The present invention also provides a method for preventing and/or treating a disease caused by bacteria , in particular Gram - negative bacteria , comprising administering to a subject a prophylactically and/or therapeutically effective amount of the anhydrous crystal of polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof .


Figure 1A shows the crystal of polymyxin B1 sulfate in an ethanol - n - butanol aqueous solution;Figure 1B shows a sample of crystal of polymyxin B1 sulfate after drying;Figure 2A is an XRD pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 2B is a peak list of the XRD pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 2C is a hot stage XRD pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 3A is an IR spectrum of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 3B is a peak list of the IR spectrum of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 4 is a DSC pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 5 is a DVS pattern of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 6 is a TGA spectrum of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 7 is an isotherm diagram of the crystal 1 of polymyxin B1 sulfate of the present invention;Figure 8A is an XRD pattern of the crystal A of polymyxin B1 sulfate of the present invention;Figure 8B is a peak list of the XRD pattern of the crystal A of polymyxin B1 sulfate of the present invention;Figure 8C is a hot stage XRD pattern of the crystal A of polymyxin B1 sulfate of the present invention;Figure 9A is an IR spectrum of the crystal A of polymyxin B1 sulfate of the present invention;Figure 9B is a peak list of the IR spectrum of


Best Mode for Carrying Out the Invention


The present invention will be further described in conjunction with the accompanying drawings and the embodiments to achieve better illustration of the present invention and to facilitate understanding of the technical solution of the present invention . It is to be understood that the specific examples of the present invention are only intended to illustrate the present invention and are not intended to limit the scope of the present invention .


Ethanol - water system : 100 ml aqueous solution of polymyxin B1 sulfate at a concentration of 50 g / L was taken and stirred at room temperature , to which was added 3 volumes of a 95 % ( v / v ) ethanol aqueous solution to precipitate a polymyxin B1 sulfate crystal . After stirring at a temperature of 0 - 5 ° C for 3 - 5 h , the mixture was filtered , and dried under vacuum for 8 hours to obtain a crystalline polymyxin B1 sulfate , and the crystal form was crystal form A.


Isopropanol - water : 26 ml aqueous solution of polymyxin B1 sulfate and polymyxin B2 sulfate at a concentration of 50 g / L was taken , and slowly added dropwise to 260 ml ( 10 volumes ) isopropanol under stirring . The temperature of isopropanol was always controlled within the range of 5 - 10 ° C . The crystals of polymyxin B1 sulfate and polymyxin B2 sulfate precipitated during the dropwise adding gradually increased , which was in a uniform dispersion state without adhesion . Stirring was continued for 1 hour , the mixture was filtered to obtain a solid , which was dried under vacuum for 10 hours to obtain a crystal powder of polymyxin B1 sulfate and polymyxin B2 sulfate ( 1.23 g ) , crystal yield : 94.6 % . The crystal form of polymyxin B1 sulfate was crystal form 1 .


N - butanol - water : 13.5 g polymyxin B2 sulfate was dissolved in 150 ml pure water , and slowly added dropwise to 1500 ml ( 10 volumes ) n - butanol under stirring with the temperature being always controlled within the range of 25 - 30 ° C . The crystal of polymyxin B2 sulfate precipitated during the dropwise adding gradually increased , which was in a uniform dispersion state without adhesion and had a good granularity . After the dropwise addition of the aqueous solution of polymyxin B2 sulfate was completed , stirring was continued for 30 min , the mixture was filtered , and the filtered solid was dried under vacuum for 20 hours to obtain a crystal powder of polymyxin B2 sulfate ( 13.2 g ) , crystal yield : 97.8 % .


According to the results of this competitive experiment , it was confirmed that among the two crystal forms of polymyxin B1 sulfate , the most stable crystal form in ethanol and in a mixed solvent of ethanol / water at room temperature is crystal form A.


According to the results of crystal mush experiment at room temperature , it was confirmed that the most stable crystal form of polymyxin B1 sulfate in ethanol and in a mixed solvent of ethanol / water at room temperature is crystal form A.


Based on the above results , when polymyxin B1 sulfate is handled at room temperature , polymyxin B1 sulfate can be stably present as the crystal form A using ethanol or a mixed solvent of ethanol / water .


1 . An anhydrous crystal 1 of polymyxin B1 sulfate , characterized in that , said anhydrous crystal 1 has an X - ray powder diffraction pattern having diffraction peaks at 3.396 , 4.895 and 6.903 expressed by 2θ degree using Cu - Ka radiation ; and preferably , said crystal 1 has an X - ray powder diffraction pattern expressed by 2θ degree using Cu - Ka radiation as shown in Figure 2A ; preferably , said anhydrous crystal 1 has an infrared absorption spectrum having characteristic bands at 1071.93 cm -1 , 1242.91 cm -1 , 1384.25 cm -1 , 1457.69 cm -1 , 1524.29 cm -1 , 1639.38 cm -1 , 2957.69 cm -1 , 3064.55 cm -1 and 3270.53 cm -1 as measured by KBr tableting method ; and more preferably , said anhydrous crystal 1 has an infrared absorption spectrum as measured by KBr tableting method as shown in Figure 3A ; and more preferably , said anhydrous crystal 1 has a melting point of 226.97 ° C , and has a differential scanning calorimetry pattern as shown in Figure 4 .


2 . An anhydrous crystal A of polymyxin B1 sulfate , characterized in that , said anhydrous crystal A has an X - ray powder diffraction pattern having a diffraction peak at 3.401 expressed by 2θ degree using Cu - Ka radiation ; and preferably , said anhydrous crystal A has an X - ray powder diffraction pattern expressed by 2θ degree using Cu - Ka radiation as shown in Figure 8A ; preferably , said anhydrous crystal A has an infrared absorption spectrum having characteristic bands at 1071.93 cm -1 , 1242.91 cm -1 , 1384.25 cm -1 , 1457.69 cm -1 , 1524.29 cm -1 , 1639.38 cm -1 , 2957.69 cm -1 , 3064.55 cm -1 and 3270.53 cm -1 as measured by KBr tableting method ; and more preferably , said anhydrous crystal A has an infrared absorption spectrum as measured by KBr tableting method as shown in Figure 9A ; and more preferably , said anhydrous crystal A has a melting point of 225.00 ° C , and a differential scanning calorimetry pattern as shown in Figure 10 .


