CN105111305B - The chromatographic purification method of acylated insulin - Google Patents
The chromatographic purification method of acylated insulin Download PDFInfo
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Abstract
The invention discloses a kind of chromatographic purification methods of acylated insulin, belong to the preparation field of Recombulin or insulin analog.The chromatographic purification method of acylated insulin of the present invention is using advanced silica-gel carrier (such as C4, C8 and C18 alkane is the reverse phase filler of aglucon) as chromatographic stuffing; acylated insulin is finely isolated and purified using high pressure chromatographic system; in the case where pH is the alkaline eluant environment of 6.5-8.0; can a step crude samples purity is improved by 50-60% to 99.0% or more, sample stable yield is 90% or more after purification.In addition, the method for the present invention study sample applied sample amount is big, it is greatly saved purifying cost；Linear flow rate is high in chromatographic purification process, can accomplish the fast purifying to insulin analog.The method of the present invention is easy to operate, reliable and stable, have it is stronger can amplification, be suitable as the chromatographic purification method of preparation of industrialization insulin analog.
Description
Technical field
The present invention relates to a kind of Reverse phase chromatography methods of acylated insulin, belong to Recombulin or insulin type seemingly
The preparation field of object.
Background technique
Diabetes are a kind of common metabolism endocrine system diseases, seriously endanger human health.Acylated insulin is in one kind
Property, soluble, Recent Development of Long-acting Insulin Analogs, such as insulin detemir, i.e. lysine B29N (ε-myristoyl) remove (B30) people's pancreas
Island element is first method using chemical modification, with can be with the fatty-acylation peptide chain of the albumin Reversible binding in blood
On amino acid and generate.Acylated fatty acid can stablize the self assemble of insulin molecule, and in conjunction with albumin invertibity,
Acidylate insulin to absorb slowly from subcutaneous infusion sites, acting duration extends, and can be reduced while controlling blood glucose low
The risk of blood glucose.
In the development history of insulin, the promotion for being introduced as insulin quality of preparative efficient liquid phase technology is played very
Crucial effect so that the insulin that is extracted from animal and using gene recombination technology preparation actrapid monotard purity from
90% or so promotes property and the very close related substances of insulin even higher to 98%, including deamination products
Content is also well controlled, and the safety of insulin is further protected.Current main chromatogram purification technique has two
Kind: first is that by selecting organic polymer fillers to carry out the purifying of insulin or insulin analog, such as Chinese patent
Source 15RPC, Source 30RPC organic polymer fillers are used in CN103275210A, in eluent pH 2-4 environment
Under effectively a kind of monoacylated insulin of fatty acid can be purified, sample purity can reach 98% after purification, sample yield
It is 71.9%；Second is that by selecting advanced silica-gel carrier filler to carry out the purifying of insulin or insulin analog, such as China specially
In sharp CN1171742A using C4 filler 50mm prepare carried out on column it is acylated after insulin purifying, sample purity is not after purification
Know, sample yield is 40.2%.
Organic polymer fillers generally use medium/low-voltage equipment, and advanced silica-gel carrier generally uses high pressure tomography devices.
From the point of view of comparing, although lower using the cost of equipment maintenance that organic polymer fillers are purified, have many apparent
Disadvantage: first, the partial size (generally at 30 microns or more, big to several hundred microns) of organic polymer fillers is carried than common advanced silica gel
Body packing material size (generally between 5-20 microns) is much larger, since the pressure-resistant performance of medium itself is poor, fills column process pressure-bearing energy
Power is limited, the column interior medium filled will not dense uniform, the theoretical cam curve of measurement do not exceed 20000 generally, newly
Filling chromatographic column column effect it is not high, it is lower so as to cause sample separating degree, after purification sample purity be relatively inaccessible to 98% or more and
Yield is lower, this will obviously increase the production cost of product；Second, organic polymer fillers partial size generally between 5-300 μm,
Particle uniformity is poor, and resistance to pressure is lower, and the broken of filler can be all caused in filling chromatographic column and purification process, so as to cause
It generates to collapse inside chromatographic column and causes column effect decline, separating degree reduces, and further results in yield decline.
