CN104101656A - Purity determination method for gonadotropin - Google Patents
Purity determination method for gonadotropin Download PDFInfo
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Abstract
The invention discloses an HPLC purity determination method for gonadotropin. Specifically, the invention provides a high pressure liquid chromatography determination method for raw powder and/or bulk drugs by a molecular exclusion chromatography column filled by sephadex or cross-linked agarose or a mixed cross-linked material of the two, wherein the main peak USP theoretical plate number of the molecular exclusion chromatography column is not less than 1600. The method has good specificity, accuracy and repeatability.
Description
Technical field
The present invention relates to the purity analysis of promoting sexual gland hormone.Relate in particular to the purity analysis of people's urinary fsh.
Background technology
The promoting sexual gland hormone for the treatment of infertility is the approaching glycoprotein material of a class formation, comprise human chorionic gonadtropin (HCG), HMG (HMG), follicular stimulating hormone (FSH), lutropin (LH), wherein HMG is the potpourri that contains follicular stimulating hormone and lutropin and both proportional (2:1-1:2).
HCG, FSH, LH pass through the form combination of non-covalent bond by α chain and two subunits of β chain, wherein their α subunit is identical, have 92 amino acid, molecular weight is about 14500D, and the 52nd and 78 locational asparagines are that the glycosylated amino acid of N-occurs.The β subunit of HCG has 145-147 amino acid, molecular weight 22200-39000D, and wherein the 13rd, 30 locational asparagines and the 121st, 127,132,145 positions are that glycosylated place occurs.The β subunit of FSH is comprised of 111 amino acid, and molecular weight is about 18000D, and wherein the 7th and 24 locational asparagines are that the glycosylated amino acid of N-occurs.And the β subunit of LH is comprised of 121 amino acid, molecular weight is about 14800D.
HCG, HMG, FSH, LH are mainly used in treating sterility and external supplementary reproduction clinically.They can extract from the specific women urine in (pregnancy period or climacteric), also can prepare by DNA recombinant technique.
The first generation product of the promoting sexual gland hormone for the treatment of infertility is Profasi (HCG), the Pergonal (HMG) of the Serono company sixties in last century, and their purity is all lower, contains a large amount of impurity.After the eighties in last century, Serono company has released respectively Metrodin-HP, and it is the highly purified FSH preparation that a kind of impurity content is very low; Released again afterwards Gonal-F (rFSH), the Luveris (rLH) etc. that utilize DNA recombinant technique to produce, in addition, Ferring company has also released high-purity menopausal gonadotropic hormone-Menopur.
The product of low-purity is because foreign protein content is large, human body is had to larger spinoff, main clinical adverse shows as allergy, fash, heating, arthralgia and lower abdomen is uncomfortable or swollen sense, stomachache, feel sick, vomiting etc., safety in utilization is brought to certain risk.In addition, because low-purity product impurity content is large, pungency is larger, so administering mode also can only be confined to intramuscular injection.Because intramuscular injection needs thrusting of certain depth, therefore be all generally to select gluteus maximus or upper arm deltoid muscle position, the shortcoming of this injection system is that pain is large, injection inconvenience.Therefore, if can develop the high purity product that specific activity is high, impurity is few, just can at clinical adverse and injection system, this be greatly improved aspect two, the one, greatly reduce spinoff, improve the security of product, the 2nd, can carry out hypodermic injection, alleviate the painful and easy to operate of patient, carry out now hypodermic patient and generally all oneself carry out drug administration by injection at home.But especially high to the requirement of product purity during hypodermic injection, the existence of trace impurity more easily causes local anaphylaxis.The high-purity gonadotrophins glycoprotein having put goods on the market is at present still had anaphylactoid report is occurred by hypodermic injection.Therefore be necessary further to improve and control the purity of gonadotrophins medicine, yet existing detection method can not detect all impurity in product very accurately, cannot really effectively control the content of impurity thus, to guarantee the security of medication.
Therefore, how to improve the assay method of the purity of this type of promoting sexual gland hormone, improve the accuracy of measuring, be all the constantly problems of research of people all the time.The medicine of protide and general chemicals are being very different in nature, and general chemicals detection method is directed to the detection of the medicine of protide often can not bring into play good effect, is especially directed to gonadotrophins medicine.Main cause is that protein matter molecular weight is relatively large, and structure is very complicated, has quaternary structure, and structural difference can allow same albumen show different character.Promoting sexual gland hormone is owing to being connected with sugar chain on the skeleton of albumen, the sugar chain number being connected with on each protein molecular of same protein may be different, and the length of each sugar chain is, the monose of sugar chain forms and to be also not quite similar, this has just caused promoting sexual gland hormone character inhomogeneity very obvious.In addition, pharmaceutical grade protein after highly purified, its residual foreign protein is closely similar with major component in nature, exactly because this similar, allow conventional Protein Separation means can not effectively remove these foreign proteins, therefore, conventional purity analysis method also cannot effectively detect the purity of promoting sexual gland hormone.
At present, it is SDS-gel electrophoresis and exclusion chromatography that the purity analysis method of promoting sexual gland hormone is used maximum, although these two kinds of methods are all to carry out Protein Separation according to bulk of molecule, but its principle is not quite similar, electrophoresis is after being combined with SDS by protein, by protein, in electric field, carry out swimming and carry out separation, and gel chromatography rule is by the relativeness between the pore size of separated component molecular volume and gel and separated.Two kinds of methods are not replaced each other, complementary short length.
The purity of gel chromatography promoting sexual gland hormone is equally also subject to the inhomogenous impact of molecular size of promoting sexual gland hormone, the effect of its separated promoting sexual gland hormone is always not ideal enough, only have molecular weight difference very greatly, the character very albumen of homogeneous can reach effective separation.And the molecular weight of promoting sexual gland hormone is generally between 28KD to 50KD, difference own is little, and the impurity that may contain or the molecular weight of related substance also differ with it and be not very large, and therefore, the effect of mensuration also cannot be fully up to expectations.