3 . An anhydrous crystal of polymyxin B2 sulfate , characterized in that , said anhydrous crystal of polymyxin B2 sulfate has an X - ray powder diffraction pattern expressed by 2θ degree using Cu - Ka radiation as shown in Figure 17A.


4 . A method for preparing an anhydrous crystal of polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof , said method comprises the following steps of : ( 1 ) adding water to polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof to just completely dissolve the solid to obtain a saturated solution ; ( 2 ) slowly adding an organic solvent dropwise into said saturated solution , or slowly adding said saturated solution dropwise into an organic solvent at a controlled temperature within the range of 0 - 60 ° C to precipitate a solid ; wherein , said organic solvent is selected from one or more of C1 - C4 alcohol , C3 - C4 ketone , ethyl acetate or butyl acetate ; preferably , said C1 - C4 alcohol is selected from one or more of methanol , ethanol , isopropanol , n - propanol or n - butanol ; and still preferably , said C3 - C4 ketone is selected from one or more of acetone or 2 - butanone ; and ( 3 ) filtering off the solid and drying it under vacuum to obtain an anhydrous crystal of polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof .


5 . A method for preparing the anhydrous crystal 1 of polymyxin B1 sulfate according to claim 1 , said method comprises the following steps of : ( 1 ) adding water to polymyxin B1 sulfate to just completely dissolve the solid to obtain a saturated solution ; ( 2 ) slowly adding an organic solvent dropwise into said saturated solution , or slowly adding said saturated solution dropwise into an organic solvent at a controlled temperature within the range of 0 - 60 ° C to precipitate a solid ; wherein , said organic solvent is selected from n - butanol , isopropanol , n - propanol , or 2 - butanol ; and ( 3 ) filtering off the solid and drying it under vacuum to obtain an anhydrous crystal 1 of polymyxin B1 sulfate .


6 . A method for preparing the anhydrous crystal A of polymyxin B1 sulfate according to claim 2 , said method comprises the following steps of : ( 1 ) adding water to polymyxin B1 sulfate to just completely dissolve the solid to obtain a saturated solution ; ( 2 ) slowly adding an organic solvent dropwise into said saturated solution , or slowly adding said saturated solution dropwise into an organic solvent at a controlled temperature within the range of 0 - 60 ° C to precipitate a solid ; wherein , said organic solvent is selected from ethanol , ethanol - n - butanol , n - butanol - isopropanol , methanol , acetone , butanone , or ethanol - ethyl acetate ; and ( 3 ) filtering off the solid and drying it under vacuum to obtain an anhydrous crystal A of polymyxin B1 sulfate .


7 . The method according to any one of claims 4 to 6 , characterized in that , in step ( 1 ) , after adding water to polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof , the solid is just completely dissolved by heating it at a temperature below 60 ° C ; preferably , in step ( 2 ) , said organic solvent is used in an amount of 0.5 - 20 volumes in terms of the volume of said saturated solution ; and more preferably , in step ( 2 ) , after the solid is precipitated , stirring is continued for 0 - 8 hours .


8 . A pharmaceutical composition comprising the anhydrous crystal 1 of polymyxin B1 sulfate according to claim 1 , the anhydrous crystal A of polymyxin B1 sulfate according to claim 2 , the anhydrous crystal of polymyxin B2 sulfate according to claim 3 , or the anhydrous crystal of polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof prepared according to the method according to any one of claims 4 to 7 .


9 . The pharmaceutical composition according to claim 8 , comprising a pharmaceutically acceptable carrier or excipient , and optionally an antibacterial active ingredient other than the anhydrous crystal of polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof .


10 . Use of the anhydrous crystal 1 of polymyxin B1 sulfate according to claim 1 , the anhydrous crystal A of polymyxin B1 sulfate according to claim 2 , the anhydrous crystal of polymyxin B2 sulfate according to claim 3 , or the anhydrous crystal of polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof prepared according to the method according to any one of claims 4 to 7 in the preparation of a medicament for preventing and/or treating a disease caused by bacteria , in particular Gram - negative bacteria .


11 . The use according to claim 10 , wherein said disease is selected from various infections caused by Gram - negative bacteria , in particular Pseudomonas aeruginosa and Escherichia coli ; and preferably , said disease is respiratory system infection , peritonitis , bile duct infection , urinary tract infection , burn infection , corneal infection and sepsis .


12 . A method for preventing and/or treating a disease caused by bacteria , in particular Gram - negative bacteria , comprising administering to a subject a prophylactically and/or therapeutically effective amount of the anhydrous crystal 1 of polymyxin B1 sulfate according to claim 1 , the anhydrous crystal A of polymyxin B1 sulfate according to claim 2 , the anhydrous crystal of polymyxin B2 sulfate according to claim 3 , or the anhydrous crystal of polymyxin B1 sulfate , polymyxin B2 sulfate or a mixture thereof prepared according to the method according to any one of claims 4 to 7


POLYMYXIN B SULPHATE CRYSTAL AND PREPARATION METHOD THEREFOR


The present invention belongs to the technical field of pharmacy , and in particular relates to a polymyxin B sulfate crystal and a preparation method thereof .


The present invention provides a polymyxin B sulfate crystal and a preparation method thereof . Said method comprises the following steps of : adsorbing a polymyxin B fermentation liquid with a resin , and adding sulfuric acid or acidic ethanol for desorbing ; concentrating to obtain a saturated solution of polymyxin B sulfate ; and using an organic solvent to precipitate a crystal from the saturated solution , filtering and drying to obtain the polymyxin B sulfate crystal .