From the point of view of comparing, the pillar of advanced silica-gel carrier filler filling, column effect generally can achieve 40000 or more, and filler
It is resistant to 10MPa pressure, non-friable, long-term purification experiment rear pillar effect is more stable, will not reduce；And in higher proof pressure
Under, the linear flow rate in purification process can be higher, can accomplish the fast purifying to insulin analog, preferably avoids in color
It composes purpose product in purification process and adverse effect, such as deamination reaction occurs, to influence sample purity and yield.
Therefore, establish that a kind of method is simple and direct, the purity and high income of product, have it is stronger can amplification, be suitable as
The chromatographic purification method of industrialized purification acylated insulin or its analog, will be with important value and application prospect.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of chromatographic purification method of acylated insulin, this method can one
Step is improved the purity of crude samples to 99.0% or more by 50-60%, and sample stable yield has 90% or more after purification
It is stronger can amplification, be suitable as industrialized purification acylated insulin or the chromatographic purification method of its analog.
In order to solve the above technical problems, the technical solution used in the present invention is:
The present invention discloses a kind of chromatographic purification method of acylated insulin first, comprising the following steps: (1) with advanced silicon
Glue carrier is balanced as chromatographic stuffing, by chromatographic column with equilibrium liquid, by acylated insulin crude product loading to be purified, with flat
The liquid that weighs balances chromatographic column；(2) it is eluted with elution with mobile phase, collects main peak, obtain purpose product acylated insulin sterling；
Wherein, elution mobile phase includes A phase and B phase；The elution is 6.5-8.0 with the pH of mobile phase A phase, it is preferred that described to wash
Taking off with the pH of mobile phase A phase is 7.0-8.0, most preferably pH 7.0.
Wherein, the A phase is that phosphate buffer, disodium hydrogen phosphate-citrate buffer solution, trishydroxymethylaminomethane are slow
Any one or more in fliud flushing, boric acid-borate buffer solution, preferably phosphate buffer.The present invention is by adjusting phosphoric acid
The ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate in salt buffer, reaches pH described in A phase；Either by the way that hydrochloric acid or hydrogen-oxygen is added
Change the pH value that sodium adjusts A phase.
The B phase includes organic solvent and water；Wherein the organic solvent is acetonitrile or carbon atom number is in the alcohol of C1-C4
Any one or more, preferably acetonitrile or ethyl alcohol；It is furthermore preferred that the B phase is by acetonitrile and water, 9:1 is formed by volume；
Alternatively, by second alcohol and water, 9:1 is formed by volume.
Chromatographic purification method of the present invention, the aglucon of step (1) the advanced silica-gel carrier (i.e. chromatographic stuffing) are C4-C18
Alkane, preferably C8 alkane；Wherein, the aglucon is the alkane being bonded on the surface of advanced silica-gel carrier.Institute of the present invention
The filling pressure for stating advanced silica-gel carrier filler is 7-10MPa.The average particle size of the chromatographic stuffing is 5-30 μm, preferably 5-
10μm.Filler particle size is smaller, and column effect is higher, and Sample Purification on Single separating degree is higher.Step (1) described equilibrium liquid includes A phase and B phase,
With in elution mobile phase A phase and B phase it is identical；According to volume basis, step (1) equilibrium liquid by 75%-90% A phase
With the B phase composition of 10%-25%, preferably by 80% A phase and 20% B phase composition.
According to volume basis, step (2) the elution mobile phase is by the A phase of 50%-60% and the B phase of 40%-50%
Composition, preferably by 55% A phase and 45% B phase composition.The mode of step (2) described elution be isocratic elution mode (i.e.
The constant rate of A phase and B phase during elution) or linear gradient elution mode any one.
The present invention has investigated influence of the different aglucons of chromatographic stuffing to acylated insulin separating effect, the results showed that C4,
Tri- kinds of aglucons of C8, C18 can reach preferable purification effect, and sample purity reaches 97.89%-99.28% after purification, and purifying is received
Rate is 85.5%-91.5%；Wherein C8 aglucon is better than other two kinds of filler aglucons for the purification effect of acylated insulin, and has
Significant difference on statistically significant.