Existing document also has report to adopt the method for gel chromatography to detect the method for promoting sexual gland hormone purity, as document: Claudio Wolfenson and others, ' Article Batch-to-batch Consistency of Human-derived Gonadotrophin Preparations Compared with Recombinant Preparations ', Reproductive biomedicine online, 10 (2005), 442 – 454. and other many pieces of documents were all reported the Biosep S2000(TSKG2000 that adopts Tosoh Bioscience company) gel column is to follicular stimulating hormone, human menopausal gonadotropin (HMG) or human chorionic gonadtropin carry out purity analysis.Yet this analytical approach is to follicular stimulating hormone, small molecular weight impurity in high-purity menopausal promoting sexual gland hormone sample cannot be effectively separated, its small molecular weight impurity peak is covered by major component peak, cannot calculate small molecular weight impurity content, it is also undesirable to the separating effect of the small molecular weight impurity of human chorionic gonadtropin.
In addition, document: Bert HM van de Weijer and others, ' Compositional Analyses of a Human Menopausal Gonadotrophin Preparation Extracted from Urine (menotropin) .Identification of Some of Its Major Impurities. ', Reproductive biomedicine online, 7 (2003), 547 – 57. also reported employing Superdex7510/30 gel column, phosphate buffer with the 0.2mol/L that contains 20% acetonitrile, pH7.0 is mobile phase, the method that 210nm place ultraviolet detects is to high-purity menopausal promoting sexual gland hormone, rFSH, restructuring human chorionic gonadtropin and restructuring interstitialcellstimulating hormone (ICSH) etc. carry out purity analysis.Yet, because the separating effect of the method is not very desirable, it cannot be effectively separated to the impurity of high-purity menopausal promoting sexual gland hormone, cause main peak to protract and trail, cause the USP degree of separation at large molecular impurity and small molecular weight impurity and major component peak to be all less than 1, therefore far can not meet the requirements, the impurity level in working sample more accurately.Inventor carries out the purity analysis of follicular stimulating hormone with reference to document analytical approach, although find the method large molecule and small molecular weight impurity in good separated follicular stimulating hormone simultaneously, but all not fully up to expectations in degree of separation, especially in the situation that impurity (or related substance) amount is lower, as impurity level is less than in 2% situation, its degree of separation is poor.
Document: Zheng Luxia etc., the research of injection chorionic gonadotrophin moderate purity assay method, Pharmaceutical Analysis magazine, 32(2012), 333-336. has reported that adopting two TSK G3000SWXL to connect carries out purity analysis to injection chorionic gonadotrophin, although near the separation case of the different material for molecular weight 40KD is improved, but for small molecular weight impurity, especially in follicular stimulating hormone, the separation case of the albumen impurity of molecular weight is not improved, and its examination different model be directed to the TSK chromatographic column of different molecular weight section, all be not so good as the effective of TSK G3000SWXL.
As can be seen here, the purity liquid phase analysis method that is directed at present the promoting sexual gland hormone for the treatment of infertility is desirable not enough, its main cause is: first, it not is very large due to the impurity of such glycoprotein and the molecular weight of principal goods matter, differing, cannot very effective separation on the filler of existing molecular exclusion.Secondly, owing to containing a plurality of sugar chains on such glycoprotein, and each sugar chain has diversity to a great extent, caused same glycoprotein also to there is the different on molecular weight, therefore, what in the liquid phase of molecular exclusion, reflect is exactly peak broadening, thereby easily causes main peak and the impurity peaks cannot be effectively separated.Again, because glycoprotein is in qualitative diversity, and the character of impurity is similar to main active component, therefore cannot carry out effective purity analysis by the method for separating and analyzing of other principles.
People have also attempted connecting to increase column length by two gel chromatographic columnses, improve theoretical cam curve, thereby improve the method for the separating effect of impurity, although the separating effect of impurity makes moderate progress really, the impurity that is directed to chorionic gonadotrophin detects and to improve significantly, but the detection that is directed to highly purified FSH small molecular impurity does not have good change.
In addition, in the preparation process of gonadotrophins bulk drug and gonadotrophins preparation, it prepares the raw material of use, the quality of powder as former in gonadotrophins and gonadotrophins bulk drug is directly connected to the quality of final products, if raw materials used non-conformity of quality closes requirement, the product of preparing will not meet drug standard yet.Therefore, in the urgent need to finding a kind of solid detection method, the quality of the final formulation products obtaining of effective guarantee.
Therefore, this area is in the urgent need to developing a kind of analytical approach that can better measure such promoting sexual gland hormone purity.
Summary of the invention
One object of the present invention is to provide a kind of analytical approach of measuring promoting sexual gland hormone purity.
Another object of the present invention is to provide a kind of high-purity, is applicable to the gonadotrophins bulk drug of multiple administering mode or the preparation method of gonadotrophins preparation.
A first aspect of the present invention, provide a kind of by the method for promoting sexual gland hormone purity in HPLC working sample, it is characterized in that, with molecular exclusion chromatography post, described sample is carried out to high-pressure liquid chromatography, thereby record the promoting sexual gland hormone purity in described sample, the filler of wherein said molecular exclusion chromatography post comprises cross-link dextran, Sepharose or its combination;
And, main peak USP theoretical cam curve >=1600 of described molecular exclusion chromatography post.
In another preference, described molecular exclusion chromatography post is that glucosan, agarose or its mix the chromatographic column that cross-linking agent is filler, is preferably the chromatographic column that is filler by agarose and the crosslinked Superdex forming of glucosan.
In another preference, described molecular exclusion chromatography post is Superdex7510/300GL chromatographic column.
In another preference, described chromatographic column is prepacked column or non-prepacked column.
In another preference, described theoretical cam curve is not less than 1700, is more preferably not less than 1800.
In another preference, adopt 2 molecular exclusion chromatography post series connection to detect, preferably, adopt 2 Superdex7510/300GL chromatography column in series to detect.
In another preference, the mobile phase using in described assay method is acetonitrile and buffer salt solution.
In another preference, the flow rate of mobile phase using in described assay method is 0.1～1.0ml/ minute.
In another preference, the detection wavelength using in described assay method is 200～300nm.
In another preference, in described mobile phase, acetonitrile is 0～1/2 to the volume ratio of water, is preferably 1/20～2/5.
In another preference, described buffer salt is selected from lower group: phosphate, Tris, acetate, sodium chloride, or its combination; Preferably, described buffer salt is selected from lower group: phosphate, sodium chloride, or its combination.
In another preference, described buffer salt is dihydric phosphate, is preferably sodium dihydrogen phosphate.
In another preference, described buffer salt solution concentration is 10mM～1700mM, is preferably 100mM～300mM; Described buffer salt solution concentration is the total concentration of various salt in solution.