As for the refining of polymyxin B sulfate product , it is usually carried out by a spray drying method ( patent application CN201210519331.7 ) or a lyophilization method ( patent application CN201510775580.6 ) due to its difficulty to crystallize . The current patent about polymyxin B crystal includes polymyxin B1 dihydrate crystal disclosed in the patent application CN201210379231.9 , which is obtained by precipitation using a mixed solvent of acetone and diethyl ether . But diethyl ether is extremely volatile and easily to be oxidized in air and causes an explosion , which is not suitable for industrial production . In addition , the products on the market are all mixtures of polymyxin B sulfate . Regarding the preparation of polymyxin B1 sulfate monomer , the patent application filed by the present applicant has been granted ( Patent No . ZL201110390624.5 ) , and the final product is also prepared by spray drying method . Both the polymyxin B sulfate prepared by spray drying method and the commercially available polymyxin B sulfate product have strong hygroscopicity , the solution of which is tested to exhibit a low clarity and certain turbidity , and the product quality needs to be improved . Crystallization of the mother liquor by conventional crystallization method can remove some impurities , but the precipitate of polymyxin B sulfate is very sticky and easy to agglomerate , making it difficult to carry out the crystallization operation . Therefore , it is necessary to develop a new polymyxin


B sulfate crystal and a preparation method thereof .


In view of the problems that the polymyxin B sulfate product in the prior art is not high in clarity and purity , and the crystal precipitate is easily to agglomerate , the present invention provides a polymyxin B sulfate crystal and a preparation method thereof .


The method for preparing a polymyxin B sulfate crystal provided by the present invention comprises the following steps of :


Preferably , in step 1 ) of the method of the present invention , the concentration of said sulfuric acid aqueous solution is 0.2 mol / L.


According to a specific embodiment of the method of the present invention , in step 1 ) , the desorbing process is desorbing at a rate of 0.5 column volume of 0.2 mol / L sulfuric acid aqueous solution per hour , and the desorption liquid is fractionally collected . The collection is started when the desorption solution potency is higher than 500 µg / ml , and the collection is stopped when the potency is less than 500 µg / ml .


Preferably , in step 1 ) of the method of the present invention , before adsorbing a polymyxin B fermentation liquid with a resin , said polymyxin B fermentation liquid is subjected to a pretreatment comprising the following steps of :


Preferably , in step 1 ) of the method of the present invention , said adsorbing a polymyxin B fermentation liquid with a resin comprises :


( i ) ion exchange treatment;(ii ) macroporous adsorption resin treatment .


According to a specific embodiment of the method of the present invention , said ion exchange treatment comprises adjusting the pH of the filtrate of the polymyxin B fermentation liquid treated by acidifying to 6.7 , and then adsorbing with a weakly acidic ion exchange resin , and after completely adsorbing , washing with 2 - 3 volumes of water in terms of column volume ( i.e. the volume of the weakly acidic ion exchange resin in the column ) until the effluent is colorless , and then desorbing with a sulfuric acid aqueous solution . Preferably , said weakly acidic ion exchange resin is LXD-135 weakly acidic ion exchange resin . More specifically , said ion exchange treatment comprises adjusting the pH of the filtrate of the polymyxin B fermentation liquid pretreated by acidifying to 6.7 with 2 M sodium hydroxide solution , then adsorbing with a weakly acidic ion exchange resin , and after completely adsorbing , washing with 2 - 3 column volumes of water until the effluent is colorless , and then desorbing with0.2 M sulfuric acid aqueous solution ; and preferably , said weakly acidic ion exchange resin is LXD-135 weakly acidic ion exchange resin .


Preferably , the method of the present invention further comprises in step 1 ) , concentrating the obtained desorption liquid , decolorizing it by activated carbon , and filtering .


Preferably , in step 2 ) of the method of the present invention , the pH of the desorption liquid is adjusted to 5.0 - 7.0 with a 0.5M-10 M sodium hydroxide solution .


Preferably , in step 3 ) of the method of the present invention , said organic solvent is selected from one or more of C1 - C4 alcohol , C3 - C4 ketone , ethyl acetate or butyl acetate ; more preferably , said C1 - C4 alcohol is selected from one or more of methanol , ethanol , isopropanol , n - propanol or butanol ; and more preferably , said C3 - C4 ketone is selected from acetone or 2 - butanone .


Preferably , in step 3 ) of the method of the present invention , the process of precipitating the crystal is carried out at a temperature of 0 - 40 ° C .


According to a specific embodiment of the method of the present invention , the method for preparing a polymyxin B sulfate crystal comprises the following steps of :


1 ) acidifying a polymyxin B fermentation liquid , adding celite and filtering , ion exchange treating , macroporous adsorption resin treating , followed by adding a sulfuric acid aqueous solution for desorbing to obtain a desorption liquid ; and concentrating the obtained desorption liquid , decolorizing it by activated carbon , and then filtering to obtain a filtrate;2 ) adjusting the pH of the filtrate to 5.0 - 7.0 with a sodium hydroxide aqueous solution , and concentrating until a solid precipitates to obtain a saturated solution of polymyxin B sulfate ; and3 ) adding an organic solvent dropwise into the saturated solution of polymyxin B sulfate obtained from step 2 ) while stirring until a crystal precipitates , and holding the temperature during adding dropwisein the range of 0 - 10 ° C ; and continuing adding 0.5 - 5 volumes of the organic solvent in terms of the volume of the saturated solution of polymyxin B sulfate dropwise and stirring for 0 - 8 hours , filtering , discarding the filtrate , and drying the filter cake under vacuum for 3 - 20 hours to obtain a crystalline polymyxin B sulfate .


The present invention also provides a crystalline polymyxin B sulfate prepared according to the above method , that is , the present invention also provides a polymyxin B sulfate crystal , wherein the crystal has an X - ray powder diffraction pattern expressed by 2θ degree using Cu - Ka radiation as shown in Figure 3 . As can be seen from Figure 3 , the polymyxin B sulfate prepared according to the method of the present invention is crystalline .


Further , the present invention also provides a pharmaceutical composition , said pharmaceutical composition comprises the polymyxin B sulfate crystal and a pharmaceutically acceptable carrier . Optionally , the pharmaceutical composition further comprises one or more antibacterial active ingredients other than polymyxin B sulfate . Preferably , said pharmaceutical composition is in the form of tablet , capsule , or granule ; more preferably , said tablet is selected from rapid - release tablet , chewable tablet , dispersible tablet , effervescent tablet , sustained release tablet , controlled release tablet or enteric coated tablet , said capsule is selected from hard capsule , soft capsule , sustained release capsule , controlled release capsule or enteric coated capsule , and said granule is selected from suspension granule , effervescent granule , enteric coated granule , sustained release granule or controlled release granule .