The present invention further investigates the influence under the different pH elution requirement of A phase to sample separating effect, the results showed that,
Item is eluted significantly better than slant acidity (pH3.5) to the purification effect of destination protein under the elution requirement of A phase meta-alkalescence (pH7.5)
Part, sample purity is higher after purification, and up to 99.25%, and purifying yield is also higher, up to 90.9%.Therefore, it is considered herein that for
Acylated insulin is purified using advanced silica-gel carrier filler, is easier to obtain purity and yield all using the elution requirement of meta-alkalescence
Higher sample.Meanwhile the present invention optimizes pH condition around the elution requirement of A phase pH7.5 again, the results showed that when A phase
When pH6.5 to 8.0, destination protein purity after purification and yield are all higher, reach 98.89%-99.55% and 88.1%- respectively
93.9%, especially when A phase pH is 7.0, the destination protein purity highest eluted, and purify yield also highest.
It is generally acknowledged that advanced silica-gel carrier filler can achieve best purification effect under the conditions of acidic elution in this field.
Chinese patent CN 1313866A is disclosed can achieve under acid (pH3.5) elution requirement using advanced silica-gel carrier filler
Best purification effect, sample purity is greater than 98.5% after purification, yield 68%；And use organic polymer for stationary phase,
It is eluted under the conditions of alkaline (pH9.0), sample purity is greater than 98.5% after purification, yield 64%-73%.It is pure by the way of chromatography
When changing acylated insulin crude product, when purity reaches 98% or more, under the premise of not influencing yield, even if purity is improved
0.5 percentage point is also very difficult.Compared with published related Chinese patent is in main technologic parameters and purification effect
Compared with the chromatographic purification method of acylated insulin of the present invention, by using advanced silica-gel carrier filler, (aglucon is the alkane of C4-C18
Reverse phase silica gel filler), under alkaline (pH 6.5-8.0) eluent environment, can a step by the purity of study after acylation by 50-
60% raising is to 99.0% or more and sample stable yield is 90% or more after purification, and sample purity and yield are all remote high after purification
In prior art achievement obtained.In addition, the applied sample amount of study sample of the present invention is big, it is long-range more than 16 grams per liter bed volumes
The insulin analog chromatogram purification applied sample amount mentioned in published patent or technology；Linear flow in chromatographic purification process
Speed is up to 250cm/h, can accomplish the fast purifying to insulin analog.
Sample to be purified of the present invention is by going the actrapid monotard of B30 (will to be naturally arranged on actrapid monotard's B chain
30 threonines remove) and selected from any one following aliphatic ester react acylated insulin solution obtained: palmitinic acid
N- succinimide base ester, octanoic acid N- succinimide base ester or myristic acid N- succinimide base ester.Purpose after reaction
Product (i.e. acylated insulin) is that aliphatic ester is reacted with the epsilon-amino of the B29-Lys for the actrapid monotard for removing B30 and with amido bond
The product being connected to form, while there is also many impurity in reaction solution, such as product of non-purpose site modification and incomplete
Raw material (aliphatic ester and the actrapid monotard for removing B30) of reaction etc..Actrapid monotard is made of small point acid 51 amino acid
Sub- protein, molecular formula C257H383N65O77S6, molecular weight 5807.69.And actrapid monotard of the present invention includes: organic synthesis
Actrapid monotard or by genetic engineering prepare rh-insulin；Wherein, the actrapid monotard of organic synthesis is according to people's pancreas
The structure artificial of island element synthesizes；Rh-insulin is expressed using genetically engineered biological fermentation (such as Escherichia coli, saccharomycete)
Actrapid monotard.
The method for synthesizing acylated insulin is known in the art, such as may refer to Chinese patent CN
1171742A or paper " technical study that Recombulin precursor is converted to actrapid monotard and insulin detemir ", Liu Haifeng, China
Eastern Polytechnics, method disclosed in Ph.D. Dissertation (2013).
The chromatographic purification method of acylated insulin of the present invention be using purpose product acylated insulin in sample after acylated with
Other impurities relative hydrophobic sex differernce in specific stationary phase and mobile phase is purified.The method of the present invention simply may be used
Row, study sample applied sample amount is big, after purification sample purity height and high income, and purifying is at low cost, and process stabilizing is reliable, is suitable for work
The amplification of industry produces.