In another preference, the pH of described buffer salt is 5.0～10.0.
In another preference, described promoting sexual gland hormone comprises the one or more compositions that are selected from lower group: human chorionic gonadtropin, HMG, follicular stimulating hormone, lutropin; Preferably, described promoting sexual gland hormone comprises follicular stimulating hormone.
In another preference, described method also comprises following one or more conditions:
A, high pressure liquid chromatography system comprise the Superdex7510/300GL post of two series connection;
B, mobile phase: acetonitrile: buffer salt solution=1:20～2:5, wherein said ratio is volume ratio;
C, flow velocity: 0.2～0.8ml/min,
D, column temperature: 25～45 ℃;
E, detection wavelength: 210～235nm;
F, sample introduction concentration: 3000～4500IU/ml.
In another preference, described sample is the former powder of gonadotrophins or gonadotrophins bulk drug.
A second aspect of the present invention, provides a kind of method of preparing gonadotrophins bulk drug, and the preparation process of described gonadotrophins bulk drug comprises following steps:
A, use the method for detecting purity as described in first aspect present invention, measure the former powder purity of gonadotrophins;
If the measurement result of B step (A) shows total impurities content > 2% in former powder, described former powder is re-started to purifying, until measurement result shows total impurities content≤2%;
If the measurement result of C step (A) shows total impurities content≤2% in this former powder, in the former powder of this gonadotrophins, add the excipient of pharmaceutically acceptable amount, make gonadotrophins bulk drug.
A third aspect of the present invention, provides a kind of method of preparing gonadotrophins pharmaceutical preparation, and described gonadotrophins pharmaceutical preparation preparation process comprises following steps:
A, use the method for detecting purity as described in first aspect present invention, measure the purity of gonadotrophins bulk drug;
If the measurement result of B step (A) shows total impurities content≤2% in this bulk drug, in this gonadotrophins bulk drug, add the excipient of pharmaceutically acceptable amount, make gonadotrophins pharmaceutical preparation.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, at this, tire out and state no longer one by one.
Accompanying drawing explanation
Fig. 1 has shown the chromatogram of embodiment 1 method one gained;
Fig. 2 has shown the chromatogram of embodiment 1 method two gained;
Fig. 3 has shown the chromatogram of embodiment 1 method three gained;
Fig. 4 has shown the chromatogram of embodiment 1 method four gained;
Fig. 5 has shown the chromatogram of embodiment 2 method one gained;
Fig. 6 has shown the chromatogram of embodiment 2 method two gained;
Fig. 7 has shown the chromatogram of embodiment 3 sequence number 1 experiment gained.
Embodiment
The inventor finds through extensive and deep research, be surprised to find that first, when the filler that adopts cross-link dextran and/or Sepharose as molecular exclusion chromatography post, and the theoretical cam curve of molecular sieve reaches 1600 when above, can effectively impurity and principal goods matter be separated, thereby can measure exactly the content of a small amount of impurity in high-purity promoting sexual gland hormone sample.On this basis, inventor has completed the present invention.
Test of the present invention shows, when reaching more than 1600, the theoretical cam curve of molecular sieve (can adopt the molecular sieve column of two series connection, as two Superdex7510/300GL), while carrying out promoting sexual gland hormone purity testing by HPLC, take a certain proportion of acetonitrile as mobile phase, with UV-detector, at 200～300nm place, monitor, the promoting sexual gland hormone that contains utmost point low impurity content for mensuration, its impurity accesses separated preferably with principal goods mass-energy, for the impurity content of measuring more accurately high-purity promoting sexual gland hormone, play a part positive, for the safety evaluatio of such material and the further raising of quality provide strong means.
Term
As used herein, " promoting sexual gland hormone " is selected from one or more following mixing: human chorionic gonadtropin (chorionic gonadotropin, CG), follicular stimulating hormone (follicule-stimulating hormone, FSH), lutropin (luteotropic hormone, LH), HMG (human menopausal gonadotropin, HMG).Described " the former powder of promoting sexual gland hormone " to be promoting sexual gland hormone adding the xeraphium of thinning agent before being prepared into bulk drug.The former powder of described gonadotrophins can, with the method preparation of prior art, as extracted from the specific women urine in (pregnancy period or climacteric), also can be prepared by DNA recombinant technique.
In another preference, described prior art includes, but is not limited to, the highly purified HMG that the disclosed preparation method of Chinese patent CN1246332C obtains; The HCG that the disclosed preparation method of Chinese patent CN1958603B obtains; And the FSH of the disclosed method acquisition of CN101307103A.
As used herein, " gonadotrophins bulk drug " refers to the composition that contains the former powder of gonadotrophins, and it consists of acceptable carrier in promoting sexual gland hormone and pharmaceutically/bromatology.Can be solid or liquid, can obtain by the existing method of preparing promoting sexual gland hormone composition in this area, it can directly be used for being prepared into pharmaceutical preparation, and it is pharmaceutically defined as bulk drug.Such as but not limited to, the gonadotrophins bulk drug that the disclosed method of Chinese patent CN101269215B obtains.
The adquisitiones of above bulk drug is only that bulk drug of the present invention should be not limited for example.
In the preferred embodiment of the present invention, the former powder content of FSH in described FSH bulk drug is for being not less than 10 μ g/mg bulk drugs or being not less than 100 international units/mg bulk drug.
As used herein, term " gonadotrophins pharmaceutical preparation " refers to and contains the gonadotrophins bulk drug for the treatment of effective dose, and pharmaceutically acceptable carrier, for example (but being not limited to), the FSH composition that the FSH composition that the disclosed method of Chinese patent CN1795012A obtains or the disclosed method of European patent EP 0618808 obtain.
In the preferred embodiment of the present invention, the FSH bulk drug in described FSH preparation accounts for 0.001～99.9wt% of total formulation weight amount; Being preferably 0.01～99wt% of composition total weight, is more preferably 0.02～95wt%, more preferably 0.05～90wt%.Surplus is the materials such as pharmaceutically acceptable carrier and other adjuvant.
" total impurities content " as used herein refers to the summation of each impurity content except main composition, the summation that has comprised " large molecular impurity " as herein described, " small molecular weight impurity " and other impurity content that may exist.
" large molecular impurity " as used herein refers to that molecular weight is greater than the impurity of main composition, and " small molecular weight impurity " refers to that molecular weight is less than the impurity of main composition.