The present invention also provides the use of the polymyxin B sulfate crystal in the preparation of an antibacterial drug , preferably in the preparation of a medicament against Gram - negative bacteria . The present invention also provides the use of the polymyxin B sulfate crystal in the preparation of a medicament against drug - resistant bacteria , in particular Gram - negative drug - resistant bacteria , such as Gram - negative super drug - resistant bacteria .


In other words , the present invention also provides a polymyxin B sulfate crystal for use in resisting bacteria , in particular Gram - negative bacteria . The present invention also provides a polymyxin B sulfate crystal for use in resisting drug - resistant bacteria , in particular Gram - negative drug - resistant bacteria , such as Gram - negative super drug - resistant bacteria .


The present invention also provides a method for preventing and/or treating a bacterial infection , in particular a disease or condition caused by Gram - negative bacteria , the method comprises administering to a subject or patient in need thereof a prophylactically and/or therapeutically effective amount of pharmaceutical preparation comprising the polymyxin B sulfate crystal .


The present invention also provides a method for preventing and/or treating drug - resistant bacteria infection , in particular a disease or condition caused by Gram - negative drug - resistant bacteria , such as Gram - negative super drug - resistant bacteria , the method comprises administering to a subject or patient in need thereof a prophylactically and/or therapeutically effective amount of pharmaceutical preparation comprising the polymyxin B sulfate crystal .


The present invention provides a polymyxin B sulfate crystal product , and the experimental results show that the product exhibits a crystal form , has better clarity and purity , and has low hygroscopicity . In addition , the preparation method provided by the present invention is easy to operate , and the solvent used for crystallization is low in cost and easy to obtain , which is suitable for large - scale industrial production , and lays a foundation for extending clinical application of polymyxin B sulfate crystal .


Best Mode for Carrying Out the Invention


The present invention will be further described in conjunction with the accompanying drawings and the embodiments to achieve better illustration of the present invention and to facilitate understanding of the technical solution of the present invention . It is to be understood that the specific examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention .


In the present application , the polymyxin B fermentation liquid used is prepared by pure strain fermentation for 40 - 50 hours using bacillus polymyxa with flour and cottonseed meal as main raw materials to obtain the polymyxin B fermentation liquid . The control sample is a polymyxin B sulfate raw material sample and a commercially available sample .


crystal product of the present invention is great in terms of product quality . The chromatographic purity of main components ( B1 , B2 , B3 , B1 - I ) in the HPLC result of the commercially available product ( prepared by spray drying ) is 89.99 % ( Figure 4A ) , while the chromatographic purity of active ingredients ( B1 , B2 , B3 , B1 - I ) in the HPLC result of the crystal sample of the present invention is 92.62 % ( Figure 4B ) , which is much higher than the requirement that the sum of the four components ≥80.0 % in the European Pharmacopoeia . Compared with the spray drying process currently commonly used , the method of the present invention can significantly improve the chromatographic purity and content of the crystal product , thereby improving the product quality .


4L Polymyxin B fermentation liquid with a potency of 1.5 g / L was taken , and then a desorption liquid was prepared according to the procedure of Example 1 . The desorption liquid of this example was concentrated to a saturated solution with a volume of 25 ml ( containing 2.60 g polymyxin B sulfate , measured by sampling ) . Then , the saturated solution was slowly added dropwise into 250 ml ( 10 volumes in terms of the volume of the saturated solution of polymyxin B sulfate ) isopropanol under stirring . The temperature of isopropanol during adding was always controlled within the range of 5 - 10 ° C . The polymyxin B sulfate crystals precipitated during adding gradually increased , which was in a uniform dispersion state without adhesion . Stirring was continued for 1 hour , the mixture was filtered , and the filtered solid was dried under vacuum at a temperature of 50 ° C for 10 hours to obtain a crystal powder of polymyxin B sulfate ( 2.40 g ) , crystal yield : 92.3 % .


10L Polymyxin B fermentation liquid with a potency of 2.0 g / L was taken , and then a desorption liquid was prepared according to the procedure of Example 1 . The desorption liquid of this example was concentrated to a saturated solution with a volume of 140 ml ( containing 13.50 g polymyxin B sulfate , measured by sampling ) . Then , the saturated solution was slowly added dropwise into 700ml ( 5 volumes in terms of the volume of the saturated solution of polymyxin B sulfate ) n - butanol under stirring . The temperature of the n - butanol solution system during adding was always controlled within the range of 25 - 30 ° C . The polymyxin B sulfate crystals precipitated during adding gradually increased , which was uniformly dispersed without adhesion and had a good granularity . After dropwise addition of the aqueous solution of polymyxin B sulfate was completed , stirring was continued for 30 min , the mixture was filtered , and the filtered solid was dried under vacuum at a temperature of 40 ° C for 20 hours to obtain a crystal powder of polymyxin B sulfate ( 12.40 g ) , crystal yield : 91.9 % .


7L Polymyxin B fermentation liquid with a potency of 1.5 g / L was taken , and then a desorption liquid was prepared according to the procedure of Example 1 . The desorption liquid was concentrated to a saturated solution with a volume of 50 ml ( containing 5.00 g polymyxin B sulfate , measured by sampling ) . Then , the saturated solution was slowly added dropwise into 400 ml ( 8 volumes in terms of the volume of the saturated solution of polymyxin B sulfate ) mixed solvent of ethanol ( 200 ml ) and n - butanol ( 200 ml ) under stirring . The temperature during adding was always controlled within the range of 0 - 5 ° C . The polymyxin B sulfate crystals precipitated during adding gradually increased , which was uniformly dispersed with a good granularity ( Figure 1 ) . After dropwise addition of the aqueous solution of polymyxin B sulfate was completed , stirring was continued for 2 hours , the mixture was filtered , and the filtered solid was dried under vacuum at a temperature of 60 ° C for 8 hours to obtain a crystal powder of polymyxin B sulfate ( 4.52 g ) , crystal yield : 90.4 % . It was observed by microscope at a magnification of 40X and 100X that the polymyxin B sulfate crystals were very obvious , and the individual crystal was uniform ( Figure 2A and Figure 2B ) .