The method of the present invention is suitable for analysis chromatography, half preparation chromatography, especially preparative scale chromatography.
Technical solution of the present invention compared with prior art, mainly have following several respects the utility model has the advantages that
It (1) can a step under alkaline (pH7.0) eluent environment as chromatographic stuffing by using advanced silica-gel carrier
Study purity after acylation is improved by 50-60% to 99.0% or more, and sample stable yield is 90% or more after purification, purifying
Sample purity and yield are all much higher than prior art achievement obtained afterwards.
(2) applied sample amount of study sample to be purified in the present invention is big, public much larger than more than 16 grams per liter bed volumes
The insulin analog chromatogram purification applied sample amount mentioned in the patent or technology opened, is greatly saved purifying cost.
(3) linear flow rate is up to 250cm/h in chromatographic purification process of the present invention, can accomplish to the quick of insulin analog
Purifying preferably avoids the purpose product in chromatographic purification process that adverse effect, such as deamination reaction occurs, to influence sample
Purity and yield.
The term definition involved in the present invention arrived
Unless otherwise defined, otherwise all technologies used herein and scientific term all have with it is of the art
Those of ordinary skill usually understands identical meaning.
Term " reverse phase silica gel " is the silica material that surface is coated with hydrophobic matrix, and wherein hydrophobic matrix can be alkane
Hydrocarbon；" coating " functional group i.e. different using the Si-OH group bonding of Silica Surface, becomes suitable for different clastotypes
Chromatograph packing material；Silica gel can be generally chemically modified by three kinds of approach to prepare silica gel bonded phase: coating, entirety
The chemical modification method of modification method and surface Si-OH.In the present invention, term " reverse phase silica gel " and term " advanced silica-gel carrier "
Meaning is identical, and the two can be interchanged.
Term " preparation chromatography " is interpreted as preparing net product at industrial scale.
Detailed description of the invention
Fig. 1 is to use in embodiment 1The chromatogram purification map of the acylated insulin of filler；
Fig. 2 is to use in embodiment 1The chromatogram purification map of the acylated insulin of filler；
Fig. 3 is to use in embodiment 1The chromatogram purification map of the acylated insulin of filler；
Fig. 4 is the chromatogram purification map for the acylated insulin that the A phase in embodiment 2 using pH7.5 is eluted；
Fig. 5 is the chromatogram purification map for the acylated insulin that the A phase in embodiment 2 using pH3.5 is eluted；
Fig. 6 is the chromatogram purification map of acylated insulin in embodiment 3；
Fig. 7 is the chromatogram purification map of acylated insulin in embodiment 4；
Fig. 8 is the chromatogram purification map of acylated insulin in embodiment 5.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1, test material and instrument
The buffer materials such as trishydroxymethylaminomethane (Tris) are purchased from Amresco company, anhydrous sodium sulfate (Na2SO4)、
Hydrochloric acid (HCl) and sodium hydroxide (NaOH) are purchased from Sinopharm Chemical Reagent Co., Ltd.；Acetonitrile (CH3CN, HPLC grades and preparation
Chromatographic grade) etc. other organic solvents be purchased from J&K Chemical company；Semi-preparative chromatographic column (50mm × 250mm), filler All it is purchased from Sweden Kromasil company；Point
Analysis type chromatographic column (4.6mm × 150mm) filler isPurchased from Kromasil company；The efficient liquid of analytic type
Phase instrument is Agilent 1260, and preparative efficient liquid phase instrument is Dalian Yi Lite P270.
Sample purity detection method:
It is detected using sample of the reversed phase chromatography method to study and purified pool, instrument is the analysis of Agilent 1260
Type efficient liquid phase, pillar use Kromasil C4-3.5 μm chromatographic column (4.6mm × 150mm).Mobile phase A phase: the anhydrous sulphur of 10mM
Sour sodium-acetonitrile (75:25 is mixed by volume), using salt acid for adjusting pH to 3.5；Mobile phase B phase: acetonitrile-water is (by volume
55:45 mixing).Flow velocity is 1.0ml/min, and column temperature is 40 DEG C, Detection wavelength 214nm.The elution of 25-50%B phase gradient
35min.Retention time is purpose product in 27-29 minutes main peaks.Purity, which calculates, uses area normalization method.
