.5～1.0cm, covers gauze feeding vacuum freeze drier (Shanghai Tofflon Science and Technology Co., Ltd. LYO-1) and presses Carry out lyophilization according to pre-designed freeze-drying curve, freeze-dried obtain Pitressin Tannate lyophilized powder solid 10.2g, press The 40g vassopressin crude product calculated yield that feeds intake is 25.5%.

Embodiment 10 (HPLC method detection octreotide crude material and purification midbody solution purity)

Instrument: Waters2695/2489 high performance liquid chromatograph

Detached dowel: Kromasil100-3.5C18column4.6 × 100mm

Flowing phase: A is 10% Tetramethylammonium hydroxide: water: acetonitrile percent by volume is the aqueous solution of 2:88:10, B is 10% Tetramethylammonium hydroxide: water: acetonitrile percent by volume is the aqueous solution of 2:38:60.

Flow velocity is 0.8mL/min, and detection wavelength is 210nm, and room temperature detects,

Gradient see table shown in 11, and percentage ratio is percent by volume.

Table 11 flows phase gradient

Elution step	Elution time	Eluent
			1	0～30min	73%A+27%B
2	30～31min	73%A+27%B → 55%A+45%B
			3	31～37min	73%A+27%B

Embodiment 11

(1) the anti-phase purification of octreotide crude material

Instrument: Varian SD-1 high-pressure liquid phase preparation system

Chromatographic column: embodiment 2 self-chambering prepare post Load&Lock400375 × 260mm, PLRP-S10 μm 10nm

Flowing phase: A be percent by volume be the trifluoroacetic acid aqueous solution of 0.1%, B be percent by volume be 0.1% trifluoro Acetic acid acetonitrile solution, octreotide crude product (using Solid phase synthesis to prepare) is molten through the acetonitrile solution of percent by volume 5% Agent is dissolved and is configured to 10g/L solution, and after filtering clarification, sample liquid is flowing phase C, measures its HPLC purity as described in Example 10 Being 85.13%, retention time is 8.59min.

The anti-phase purification condition of the present embodiment is as follows: flow velocity 200mL/min, 220nm detect, and purification gradient see table Shown in 12, it is that 86-92min target main peak is collected as octreotide-trifluoroacetic acid solution, by embodiment 10 to retention time It is 99.49% that method measures its HPLC purity, and retention time is 8.66min.

The gradient (percentage ratio is percent by volume) of the anti-phase purification of table 12

Elution step	Elution time	Eluent
			1	0～2min	95%A+5%B
2	2～22min	100%C
			3	22～40min	95%A+5%B
4	40～100min	95%A+5%B → 65%A+35%B
			5	100～110min	40%A+60%B
6	110～120min	95%A+5%B

(2) the anti-phase desalination of octreotide-trifluoroacetic acid solution

Instrument: Varian SD-1 high-pressure liquid phase preparation system

Chromatographic column: embodiment 2 self-chambering prepare post Load&Lock400375 × 260mm, PLRP-S10 μm 10nm

Flowing phase: mobile phase A is pure water, and Mobile phase B is acetonitrile, flowing phase C is 0.1M NaOH solution, flow velocity 200mL/ Min, 220nm detect, and the gradient of anti-phase desalination is shown in Table 13, and is that 142-152min target main peak is carried out to retention time Being collected as octreotide and remove TFA solution, measuring its HPLC purity by method in embodiment 10 is 99.91%, and retention time is 8.62min。

Gradient (percentage ratio is percent by volume) used by the anti-phase desalination of table 13

Elution step	Elution time	Eluent
			1	0～5min	95%A+5%B
2	5～45min	75%A+25% octreotide-trifluoroacetic acid solution
			3	45～55min	95%A+5%B

4	55～75min	75%A+5%B+20%C
			5	75～95min	95%A+5%B
6	95～155min	95%A+5%B → 65%A+35%B
			7	155～175min	40%A+60%B
8	175～180min	95%A+5%B

Embodiment 12 (octreotide becomes salt, concentration and lyophilizing)

Octreotide embodiment 11 obtained removes TFA solution 1L, adds 2mL glacial acetic acid, obtains octreotide Acetate Solution, Remove the partial acetonitrile in solution at-0.1MPa, the spin concentration that reduces pressure under the conditions of 30 DEG C, be distributed in rustless steel pallet, liquid level Altitude control, 0.5～1.0cm, covers gauze and sends into vacuum freeze drier (Shanghai Tofflon Science and Technology Co., Ltd. LYO-1) carry out lyophilization according to pre-designed freeze-drying curve, freeze-dried obtain octreotide acetate lyophilized powder solid 12.3g, by feeding intake, 40g octreotide crude product calculated yield is 30.8%, measures its HPLC purity as described in Example 10 and is 99.68%, retention time is 8.60min.Use waters micromass ZQ substance level Four bar Electrospray Mass Spectrometry (ESI-MS) Measure its molecular mass peak [M+1]⁺Measured value 1019.86, leading ion fragment peak [M+2]²⁺Measured value 510.67, all meets reason Opinion value 1019.26.Detecting octreotide acetate lyophilized powder result by embodiment 6 to show, acetic acid content is 8.35% (percent mass Than), TFA content is not for detecting (detection limit 5 μ g/g).

Claims (5)

1. a preparation method for octreotide, it comprises the steps: to use high-efficient liquid phase reverse-phase chromatography by octreotide crude product Solution carries out anti-phase purification, anti-phase desalination successively,；The filler of high-efficient liquid phase reverse-phase chromatography is stryrene divinyl base Benzene copolymer；

The condition of described anti-phase purification is as follows: mobile phase A be percent by volume be the trifluoroacetic acid aqueous solution of 0.1%, flowing Phase B be percent by volume be the trifluoroacetic acid acetonitrile solution of 0.1%, flowing phase C be described octreotide crude product solution, flow velocity is 180～220mL/min, detection wavelength is 220nm；

Carrying out online loading, eluting according to the condition of following table, percentage ratio is percent by volume；

Elution step Elution time Eluent 1 0～2min 95%A+5%B 2 2～22min 100%C 3 22～40min 95%A+5%B 4 40～100min 95%A+5%B → 65%A+35%B 5 100～110min 40%A+60%B 6 110～120min 95%A+5%B

Collect the eluent that retention time is 45～95min and obtain octreotide-trifluoroacetic acid solution；

The condition of described anti-phase desalination is as follows: mobile phase A is water, and Mobile phase B is acetonitrile, and flowing phase C is 0.1mol/L NaOH Solution, flow velocity is 180～220mL/min, and detection wavelength is 220nm；

Carrying out online loading, eluting according to the condition of following table, percentage ratio is percent by volume；

Collect the eluent that retention time is 100～155min and i.e. can get octreotide solution.

2. preparation method as claimed in claim 1, it is characterised in that described octreotide crude product is for using Solid phase synthesis system The octreotide crude product obtained, the HPLC purity of described octreotide crude product is 80～90%.

3. preparation method as claimed in claim 1, it is characterised in that described octreotide crude product solution is that octreotide crude product is molten In the 10g/L solution that the acetonitrile solution that concentration of volume percent is 5% is formed.

4. preparation method as claimed in claim 3, it is characterised in that in described anti-phase purification, flow velocity is 200mL/ min；In described anti-phase desalination, flow velocity is 200mL/min.

5. preparation method as claimed in claim 1, it is characterised in that the filling condition of described high-efficient liquid phase reverse-phase chromatography As follows: filler is Agilent PLRP-S styrene diethylene benzene copoly mer, aperture 10nm, particle diameter 10 μm, fill column density 0.33g/mL, post pressure is 650psi.