15 L Polymyxin B fermentation liquid with a potency of 1.5 g / L was taken , and then a desorption liquid was prepared according to the procedure of Example 1 . The desorption liquid was concentrated to a saturated solution with a volume of 100 ml ( containing 10.00 g polymyxin B sulfate , measured by sampling ) . Then , the saturated solution was slowly added dropwise into 1000 ml ( 10 volumes in terms of the volume of the saturated solution of polymyxin B sulfate ) mixed solvent of ethanol ( 500 ml ) + isopropanol ( 500ml ) under stirring . The temperature during adding was always controlled within the range of 5 - 10 ° C . After the crystals were precipitated , the remaining solvent was continuously added dropwise , to obtain crystals which were uniformly dispersed and had a good granularity , with solid - liquid separation being quickly achieved after standing ( 30 min ) . Stirring was continued for 30 min , the mixture was filtered , and the filtered solid was dried under vacuum at a temperature of 55 ° C for 10 hours to obtain a crystal powder of polymyxin B sulfate ( 9.23 g ) , crystal yield : 92.3 % .


While the specific embodiments of the present invention has been illustrated and described in detail , it is understood that the present invention is not limited by the specific embodiments described . Various changes , modifications and variations of the present invention are within the scope of the present invention without departing from the spirit and scope of the invention .


1 . A method for preparing a polymyxin B sulfate crystal , characterized in that , said method comprises the following steps of : 1 ) adsorbing a polymyxin B fermentation liquid with a resin , adding a sulfuric acid aqueous solution and/or an acidic ethanol aqueous solution for desorbing to obtain a desorption liquid ; 2 ) adjusting the pH of the desorption liquid obtained from step 1 ) to 5.0 - 7.0 , and then concentrating until a solid precipitates to obtain a saturated solution of polymyxin B sulfate ; and 3 ) adding an organic solvent dropwise into the saturated solution of polymyxin B sulfate obtained from step 2 ) , or adding the saturated solution of polymyxin B sulfate obtained from step 2 ) dropwise into an organic solvent while stirring to precipitate a crystal , filtering , and drying the filter cake to obtain a polymyxin B sulfate crystal .


2 . The method according to claim 1 , characterized in that , in step 1 ) , the concentration of said sulfuric acid aqueous solution is 0.2 mol / L.


3 . The method according to claim 1 or 2 , characterized in that , said method further comprises in step 1 ) , before adsorbing a polymyxin B fermentation liquid with a resin , said polymyxin B fermentation liquid is subjected to a pretreatment which comprises the following steps of : a ) acidifying ; b ) adding celite and filtering ; preferably , said adsorbing a polymyxin B fermentation liquid with a resin comprises : ( i ) ion exchange treatment ; ( ii ) macroporous adsorption resin treatment ; more preferably , said pretreatment comprises adjusting the pH of the polymyxin B fermentation liquid to 1.8 , then adding celite in step b ) , filtering after stirring homogeneously , and top washing with water until the potency of the filtrate is less than 0.2 g / L ; and still preferably , said adjusting the pH of the polymyxin B fermentation liquid is carried out by adding oxalic acid ; still preferably , said ion exchange treatment comprises adjusting the pH of the filtrate of the polymyxin B fermentation liquid treated by acidifying to 6.7 , then adsorbing with a weakly acidic ion exchange resin , and after completely adsorbing , washing with 2 - 3 volumes of water in terms of column volume until the effluent is colorless , and then desorbing with 0.2 mol / L sulfuric acid aqueous solution ; preferably , said weakly acidic ion exchange resin is LXD-135 weakly acidic ion exchange resin ; and said adjusting the pH of the filtrate of the polymyxin B fermentation liquid treated by acidifying is carried out with a 2 M s


odium hydroxide solution ; yet preferably , said macroporous adsorption resin treatment comprises adjusting the pH of the ion exchange treated polymyxin B fermentation liquid to 6.5 , and after completely adsorbing by macroporous adsorption resin , top washing with 2 volumes of water in terms of column volume , and then desorbing with an acidic ethanol aqueous solution ; preferably , said adjusting the pH of the ion exchange treated polymyxin B fermentation liquid is carried out with a 2 M sodium hydroxide solution ; and further preferably , said acidic ethanol aqueous solution is a 40 vol% ethanol aqueous solution having a pH of 3 ; further preferably , said method further comprises in step 1 ) , concentrating the obtained desorption liquid , decolorizing it by activated carbon , and filtering .


4 . The method according to any one of claims 1 to 3 , characterized in that , in step 2 ) , the pH of the desorption liquid is adjusted to 5.0 - 7.0 with a 0.5M-10 M sodium hydroxide solution .


5 . The method according to any one of claims 1 to 4 , characterized in that , in step 3 ) , said organic solvent is selected from one or more of C 1 -C 4 alcohol , C 3 -C 4 ketone , ethyl acetate or butyl acetate ; preferably , said C 1 -C 4 alcohol is selected from one or more of methanol , ethanol , isopropanol , n - propanol or butanol ; and still preferably , said C 3 -C 4 ketone is selected from one or more of acetone or 2 - butanone .


6 . The method according to any one of claims 1 to 5 , characterized in that , in step 3 ) , the process of precipitating a crystal is carried out at a temperature of 0 - 40 ° C ; preferably , the method further comprises in step 3 ) , continuing stirring for 0 - 8 hours after completion of adding dropwise ; and more preferably , in step 3 ) , said drying is drying under vacuum at a temperature of 40 - 60 ° C for 3 - 20 hours .


7 . A polymyxin B sulfate crystal , characterized in that , said polymyxin B sulfate crystal has an X - ray powder diffraction pattern expressed by 2θ degree using Cu - Ka radiation as shown in Figure 3 .