The preparation of 1 acylated insulin of Preparative Example
The preparation method of acylated insulin solution is referring to method disclosed in Chinese patent CN 1171742A.
The actrapid monotard (BHI) of biosynthesis is crystallized into (71.9mg) and is dissolved in 6.58mL DMSO.Solution is stirred at room temperature
It mixes to crystal all dissolutions (visually observing).20mg active ester solid is added in 2mL DMSO and is vigorously stirred to all work
Property all dissolutions (visually observing) of ester particle, the solution of active ester (palmitinic acid N- succinimide base ester) is made.At this point, by 1,
1,3,3- tetramethylguanidine (26.8 μ l) is added in 5mL BHI solution, and DMSO (94.4mL) and previously prepared activity is then added
Ester solution (400 μ l).Reaction carries out about 60 minutes for (20 to 25 DEG C) at room temperature.It samples after 15 minutes, is diluted with 1N acetic acid
It 20 times and is analyzed with HPLC.With the B terminated in sample29-NεThe amount of palmitoyl human insulin divided by initial BHI amount,
Calculating reaction yield is 67.1%.
Embodiment 1 is investigated different with influence of the based filler to separating effect
The study sample is Preparative Example 1 by going actrapid monotard and the palmitinic acid N- succinimide base ester of B30
Acylated insulin solution made from single step reaction, study purity are 55%；Chromatographic purification method using different silica filler ( ) in semi-preparative chromatographic column (50mm
× 250mm) on carry out, filling pressure be 7-10MPa；Elution mobile phase A phase: 20mmol/L phosphate buffer is adjusted slow
The ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate in fliud flushing makes A phase pH 7.0；Elution Mobile phase B phase: acetonitrile-water (presses body
Product is mixed than 9:1)；Keeping column temperature is 20-25 DEG C；Elution flow rate is 82.5ml/min；Detection wavelength is 280nm.
(1) 20%B phase (i.e. A phase for 80%, volume ratio), which is used, as equilibrium liquid balances pillar to baseline stability.
(2) then 8.1g acylated insulin study sample loading pillar balances pillar to baseline stability using equilibrium liquid.
(3) use the isocratic elution mode of 45% B phase (i.e. A phase for 55%, volume ratio) that will be adsorbed on each on pillar
Kind substance elutes, and is collected after 280nm UV detector monitoring appearance to main peak.
Test result is shown in Table 1.
Influence of the different fillers of table 1 to separating effect (purity and yield)
From experimental result as can be seen thatFiller is best for the purification effect of acylated insulin,
Sample purity is higher after purification for performance, reaches 99.28%, and it is also higher to purify yield, therefore preferably C8 filler is as acylation
The purifying filler of insulin.
Embodiment 2 investigates the influence under the different pH elution requirement of A phase to sample separating effect
Study sample is as made from rh-insulin and the palmitinic acid N- succinimide base ester single step reaction for removing B30
Acylated insulin solution, for preparation method with Preparative Example 1, study purity is 56%；Chromatographic purification method uses advanced silica gel
Carrier () carried out on semi-preparative chromatographic column (50mm × 250mm), filling pressure is 7-10MPa；It washes
De- mobile phase A phase: 20mmol/L disodium hydrogen phosphate-citrate buffer solution adjusts pH difference using hydrochloric acid or sodium hydroxide
For 3.5 and 7.5；Elution Mobile phase B phase: acetonitrile-water (9:1 is mixed by volume)；Keeping column temperature is 20-25 DEG C；It washes
Separation of flow speed is 82.5ml/min；Detection wavelength is 280nm.
The specific steps are the same as those in embodiment 1, will be under the various substances that be adsorbed on pillar elution using linear gradient elution mode
Come, main peak is collected after 280nm UV detector monitoring appearance.
Test result is shown in Table 2.
Influence of the table 2A phase difference pH elution requirement to sample separating effect (purity and yield)
From experimental result it can be seen that under the elution requirement of A phase meta-alkalescence (pH7.5), to the purification effect of destination protein
Significantly better than under slant acidity (pH3.5) elution requirement as a result, being mainly manifested under the elution requirement that A phase is pH7.5, after purification
Sample purity is higher, reaches 99.25%, and purifying yield is also higher, up to 90.9%.Therefore, it is considered herein that for using high
Grade silica-gel carrier filler purifies acylated insulin, is easier to obtain purity using the elution requirement of meta-alkalescence and yield is all higher
Sample.