As used herein, term " contain " or " comprising " comprised " comprising ", " substantially by ... form " and " by ... form ".As used herein, term " treatment effective dose " refers to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.
As used herein, the composition of term " pharmaceutically acceptable " or " acceptable in bromatology " is applicable to people and/or animal and without excessive bad subsidiary reaction (as toxicity, stimulation and allergic reaction), has the material of rational benefit/risk ratio.
As used herein, term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and thinning agent.This term refers to some medicament carriers like this: they itself are not necessary active component, and after using, there is no undue toxicity.Suitable carrier is well known to those of ordinary skill in the art.At < < Lei Mingdun pharmaceutical science > > (Remington's Pharmaceutical Sciences, Mack Pub.Co., N.J.1991) in can find discussing fully about pharmaceutically acceptable excipient.
Described " pharmaceutically acceptable carrier " can contain liquid, as water, salt solution, glycerine and ethanol.In addition, in these carriers, also may there is complementary material, as filling agent, disintegrant, lubricant, glidant, effervescent agent, wetting agent or emulsifying agent, flavouring, pH buffer substance etc.Conventionally, these materials can be formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably, pH is about 6-8.
The effective dose that should be understood that FSH used can change with the order of severity to be administered or object that treat.Concrete condition for example, decides according to the individual instances of object (object body weight, age, health, the required effect reaching), and this is in the scope that skilled practitioners or nutritionist can judge.
Pharmaceutical composition of the present invention can be solid-state (as granule, tablet, freeze-dried powder, suppository, capsule, sublingual lozenge) or liquid (as oral liquid, liquid drugs injection) or other suitable shape.
Press Chinese Pharmacopoeia regulation: unless otherwise specified, generally, the degree of separation between component to be measured and adjacent concurrent should be greater than 1.5.Yet because promoting sexual gland hormone belongs to biochemical drug, the separation of this type of impurity of the drug is very difficult, so builds consensus in this area: degree of separation is greater than 1 and just meets the requirements.If degree of separation is too small, show that between two peaks, separation case is very poor, it comes the error of calculated purity will be very large by integration.
In addition, during the common integral analysis of analytical approach that generally uses in this area, adopt peak valley to peak valley integration, like this, can be by artificial the raising of baseline, thus make degree of separation numerical value improve to meet the requirement that is greater than 1.Because baseline is by artificial raising, the integral area at each peak will differ greatly, and especially, when this peak area itself is very little,, when impurity content is very low, this situation can be more obvious.Therefore when impurity content is very low, if impurity content is lower than 3% time, more preferably impurity content is lower than 2% time, and this integration method is also inapplicable.In addition, when impurity content is lower, if impurity content is lower than 3% time, more preferably impurity content is lower than 2% time, major component summit is very high, and impurity peaks relatively can be very little, so, two peak-to-peak separation case will be poorer, and degree of separation is lower, and the error of purity testing will be larger.Therefore, the integral way that the present invention adopts is that the mode of vertical line split plot design is carried out detached peaks, and this kind of method can eliminate that baseline is raised and the error introduced, thereby reaches the object of accurate quantitative analysis impurity.Two kinds of integration methods can be with reference to original work Hans-Joachim Kuss, Stavros Kromidas, the < < liquid phase of Chen little Ming, Tang Yayan compiling and the detailed introduction of chromatogram ration analysis guide for use > > mono-book are carried out.
Theoretical cam curve
As used herein, " USP theoretical cam curve " or " theoretical cam curve " or " number of theoretical plate " are for evaluating the index of the separation efficiency of chromatographic column.Because the chromatographic behavior of different materials in same chromatographic column is different, while adopting number of theoretical plate as measurement column efficiency index, should indicate mensuration material, the number of theoretical plate that number of theoretical plate as herein described is main peak.The computing formula of number of theoretical plate is:
N=16 (t
r/ W)
2or n=5.54 (t
r/ W
h/2)
2
Wherein, n is number of theoretical plate,
T
rfor the retention time at peak,
W is the peak width at peak,
W
h/2peak width at half height for peak.
Conventionally, theoretical cam curve can regulate and control by controlling the retention time at peak, as changed column length, or the methods such as many root chromatogram column series connection uses is realized.
As used herein, " USP degree of separation " or " degree of separation " are for evaluating a kind of index of the separation degree of component to be measured and adjacent concurrent or difficult separate substance, and it is the key index of weighing chromatographic system usefulness.It is with reference to American Pharmacopeia, the definition of degree of separation to be come, and Chinese Pharmacopoeia also has identical computing formula to degree of separation.On the liquid-phase chromatographic analysis software Empower2 of Waters company, can automatically calculate by integral analysis and provide " USP degree of separation " value.Its computing formula is as follows:
In formula:
T
r2retention time for adjacent two Zhong Houyi peaks, peak
T
r1retention time for last peak in adjacent two peaks
W
1, W
2and W
1, h/2, W
2, h/2difference is peak width and the peak width at half height at adjacent two peaks for this reason.
The method of HPLC working sample purity
While carrying out separation by high performance liquid chromatography for difficult separated impurity, changing theoretical cam curve is conventional way.Yet, if raising theoretical cam curve, must cause post to press liter, but the conventional chromatographic column for separating of promoting sexual gland hormone is that filler is the glass column of cross-link dextran, the Superdex75 post that RuGE company produces, the post that can tolerate is pressed very low, and too high post is pressed easy compressible filler or the chromatographic column that rises brokenly, thereby causes chromatographic column to lose efficacy.In order to overcome this difficult problem, inventor is by a large amount of experiments, and by controlling suitable condition determination, especially suitable flow velocity has solved the problems referred to above.Method provided by the invention had both avoided gel column to damage because of superpressure, the time of a liquid phase analysis can be controlled in the preferred range again, and be obtained good impurity degree of separation, thereby measure more accurately the content of all kinds of impurity in this type of material.
The HPLC method for detecting purity of promoting sexual gland hormone provided by the invention comprises:
With the molecular exclusion chromatography post that cross-link dextran or Sepharose or both mixing cross-linking agents are filler, former powder and/or bulk drug are carried out to high-pressure liquid chromatography; Wherein, the main peak USP theoretical cam curve of described molecular exclusion chromatography post is not less than 1600.