8 . An antibacterial pharmaceutical composition , characterized in that , said pharmaceutical composition comprises the polymyxin B sulfate crystal prepared according to the method according to any one of claims 1 to 6 , or the polymyxin B sulfate crystal according to claim 7 , and a pharmaceutically acceptable carrier ; preferably , said pharmaceutical composition is in the form of tablet , capsule , or granule ; more preferably , said tablet is selected from rapid - release tablet , chewable tablet , dispersible tablet , effervescent tablet , sustained release tablet , controlled release tablet or enteric coated tablet , said capsule is selected from hard capsule , soft capsule , sustained release capsule , controlled release capsule or enteric coated capsule , and said granule is selected from suspension granule , effervescent granule , enteric coated granule , sustained release granule or controlled release granule .


9 . The pharmaceutical composition according to claim 8 , characterized in that , said pharmaceutical composition further comprises one or more antibacterial active ingredients other than polymyxin B sulfate .


10 . Use of the polymyxin B sulfate crystal prepared according to the method according to any one of claims 1 to 6 or the polymyxin B sulfate crystal according to claim 7 in the preparation of an antibacterial drug ; preferably in the preparation of a medicament against Gram - negative bacteria ; more preferably , use of the polymyxin B sulfate crystal prepared according to the method according to any one of claims 1 to 6 or the polymyxin B sulfate crystal according to claim 7 in the preparation of a medicament against drug - resistant bacteria , said drug - resistant bacteria are preferably Gram - negative drug - resistant bacteria .


LINKER PEPTIDE FOR CONSTRUCTING FUSION PROTEIN


A linker peptide for constructing a fusion protein . The linker peptide comprises a flexible peptide and a rigid peptide . The flexible peptide consists of one or more flexible units . The rigid peptide consists of one or more rigid units . The flexible unit comprises two or more amino acid residues selected from Gly , Ser , Ala , and Thr . The rigid unit comprises a human chorionic gonadotropin β - subunit carboxy - terminal peptide ( CTP ) bearing a plurality of glycosylation sites . The linker peptide can more effectively eliminate mutual steric hindrance of two fusion molecules , decreasing a reduction / loss of polymerization or activity resulting from improper folding of an active protein or a conformational change . On the other hand , the negatively charged , highly sialylated CTP can resist renal clearance , further prolonging a half - life of a fused molecule and enhancing bioavailability of a fused protein . Furthermore , a protective effect of a glycosylated side chain CTP can lower the protease sensitivity of the linker peptide , making a linker region of the fusion protein less degradable .


The present invention relates to the field of fusion proteins and , more specifically , to a peptide linker for the construction of fusion proteins .


Compared with other fusion strategies , the use of a peptide linker has a variety of advantages . First , the amino acids that make up the peptide linker are diverse ( 20 common amino acids ) . The length of the peptide linker is also an important tunable parameter , which can lead to rich diversity of peptide linkers ( 20n , n is the number of amino acid residues of the peptide linker ) . The peptide linkers are easy for bioengineering modifications . Second , the peptide linker provides certain spatial spacing , such that the two fused proteins fold correctly without interfering with each other . Third , the peptide linker can also provide more interaction possibilities for two fused proteins , promoting synergic interactions .


A number of literatures have reported the effects of the peptide linker sequences on the construction and expression of fusion proteins . For example , in the construction of the single chain antibody 1F7 , it was found that using a conventional Genex212 peptide linker ( GSTSGSGKSSEGKG ) to link the light and heavy chains did not achieve the original catalytic activity of the protein and the fusion protein was unstable . After a library of 18 amino acid residues with random sequences was constructed and screened , catalytically active single chain antibodies were obtained ( Tang Y et al . , 1996 , J Biol Chem . , 271:15682 - 15686 ) . In studying the helical peptide , Arai et al . found that the fluorescence resonance energy transfer from EBFP to EGFP reduced as the length of the peptide linker increased , suggesting that increasing the linker length could effectively separate the two functional regions ( Arai R. et al . , 2001 , Protein Eng . , 14:529 - 532 ) . In addition , in studying dengue virus NS2B protein , Luo et al . found that inserting a glycine residue between the original sites 173 and 174 , or replacing proline 174 with glycine , resulted in a significant decrease in protein activity of both the N- and C - terminal proteins . This indicated that the length and rigidity of the native peptide linker were the result of long - term evolution , and were of great significance to the function of the natural fusion proteins ( Luo D. et al . , 2010 , J Biol Chem . , 285:18817 - 18827 ) . The N - terminus of the NS2B prote


in was a serine protease , the C - terminus was a RNA helicase , and the peptide linker between them had 11 amino acid residues .


The present inventors found that the length and amino acid composition of the peptide linker , the presence or absence of glycosylation sites , the compatibility between the peptide linker and the two active molecules , and other factors , could affect the function and stability of the fusion proteins . The inventors believe that the peptide linker should have the following characteristics : 1 ) can make the linked proteins fold effectively into proper conformations , does not cause molecular dynamics change , and preferably comprises non - immunogenic natural amino acids . 2 ) should have the ability to prevent protease attack . 3 ) should try to avoid mutual impacts of the two fused proteins on each other .


In the absence of clear guidelines for the design of peptide linkers , the present inventors have developed new peptide linkers for the construction of fusion proteins , based on the inventors ' long - term research experience , particularly those in studying Fc fusion proteins . Amazingly , the peptide linkers have advantage for maintaining the biological activity of proteins or polypeptides and have broad applicability and portability .


The object of the present invention is to provide a novel peptide linker for constructing fusion proteins .


The first aspect of the present invention provides a peptide linker . The peptide linker comprises a flexible peptide and a rigid peptide . The flexible peptide consists of one or more flexible units , and the rigid peptide consists of one or more rigid units , wherein the flexible unit comprises two or more amino acid residues selected from Gly , Ser , Ala and Thr , and the rigid unit comprises the carboxyl terminal peptide ( CTP ) of human chorionic gonadotropin β subunit .


Preferably , the peptide linker is glycosylated . More preferably , the glycosylation site is located on CTP . Much more preferably , the glycosylation process is accomplished by expression in mammalian cells , such as Chinese hamster ovary cells , or other expression hosts having a suitable glycosylation modification system .