The present invention optimizes pH condition further around the elution requirement of A phase pH7.5 again, select pH6.5,7.0,
7.5 and 8.0 4 pH conditions carry out purification experiment, and test process is consistent with more than.
Test result is shown in Table 3.
Influence under table 3A phase difference pH elution requirement to sample separating effect (purity and yield)
In terms of 3 test result of table, as A phase pH6.5 to 8.0, destination protein purity after purification and yield are all higher,
Especially when A phase is pH7.0, the destination protein purity highest eluted up to 99.55%, and purifies yield also highest, and have
Significant difference on statistically significant.Thus, it is believed that A phase pH7.0 is optimal pH condition.
3 Fractional Collections sample of embodiment determines sample purity and yield after purification
Study sample is as made from rh-insulin and the palmitinic acid N- succinimide base ester single step reaction for removing B30
Acylated insulin solution, method is the same as Preparative Example 1；Chromatographic purification method using advanced silica-gel carrier filler () carried out on semi-preparative chromatographic column (50mm × 250mm), filling pressure is 7-10MPa；Elution is used
Mobile phase A phase: 20mmol/L phosphate buffer adjusts the ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate in buffer, makes A phase
PH is 7.0, elution Mobile phase B phase: acetonitrile-water (9:1 is mixed by volume)；Keeping column temperature is 20-25 DEG C；Elution stream
Speed is 82.5ml/min；Detection wavelength is 280nm.
The specific steps are the same as those in embodiment 1, is eluted the various substances being adsorbed on pillar using isocratic elution mode,
280nm UV detector monitors Fractional Collections after appearance, by one sample of collection per minute.
Test result is shown in Table 4.
Table 4 is summarized by the receipts peak situation and each sample analysis detection result of Fractional Collections sample per minute
Sample study carries out analysis detection, and purity 57% mixes 2# to 6# sample, carries out analysis detection, and purity is
99.49%.Meanwhile according to 4 result of table it is found that the total recovery of 2# to 6# sample is 92.4%.Illustrate the side according to embodiment 3
Method, it is 92.4% that purity, which reaches 99.49% sample yield, after purification.Meanwhile it being also known according to 4 result of table, 1# to 8# sample
Total recovery be 99.5%, illustrate that almost all of destination protein is all eluted, yield result is accurate.
The preparative scale chromatography purification process of 4 acylated insulin of embodiment
Study sample is Preparative Example 1 by going rh-insulin and the palmitinic acid N- succinimide base ester one of B30
Step reacts acylated insulin solution obtained, and method is the same as Preparative Example 1；Chromatographic purification method is chromatographed using advanced silica-gel carrier
Filler () carried out on semi-preparative chromatographic column (50mm × 250mm), filling pressure is 7-10MPa；
Elution mobile phase A phase: 40mmol/L Tris buffer, using hydrochloric acid and sodium hydroxide to adjust A phase pH is 8.0, and elution is with flowing
Dynamic phase B phase: alcohol-water (9:1 is mixed by volume)；Keeping column temperature is 20-25 DEG C；Elution flow rate is 82.5ml/min；
Detection wavelength is 280nm.
The specific steps are the same as those in embodiment 1, is eluted the various substances being adsorbed on pillar using isocratic elution mode,
Main peak is collected after 280nm UV detector monitoring appearance.
Test result are as follows: sample study carries out analysis detection, and purity 54%, sample purity is 99.17% after purification, receives
Rate is 91.3%.
The preparative scale chromatography purification process of 5 acylated insulin of embodiment
Study sample is as made from rh-insulin and the palmitinic acid N- succinimide base ester single step reaction for removing B30
Acylated insulin solution, method is the same as Preparative Example 1；Chromatographic purification method using advanced silica-gel carrier chromatographic stuffing () carried out on semi-preparative chromatographic column (50mm × 250mm), filling pressure is 7-10MPa；Elution is used
Mobile phase A phase: 200mmol/L boric acid-borate buffer solution uses hydrochloric acid and sodium hydroxide to adjust A phase pH as 7.5；Elution stream
Dynamic phase B phase: acetonitrile-water (9:1 is mixed by volume)；Keeping column temperature is 20-25 DEG C；Elution flow rate is 82.5ml/min；
Detection wavelength is 280nm.