Preferably, described molecular exclusion chromatography post is selected the chromatographic column that the mixing cross-linking agent of glucosan and agarose is filler.Above-mentioned chromatographic column can be prepared by conventional method, also can select commercially available prepacked column, comprises the chromatographic column that (but being not limited to) Superdex is filler, as Superdex75 10/300GL chromatographic column.
In another preference, described theoretical cam curve is not less than 1700, is more preferably not less than 1800.
In another preference of the present invention, adopt 2 molecular exclusion chromatography post series connection to detect, preferably, adopt 2 Superdex75 10/300GL chromatography column in series to detect.
When promoting sexual gland hormone is detected with above-mentioned chromatographic column, need suitably choose as mobile phase, flow velocity, detection wavelength etc. other experiment conditions of liquid chromatography, guarantee the parameters such as theoretical cam curve and retention time within the required range, avoid post to press too high simultaneously.In another preference, the experiment condition of liquid chromatography is:
Mobile phase: acetonitrile and buffer salt solution;
Flow rate of mobile phase: 0.1-1.0ml/ minute;
Detect wavelength: 200～300nm.
Wherein, the ratio of mobile phase and water need be controlled at suitable scope, guarantees the separating effect of each component.In another preference, in described mobile phase, acetonitrile is 0～1/2 to the volume ratio of water, is preferably 1/20～2/5.
In described liquid chromatography separating experiment, need select suitable buffer salt solution that pH is stabilized in 5.0～10.0 scopes.In another preference, described buffer salt is selected from lower group: phosphate, Tris, acetate, sodium chloride, or its combination; Preferably, described buffer salt is selected from lower group: phosphate, sodium chloride, or its combination.
In another preference, described buffer salinity is 10mM～1700mM, is preferably 100mM～300mM; Described buffer salt solution concentration is the total concentration of various salt in solution.
The inventive method can be used for measuring the sample that contains promoting sexual gland hormone, powder as former in gonadotrophins or bulk drug.Described promoting sexual gland hormone comprises that (but being not limited to) is selected from one or more compositions of lower group: human chorionic gonadtropin, HMG, follicular stimulating hormone, lutropin; Preferably, described promoting sexual gland hormone comprises follicular stimulating hormone.
In a preference of the present invention, described method comprises following one or more conditions:
A, high pressure liquid chromatography system: two Superdex75 10/300GL series connection connecting systems;
B, mobile phase: acetonitrile: buffer salt solution=1:20～2:5;
C, flow velocity: 0.2～0.8ml/min,
D, column temperature: 25～45 ℃;
E, detection wavelength: 210～235nm;
F, sample introduction concentration: 3000～4500IU/ml.
In another preference of the present invention, described method comprises following one or more conditions:
A, high pressure liquid chromatography system: two Superdex75 10/300GL series connection connecting systems;
B, mobile phase: acetonitrile: buffer salt solution=1:10～3:10;
C, flow velocity: 0.3～0.6ml/min,
D, column temperature: 25～35 ℃;
E, detection wavelength: 210～220nm;
F, sample introduction concentration: 3000～4000IU/ml.
In another preference of the present invention, described method comprises following one or more conditions:
A, high pressure liquid chromatography system: two Superdex75 10/300GL series connection connecting systems;
B, mobile phase: acetonitrile: (50mM phosphate, 150mM sodium chloride solution)=1:5;
C, flow velocity: 0.5ml/min,
D, column temperature: 30 ℃;
E, detection wavelength: 215nm;
F, sample introduction concentration: 3500IU/ml.
Said determination method can be used for detecting the purity of the sample that comprises promoting sexual gland hormone, and wherein, described sample comprises the former powder of (but being not limited to) gonadotrophins or gonadotrophins bulk drug.
When adopting two Superdex75 molecular sieve column series connection, especially when the number of theoretical plate of main peak is not less than 1600, the impurity peaks of follicular stimulating hormone and the separating effect of main peak are better, the degree of separation of small molecular weight impurity and main peak is not less than 1, and degree of separation meets the effectively separated generally acknowledged requirement of biochemical series products chromatogram.
The preparation of gonadotrophins bulk drug
The inventor has also obtained a kind of method of preparing gonadotrophins bulk drug, said method comprising the steps of:
A, use the method for detecting purity as described in first aspect present invention, measure total impurities content in the former powder of gonadotrophins;
If the measurement result of B step (A) shows total impurities content > 2% in former powder, described former powder is re-started to purifying, until measurement result shows total impurities content≤2%;
If the measurement result of C step (A) shows total impurities content≤2% in this former powder, in the former powder of this gonadotrophins, add the excipient of pharmaceutically acceptable amount, make gonadotrophins bulk drug.
By the assay method that uses inventor to provide, the purity of the former powder of promoting sexual gland hormone is analyzed to mensuration, thereby judge whether the former powder of gonadotrophins of this batch continues on for preparing gonadotrophins bulk drug.Instruct thus and control the production of gonadotrophins bulk drug.
The preparation of gonadotrophins pharmaceutical preparation
Meanwhile, inventor has also obtained a kind of method of preparing gonadotrophins pharmaceutical preparation, said method comprising the steps of:
A, use the method for detecting purity as described in first aspect present invention, measure total impurities content in gonadotrophins bulk drug;
If the measurement result of B step (A) shows total impurities content≤2% in this bulk drug, in this gonadotrophins bulk drug, add the excipient of pharmaceutically acceptable amount, make gonadotrophins pharmaceutical preparation.
By the assay method that uses inventor to provide, the purity of promoting sexual gland hormone in gonadotrophins bulk drug is analyzed to mensuration, thereby judge whether the gonadotrophins bulk drug of this batch continues on for the preparation of gonadotrophins pharmaceutical preparation.Instruct thus and control the production of gonadotrophins pharmaceutical preparation.
Major advantage of the present invention is:
1, the invention provides a kind of analytical approach of new mensuration promoting sexual gland hormone purity simple to operate, with strong points, and methods and results provided by the invention is accurate, highly sensitive, thereby be suitable for very much the test of promoting sexual gland hormone purity.
2, method provided by the invention is particularly suitable for low content impurity in promoting sexual gland hormone, if impurity content is lower than 3% time, and the more preferably analysis of impurity content lower than 2% time.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer, unless otherwise indicated, otherwise number percent and umber are calculated by weight.
Unless otherwise defined, the familiar meaning of all specialties of using in literary composition and scientific words and one skilled in the art is identical.In addition, any method sill similar or impartial to described content all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
Instrument and material
Waters2695 separative element, 2489 dual wavelength ultraviolet detecting devices, are produced by Waters company;
Superdex7510/300GL, YouGE company produces.