Further , the rigid peptide in the peptide linker of the present invention is located at the N - terminus or C - terminus of the flexible peptide . Preferably , the rigid peptide is located at the C - terminus of the flexible peptide . Specifically , the structural formula of the peptide linker of the present invention can be represented as F - R or R - F , wherein F and R represent the flexible peptide and the rigid peptide , respectively . Further , the flexible peptide preferably comprises 1 , 2 , 3 , 4 or 5 flexible units , and the rigid peptide preferably comprises 1 , 2 , 3 , 4 or 5 rigid units .


More preferably , the flexible unit comprises two or more G and S residues . Much more preferably , the amino acid sequence of the flexible unit has the general formula ( GS)a(GGS)b(GGGS)c(GGGGS)d , wherein a , b , c and d represent the number of structural units composed of G and S residues , and are integers greater than or equal to 0 , and a + b + c + d ≥ 1 .


Illustratively , each flexible unit is represented by Fi and i = 1 , 2 , 3 , 4 , 5 , ... n. In some embodiments of the invention , the flexible peptide preferably comprises , but is not limited to the following flexible units :


The rigid unit is selected from the full length or fragment sequence of the C - terminal amino acids 113 to 145 of human chorionic gonadotropin β subunit . Specifically , the rigid unit comprises the amino acid sequence of SEQ ID no : 1 or its truncated form .


Preferably , CTP contains at least 2 glycosylation sites . For example , in a preferred embodiment of the present invention , CTP contains 2 glycosylation sites . Illustratively , CTP contains the N - terminal 10 amino acids of SEQ ID no : 1 , i.e. SSSS*KAPPPS * , or CTP contains the C - terminal 14 amino acids of SEQ ID no : 1 , i.e. S*RLPGPS*DTPILPQ . For another example , in another embodiment of the present invention , CTP contains 3 glycosylation sites . Illustratively , CTP contains the N - terminal 16 amino acids of SEQ ID no : 1 , i.e. SSSS*KAPPPS*LPSPS*R. For other examples , in other embodiments of the present invention , CTP contains 4 glycosylation sites . Illustratively , CTP contains 28 , 29 , 30 , 31 , 32 , or 33 amino acids , starting from position 113 , 114 , 115 , 116 , 117 , or 118 and ending at position 145 of the human chorionic gonadotropin beta subunit . Specifically , CTP contains the N - terminal 28 amino acids of SEQ ID no : 1 , i.e. SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ . In this text , * represents a glycosylation site . Each possibility represents a separate embodiment of the present invention .


In other embodiments , the rigid units provided by the present invention have at least 70 % amino acid sequence identity to native CTP . In other embodiments , the rigid units provided by the present invention have at least 80 % amino acid sequence identity to native CTP . In other embodiments , the rigid units provided by the present invention have at least 90 % amino acid sequence identity to native CTP . In other embodiments , the rigid units provided by the present invention have at least 95 % amino acid sequence identity to native CTP .


Illustratively , each rigid unit is represented by Ri and i = 1 , 2 , 3 , 4 , 5 , ... , n. The rigid peptides described in some embodiments of the present invention may preferably comprise , but are not limited to , the following CTP rigid units .


( i ) R1 : SSSSKAPPPSLPSPSRLPGPSDTPILPQ;(ii ) R2 : PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ;(iii ) R3 : SSSSKAPPPS;(iv ) R4 : SRLPGPSDTPILPQ;(v ) R5 : SSSSKAPPPSLPSPSR .


In another preferred embodiment of the present invention , the peptide linker has the amino acid sequence as shown in SEQ ID no : 5(GGGSGGGSGGGSGGGSGGGSSSSSKAPPPSLPSPSR ) ( represented as F5 - R5 ) .


In another preferred embodiment of the present invention , the peptide linker has the amino acid sequence as shown in SEQ ID no : 6(GGGSGGGSGGGSGGGSGGGSSSSSKAPPPSLPSPSR ) ( represented as F5 - R5 ) .


Yet another aspect of the invention provides a fusion protein containing the peptide linker . The fusion protein comprises two biologically active molecules and a peptide linker linking the two active molecules . The structural formula of the fusion protein is expressed as K1 - L - K2 or K2 - L - K1 , wherein K1 is the first biologically active molecule , L is the above - mentioned peptide linker , and K2 is the second biologically active molecule . The components of the fusion protein are sequentially linked from the N - terminus to C - terminus . Further , the active molecules may be selected from protein or protein domain , polypeptide , antibody or antibody fragment , preferably protein or protein domain , antibody or an antibody fragment .


Illustratively , the active molecule K1 of the fusion protein comprises protein or protein domain having biological function , polypeptide ( e.g. , especially soluble or membrane signal molecule ) , cytokine , growth factor , hormone , costimulatory molecule , enzyme , receptor , protein or polypeptide having ligand function for the receptor . The active molecule K2 is serum protein or protein domain that prolongs the circulation half - life , for example , human serum albumin ( HSA ) , transferrin ( TF ) , antibody / immunoglobulin Fc fragment , and so on .


Illustratively , the active molecule K1 of the fusion protein comprises toxin , enzyme , cytokine , membrane protein , or immunomodulatory cytokine . The active molecule K2 comprises antibody or antibody fragment . K1 and K2 are linked via the peptide linker to form an antibody fusion protein . For example , K2 is an antibody Fv fragment ( VL or VH ) , and for another example , K2 is a single chain antibody ( scFv ) .