TSK G2000SWXL (7.8 * 300mm), Cao You Dong company produces.
TSK G2000SWXL (7.8 * 300mm), Cao You Dong company produces.
Embodiment 1
The HPLC purity analysis of FSH bulk drug
Sample to be analyzed: FSH bulk drug (by Shanghai Tianwei Biological Pharmaceutical Corp., produced lot number: 111201, biological value is in FSH 256.8IU/mg)
Method one: this method adopts the liquid-phase chromatography method of document " Compositional Analyses of a Human Menopausal Gonadotrophin Preparation Extracted from Urine (menotropin) " to carry out the HPLC purity analysis of FSH.
Mobile phase: get sodium hydrogen phosphate 71.6g, add water 700ml and dissolve, with phosphorus acid for adjusting pH value to 7.0, add water to 800ml, then add acetonitrile 200ml, mix, filtration, ultrasonic degas and get final product.
Get FSH bulk drug 28.90mg, with mobile phase, dissolve and make the solution that calculates Mei1mlZhong Han 500 units by labelled amount, as need testing solution.According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.With a Superdex75 post (30cm * 10mm), for analyzing chromatographic column, flow velocity is per minute 0.5ml, and column temperature is 30 ℃; Detection wavelength is 210nm.Get need testing solution 100 μ l injection liquid chromatographies, record chromatogram to 60 minute, chromatogram is analyzed.
Method two: this method adopts the liquid-phase chromatography method of document " Article Batch-to-batch Consistency of Human-derived Gonadotrophin Preparations Compared with Recombinant Preparations " to carry out the HPLC purity analysis of FSH.
Mobile phase: 100mM phosphate, 100mM sulfate, pH6.7.
Get FSH bulk drug 31.68mg, with mobile phase, dissolve and make the solution that calculates Mei1mlZhong Han 3500 units by labelled amount, as need testing solution.According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.With a TSK G2000SWXL (7.8 * 300mm), for analyzing chromatographic column, flow velocity is per minute 0.7ml, and column temperature is 30 ℃; Detection wavelength is 210nm.Get need testing solution 20 μ l injection liquid chromatographies, record chromatogram to 50 minute, chromatogram is analyzed.
Method three: this method adopts the liquid-phase chromatography method of document " research of injection chorionic gonadotrophin moderate purity assay method " to carry out the HPLC purity analysis of FSH.
Mobile phase: 50mM sodium dihydrogen phosphate, pH7.0-acetonitrile (80:20).
Get FSH bulk drug 24.14mg, with mobile phase, dissolve and make the solution that calculates Mei1mlZhong Han 3500 units by labelled amount, as need testing solution.According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.With two TSK G3000SWXL (7.8 * 300mm, 5 μ m), as analyzing chromatographic column, TSK SWXL (6.0mm * 40mm, 5 μ m) is as pre-column, flow velocity per minute 0.6ml, and column temperature is 25 ℃; Detection wavelength is 214nm.Get need testing solution 20 μ l injection liquid chromatographies, record chromatogram to 60 minute, chromatogram is analyzed.
Method four:
Mobile phase: (50mM phosphate, 150mM NaCl, pH7.0): acetonitrile=5:2
Get FSH bulk drug 22.54mg, with mobile phase, dissolve and make the solution that calculates Mei1mlZhong Han 3500 units by labelled amount, as need testing solution.According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.With two Superdex75 10/300GL series connection, be to analyze chromatographic column, flow velocity per minute 0.5ml, column temperature is 30 ℃; Detection wavelength is 215nm.Get need testing solution 100 μ l injection liquid chromatographies, record chromatogram to 90 minute, chromatogram is analyzed.Chromatogram as shown in Figure 1 to 4.
The HPLC purity testing result of above-mentioned four kinds of method FSH bulk drugs is:
Note: "---" mark represent this value because of peak shape too poor failing calculate.
From above-mentioned experimental result: method one and method two all have respectively impurity peaks to fail to detect, although method three is pressed the result of document, two TSK G3000SWXL series connection have had very large improvement to the detection of chorionic gonadotrophin purity, especially the separation of large molecular impurity is improved, but because the molecular weight (about 32KD) of follicular stimulating hormone is little compared with the molecular weight of chorionic gonadotrophin (about 44KD), and the chromatographic column of this kind is poor to the separating effect of the material of molecular weight, therefore, it is for the purity analysis of follicular stimulating hormone, its result is unsatisfactory, its content that detects small molecular weight impurity peak is 1.27%, and very poor with the degree of separation of main peak, cannot calculate USP degree of separation at all, therefore it is insincere that it detects data.This shows, method provided by the invention is optimal method.
Embodiment 2
The HPLC purity analysis of FSH bulk drug
Sample to be analyzed: FSH bulk drug (lot number is provided by Shanghai Tianwei Biological Pharmaceutical Corp.: 100401, biological value is in FSH 289.5IU/mg)
Method one: the liquid-phase chromatography method that this method adopts the chromatographic column of TSK G2000SWXL (7.8 * 300mm) and the chromatographic column of Superdex75 10/300GL to connect carries out the HPLC purity analysis of FSH.
Mobile phase: 50mM sodium dihydrogen phosphate, pH7.0-acetonitrile (80:20).
Get FSH bulk drug 28.72mg, with mobile phase, dissolve and make the solution that calculates Mei1mlZhong Han 3500 units by labelled amount, as need testing solution.With reference to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.With TSK G2000SWXL (7.8 * 300mm) and Superdex75 10/300GL series connection, be analysis chromatographic column, flow velocity per minute 0.5ml, column temperature is 25 ℃; Detection wavelength is 214nm.Get need testing solution 100 μ l injection liquid chromatographies, record chromatogram to 80 minute, chromatogram is analyzed.Chromatogram as shown in Figure 5.
Method two: mobile phase: 200mM Tris-HCl, 1500mM NaCl, pH7.0
Get FSH bulk drug 24.83mg, with mobile phase, dissolve and make the solution that calculates Mei1mlZhong Han 4000 units by labelled amount, as need testing solution.With reference to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.With two Superdex75 10/300GL series connection, be to analyze chromatographic column, flow velocity per minute 0.3ml, column temperature is 30 ℃; Detection wavelength is 215nm.Get need testing solution 100 μ l injection liquid chromatographies, record chromatogram to 90 minute, chromatogram is analyzed.Chromatogram as shown in Figure 6.
Above-mentioned two kinds of methods to the HPLC purity testing result of FSH bulk drug are:
Note: "---" mark represent this value because of peak shape too poor failing calculate.
From above-mentioned experimental result: TSK G2000SWXL and the detection of Superdex75 10/300GL series connection to FSH purity, although the separation to large molecular impurity is better, but the separating effect to the material of molecular weight is poor, although close with the measurement result value of method provided by the invention to the measurement result of the little molecule content of same batch sample, but this value is because degree of separation is bad, can only be as an estimated value, the meaning without quantitative test, therefore also cannot be for the purity analysis of follicular stimulating hormone.
Embodiment 3
The HPLC purity analysis of the former powder of FSH and bulk drug
Get the former powder of FSH or bulk drug, with mobile phase corresponding in following form, dissolve to make by labelled amount respectively and calculate the solution that contains a certain amount of unit in every 1ml, as need testing solution.With reference to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.With two Superdex75 10/300GL series connection, be to analyze chromatographic column, by changing some other liquid phase chromatogram condition, carry out liquid phase analysis.Record chromatogram, and chromatogram is analyzed.Wherein, the chromatogram of sequence number 1 experiment gained as shown in Figure 7.
Note: the experiment of comparative example 7 adopt two service time long Superdex75 10/300GL post, its post effect and theoretical cam curve have decline to a certain degree.
Each time HPLC purity testing result is:
Note: "---" mark represent this value because of peak shape too poor failing calculate.
By above-mentioned experiment, can find out, when adopting two Superdex75 10/300GL post series connection, carry out follicular stimulating hormone purity analysis, and the theoretical cam curve that guarantees main peak is not less than at 1800 o'clock, impurity degree of separation is higher than 1, thereby can determine more accurately the impurity content in follicular stimulating hormone.
In addition, by sequence number, be 8 and 9 measurement result contrast discovery, twice measurement result is identical, but because the flow velocity of sequence number 9 is slower, its main peak appearance time is 5 times of left and right of sequence number 8, is about 180min left and right, each minute is long.Therefore, too low flow velocity is not too suitable for conventional sense.
Embodiment 4
The HPLC purity analysis of FSH bulk drug
Sample to be analyzed: FSH bulk drug (by Shanghai Tianwei Biological Pharmaceutical Corp., produced lot number: 100201, biological value is in FSH 225.6IU/mg)
Method one: adopt the assay method of embodiment 1 method one, get FSH bulk drug 23.65mg, dissolve and make the solution that calculates Mei1mlZhong Han 500 units by labelled amount with mobile phase, measure as need testing solution.
Method two: adopt the assay method of embodiment 1 method four, get FSH bulk drug 27.42mg, dissolve and make the solution that calculates Mei1mlZhong Han 3500 units by labelled amount with mobile phase, measure as need testing solution.
The HPLC purity testing result of above-mentioned two kinds of method FSH bulk drugs is:
From above-mentioned experimental result: method one and method two differ less to the measurement result with the higher bulk drug of a collection of impurity content, this be because, when in FSH, impurity content is higher, as be greater than 3%, and while being less than 5%, method provided by the invention is equally effective with original assay method, only have when in FSH, impurity content is lower, if impurity content is lower than 3% time, more preferably impurity content is lower than 2% time, the measurement result of original assay method is obviously on the low side, is no longer suitable for the mensuration of such material.
The preparation of embodiment 5 FSH bulk drugs
Get respectively the former powder of FSH of 120601 and 111,201 two batches in embodiment 1, embodiment 3, with reference to the analytical approach of embodiment 3, carry out purity detecting, its impurity content and corresponding quality requirements are contrasted:
By contrast, find, 120601 batches of former powder of FSH meet our quality requirements, can be for the preparation of FSH bulk drug.And the 111201 batches of former powder of FSH do not meet our quality requirements, need to again carry out purifying further to remove impurity, thereby reach quality requirements.
According to the method for Chinese patent CN101269215B embodiment 1, with 120601 batches of former powder of FSH, be that raw material is prepared FSH bulk drug.
With reference to the analytical approach of embodiment 3, measure the impurity content of the FSH bulk drug after freeze-drying.Measurement result is as follows:
| Peak title | Quality requirements | FSH bulk drug after freeze-drying |
| Large molecular impurity | ≤1% | 0.24% |
| Small molecular weight impurity | ≤1.5% | 1.17% |
| Total impurities | ≤2% | 1.41% |
By the present embodiment, can find out: the former powder of FSH after highly purified, the content of its impurity still likely exceeds our quality requirements, if directly feeding intake, underproof former powder product carries out the preparation of bulk drug, the final product obtaining is also by defective, this will cause significant wastage, increase production cost.Therefore, adopt method for detecting purity provided by the invention to carry out quality control, thereby reprocess immediately the appearance that can avoid underproof finished product after finding that there is underproof product.
, 111201 batches of former powder of FSH again after purifying, are measured with reference to the analytical approach of embodiment 3, measurement result is as follows meanwhile:
By contrast, find, the 111201 batches of former powder of FSH, after purifying again, can conform to quality requirements, and again according to the method for Chinese patent CN101269215B embodiment 1, using 111201 batches of former powder of FSH after purifying is again that raw material is prepared FSH bulk drug.
With reference to the analytical approach of embodiment 3, measure the impurity content of the FSH bulk drug after freeze-drying.Measurement result is as follows:
| Peak title | Quality requirements | FSH bulk drug after freeze-drying |
| Large molecular impurity | ≤1% | 0.62% |
| Small molecular weight impurity | ≤1.5% | 0.67% |
| Total impurities | ≤2% | 1.29% |
As can be seen here, by method provided by the invention, can avoid the generation of underproof FSH bulk drug.
Embodiment 6 is containing the preparation of FSH lyophilized injection
The FSH bulk drug that embodiment 5 is obtained is measured purity according to the analytical approach of embodiment 3, and its impurity content and corresponding quality requirements are contrasted:
| Peak title | Quality requirements | The actual content of this crowd of FSH |
| Large molecular impurity | ≤1% | 0.25% |
| Small molecular weight impurity | ≤1.5% | 1.16% |
| Total impurities | ≤2% | 1.41% |
By contrast, find, this batch of FSH bulk drug meets our quality requirements, can be for the preparation of FSH composite injection.