Further , the biologically active molecule K1 comprises but is not limited to adenosine A1 receptor , angiotensin converting enzyme ACE , activin family , ADAM family , ALK family , α-1 - antitrypsin , programmed cell death associated protein family , nerve growth factor and receptor family , bone morphogenetic protein BMP and receptor family , complement factor , calcitonin , cancer associated antigen , cathepsin family , CCL chemokine and receptor family , CD superfamily , CFTR , CXCL chemokine and receptor family , EGF , epidermal growth factor EGF and receptor family , coagulation factor IIa , factor VII , factor VIII , factor IX , ferritin , fibroblast growth factor FGF and receptor family , follicle stimulating hormone , FZD family , HGF , glucagon , cardiac myosin , growth hormone , Ig , IgA receptor , IgE , insulin - like growth factor IGF and binding protein family , interleukin IL superfamily and its receptor superfamily , interferon INF family , iNOS , integrin family , kallikrein family , laminin , L - selectin , luteinizing hormone , MMP family , mucin family , cadherin superfamily , platelet - derived growth factor PDGF and receptor family , parathyroid hormone , serum albumin , T - cell - related receptor superfamily , TGF - α , transforming growth factor TGF - β superfamily , thyroid stimulating hormone , parathyroid stimulating hormone , tumor necrosis factor TNF superfamily and its receptor TNFRSF superfamily , urokinase , WNT signaling pathway family , thymosin α1 , thymosin β4 , VEGF , vascular endothelial growth factor VEGF and it


Met428Leu mutations ( the amino acid sequence is as shown in SEQ ID no : 11 ) ; ( v ) vFcγ4 : human IgG4 hinge , CH2 , and CH3 regions containing Ser228Pro and Leu235Ala mutations ( the amino acid sequence is as shown in SEQ ID no : 12 ) .


More preferably , in one embodiment of the present invention , the FVII - Fc fusion protein comprises , sequentially from the N- to C - terminus , FVII ( having the amino acid sequence as shown in SEQ ID no : 13 ) , the peptide linker ( having the amino acid sequence as shown in SEQ ID no : 2 ) , and human IgG Fc ( having the amino acid sequence as shown in SEQ ID no : 11 ) .


More preferably , in one embodiment of the present invention , the FVIII - Fc fusion protein comprises , sequentially from the N- to C - terminus , FVIII ( having the amino acid sequence as shown in SEQ ID no : 14 ) , the peptide linker ( having the amino acid sequence as shown in SEQ ID no : 2 ) , and human IgG Fc ( having the amino acid sequence as shown in SEQ ID no : 11 ) .


More preferably , in one embodiment of the present invention , the Exendin-4 - Fc fusion protein comprises , sequentially from the N- to C - terminus , Exendin-4 ( having the amino acid sequence as shown in SEQ ID no : 15 ) . the peptide linker ( having the amino acid sequence as shown in SEQ ID no : 2 , 3 , 5 or 7 ) , and human IgG Fc ( having the amino acid sequence as shown in SEQ ID no : 11 ) .


More preferably , in one embodiment of the present invention , the hGH - Fc fusion protein comprises , sequentially from the N- to C - terminus , hGH ( having the amino acid sequence as shown in SEQ ID no : 17 ) , the peptide linker ( having the amino acid sequence as shown in SEQ ID no : 2 ) , and human IgG Fc ( having the amino acid sequence as shown in SEQ ID no : 11 ) .


In other preferred embodiments of the invention , the active molecule K1 of the fusion protein is the antibody heavy chain variable region ( VH ) , and K2 is the antibody light chain variable region ( VL ) . K1 and K2 are linked by the peptide linker to form a single chain antibody ( scFv ) .


In some preferred embodiments of the invention , the active molecule K1 of the fusion protein comprises a first antibody or antibody fragment , and the active molecule K2 comprises a second antibody or antibody fragment . K1 and K2 are linked by the peptide linker to form a bispecific antibody .


Preferably , in one embodiment of the present invention , K1 is a full - length double - chain anti - CD20 antibody , K2 is a single chain anti - CD3 antibody , and K1 and K2 are linked by the peptide linker to form a bispecific antibody . More preferably , the heavy chain of the anti - CD20 double - chain antibody contained in the bispecific antibody has an amino acid sequence as shown in SEQ ID no : 18 and the corresponding light chain has an amino acid sequence as shown in SEQ ID no : 19 . The anti - CD3 single chain antibody contained in the bispecific antibody has an amino acid sequence as shown in SEQ ID no : 20 . The peptide linker has an amino acid sequence as shown in SEQ ID no : 4 or 6 . The heavy chain of the anti - CD20 double - chain antibody is linked to the anti - CD3 single chain antibody by the peptide linker .


Yet another aspect of the present invention also provides a method for preparing a fusion protein having the structural formula expressed as : K1 - L - K2 or K2 - L - K1 , wherein K1 is the first biologically active molecule , L is the peptide linker , and K2 is a second biologically active molecule . The components that make up the fusion protein are sequentially linked from the N- to the C - terminus . The active molecules may be selected from the groups including protein or protein domain , polypeptide , antibody or antibody fragment , preferably protein or protein domain , antibody or antibody fragment . The preparation method includes the step of allowing K1 and K2 to be linked by L. In the preferred embodiments of the present invention , the method includes the following steps :


In this present invention , technical novelities can be summarized as follows :


eters , such as reducing the clearance rate , reducing the apparent distribution of volume , increasing AUC(0 - t ) , such that the bioavailability of the fusion protein increases . It is expected that the clinical dose will be reduced.4 . The glycosyl side chains of CTP has protective effect , which can reduce the sensitivity of the peptide linker to proteases . The fusion protein is not easy to be degraded in the linking region .


" Antibody fragment " : refers to an antigen - binding fragment of an antibody or antibody analogue , which typically comprises at least a portion of the antigen - binding region or variable region of the parental antibody , for example , one or more CDRs .


The " Fc " region : includes two heavy chain fragments , each of which comprises the CH2 and CH3 domains of an antibody . The two heavy chain fragments are held together by two or more interchain disulfide bonds and the inter - CH3 domain hydrophobic interactions .


The " Fv " region comprises the variable regions from both the heavy and light chains , but lacks constant regions .


" Single chain Fv antibody " ( or scFv antibody ): refers to an antibody comprising the VH and VL domains of an antibody , wherein these domains are present in a single polypeptide chain . In general , the Fv polypeptide additionally contains a polypeptide linker between the VH and VL domains that allows scFv to form the desired structure for antigen binding .


" Bispecific antibody " : refers to an antibody that comprises two Fv domains or scFv units such that the resulting antibody recognizes two different antigenic determinants .