With water for injection preparation, contain in phosphate (pH7.0) damping fluid of 10mM, by 30g sucrose dissolved in this phosphate buffer of 400mL, if needed, with HCl or NaOH, regulate pH7.0 ± 0.2, then add FSH medicine material medicine in above-mentioned a certain amount of embodiment 4 in this solution, with water for injection, be settled to 500mL, mix, with 0.22 μ m filtrator, carry out aseptic filtration.
Above-mentioned solution is distributed in injection bottle made, and every bottle of 0.5mL, carries out freeze drying.
In resulting injection bottle made, every bottle containing 75IU FSH and 30mg sucrose.
By above-mentioned comparative example and embodiment, can find out, method provided by the invention not only can detect more impurity than existing method, and the degree of separation of impurity is also significantly improved, so to impurity quantitatively and can be more accurate to the purity analysis of major component.
The foregoing is only preferred embodiment of the present invention, not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is to be broadly defined in the claim scope of application, any technology entity or method that other people complete, if defined identical with the claim scope of application, also or a kind of change of equivalence, be all regarded as being covered by among this claim scope.
Claims (15)
1. one kind by the method for promoting sexual gland hormone purity in HPLC working sample, it is characterized in that, with molecular exclusion chromatography post, described sample is carried out to high-pressure liquid chromatography, thereby record the promoting sexual gland hormone purity in described sample, the filler of wherein said molecular exclusion chromatography post comprises cross-link dextran, Sepharose or its combination;
And, main peak USP theoretical cam curve >=1600 of described molecular exclusion chromatography post.
2. method for detecting purity as claimed in claim 1, it is characterized in that, described molecular exclusion chromatography post is that glucosan, agarose or its mix the chromatographic column that cross-linking agent is filler, is preferably the chromatographic column that is filler by agarose and the crosslinked Superdex forming of glucosan.
3. method for detecting purity as claimed in claim 1, is characterized in that, described theoretical cam curve is not less than 1700, is more preferably not less than 1800.
4. the method as described in as arbitrary in claim 1～3, is characterized in that, the mobile phase using in described assay method is acetonitrile and buffer salt solution.
5. the method as described in as arbitrary in claim 1～3, is characterized in that, the flow rate of mobile phase using in described assay method is 0.1～1.0ml/ minute.
6. the method as described in as arbitrary in claim 1～3, is characterized in that, the detection wavelength using in described assay method is 200～300nm.
7. method as claimed in claim 4, is characterized in that, in described mobile phase, acetonitrile is 0～1/2 to the volume ratio of water.
8. method as claimed in claim 4, is characterized in that, described buffer salt is selected from lower group: phosphate, Tris, acetate, sodium chloride, or its combination; Preferably, described buffer salt is selected from lower group: phosphate, sodium chloride, or its combination.
9. method as claimed in claim 4, is characterized in that, described buffer salt solution concentration is 10mM～1700mM, is preferably 100mM～300mM.
10. method as claimed in claim 4, is characterized in that, the pH of described buffer salt solution is 5.0～10.0.
11. the method for claim 1, is characterized in that, described promoting sexual gland hormone comprises the one or more compositions that are selected from lower group: human chorionic gonadtropin, HMG, follicular stimulating hormone, lutropin; Preferably, described promoting sexual gland hormone comprises follicular stimulating hormone.
12. methods as described in as arbitrary in claim 1～11, is characterized in that, described method also comprises following one or more conditions:
A, high pressure liquid chromatography system comprise the Superdex7510/300GL post of two series connection;
B, mobile phase: acetonitrile: buffer salt solution=1:20～2:5, wherein said ratio is volume ratio;
C, flow velocity: 0.2～0.8ml/min,
D, column temperature: 25～45 ℃;
E, detection wavelength: 210～235nm;
F, sample introduction concentration: 3000～4500IU/ml.
13. assay methods as claimed in claim 1, is characterized in that, described sample is the former powder of gonadotrophins or gonadotrophins bulk drug.
14. 1 kinds of methods of preparing gonadotrophins bulk drug, is characterized in that, the preparation process of described gonadotrophins bulk drug comprises following steps:
A, use method for detecting purity claimed in claim 1, measure the purity of the former powder of gonadotrophins;
If the measurement result of B step (A) shows total impurities content > 2% in former powder, described former powder is re-started to purifying, until measurement result shows total impurities content≤2%;
If the measurement result of C step (A) shows total impurities content≤2% in this former powder, in the former powder of this gonadotrophins, add the excipient of pharmaceutically acceptable amount, make gonadotrophins bulk drug.
15. 1 kinds of methods of preparing gonadotrophins pharmaceutical preparation, is characterized in that, described gonadotrophins pharmaceutical preparation preparation process comprises following steps:
A, use method for detecting purity claimed in claim 1, measure the purity of gonadotrophins bulk drug;
If the measurement result of B step (A) shows total impurities content≤2% in this bulk drug, in this gonadotrophins bulk drug, add the excipient of pharmaceutically acceptable amount, make gonadotrophins pharmaceutical preparation.
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| WO2001062773A1 (en) * | 2000-02-22 | 2001-08-30 | Applied Research Systems Ars Holding N.V. | PROCESS OF PURIFICATION OF hCG AND RECOMBINANT hCG PURIFIED BY THAT METHOD |
| CN102038649A (en) * | 2009-10-20 | 2011-05-04 | 丽珠医药集团股份有限公司 | Urine-promoted follicle stimulating hormone freeze-drying powder injection and preparation method thereof |
| CN102656182A (en) * | 2009-11-24 | 2012-09-05 | 葛莱高托普有限公司 | Process for the purification of glycoproteins |
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2013
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| WO2001062773A1 (en) * | 2000-02-22 | 2001-08-30 | Applied Research Systems Ars Holding N.V. | PROCESS OF PURIFICATION OF hCG AND RECOMBINANT hCG PURIFIED BY THAT METHOD |
| CN102038649A (en) * | 2009-10-20 | 2011-05-04 | 丽珠医药集团股份有限公司 | Urine-promoted follicle stimulating hormone freeze-drying powder injection and preparation method thereof |
| CN102656182A (en) * | 2009-11-24 | 2012-09-05 | 葛莱高托普有限公司 | Process for the purification of glycoproteins |
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