The specific steps are the same as those in embodiment 1, will be under the various substances that be adsorbed on pillar elution using linear gradient elution mode
Come, main peak is collected after 280nm UV detector monitoring appearance.
Test result are as follows: sample study carries out analysis detection, and purity 56%, sample purity is 99.11% after purification, receives
Rate is 90.8%.
1 present invention of test example is compared with related Chinese patent is in main technologic parameters and purification effect
The chromatographic purification method of acylated insulin of the present invention and published related Chinese patent in main technologic parameters and
The comparison of purification effect is shown in Table 5.
The comparison result of 5 main technologic parameters of table and purification effect
The present invention is by using advanced silica-gel carrier filler, can step general under alkaline (pH7.0-8.0) eluent environment
Study purity is improved by 50-60% to 99.0% or more after acylation, and sample stable yield is 90% or more after purification, after purification
Sample purity and yield are all much higher than prior art achievement obtained.
In addition, the applied sample amount of study sample of the present invention is big, more than 16 grams per liter bed volumes, it is much larger than published patent
Or the insulin analog chromatogram purification applied sample amount mentioned in technology, it is greatly saved purifying cost；In chromatographic purification process
Linear flow rate is up to 250cm/h, can accomplish the fast purifying to insulin analog, preferably avoid in chromatographic purification process
Adverse effect, such as deamination reaction occur for purpose product, to influence sample purity and yield.
Claims (9)
1. a kind of chromatographic purification method of acylated insulin, comprising the following steps: (1), will using silica-gel carrier as chromatographic stuffing
Chromatographic column is balanced with equilibrium liquid, by acylated insulin crude product loading to be purified, balances chromatographic column with equilibrium liquid；(2) it uses
Elution is eluted with mobile phase, is collected main peak, is obtained purpose product acylated insulin；Wherein, elution mobile phase by A phase and
B phase composition；It is characterized by: the pH value of the A phase is 7.0-8.0；
According to volume basis, step (1) equilibrium liquid is by the A phase of 75%-90% and the B phase composition of 10%-25%；
The chromatographic stuffing is selected from any one in C4-5 μm -100, C8-5 μm -100 or C18-5 μm -100；The A phase
For phosphate buffer, disodium hydrogen phosphate-citrate buffer solution, TRIS buffer or boric acid-borax buffering
Any one in liquid；The B phase is made of acetonitrile or ethyl alcohol with water；
The acylated insulin crude product to be purified is actrapid monotard by removing B30 and selected from any one following aliphatic ester
React acylated insulin solution obtained: palmitinic acid N- succinimide base ester, octanoic acid N- succinimide base ester or nutmeg
Sour N- succinimide base ester.
2. chromatographic purification method described in accordance with the claim 1, it is characterised in that: the pH value of the A phase is pH 7.0.
3. chromatographic purification method described in accordance with the claim 1, it is characterised in that: the B phase is by acetonitrile and water 9:1 by volume
Composition；Or 9:1 is formed by volume by second alcohol and water.
4. chromatographic purification method described in accordance with the claim 1, it is characterised in that: the average particle size of the chromatographic stuffing is 5-30
μm。
5. chromatographic purification method according to claim 4, it is characterised in that: the average particle size of the chromatographic stuffing is 5-10
μm。
6. chromatographic purification method described in accordance with the claim 1, it is characterised in that: the equilibrium liquid is by 80% A phase and 20% B
Phase composition.
7. chromatographic purification method described in accordance with the claim 1, it is characterised in that: according to volume basis, step (2) described elution
With mobile phase by the A phase of 50%-60% and the B phase composition of 40%-50%；
The mode of the elution is isocratic elution mode or linear gradient elution mode.
8. chromatographic purification method according to claim 7, it is characterised in that: step (2) the elution mobile phase is by 55%
A phase and 45% B phase composition.
9. chromatographic purification method described in accordance with the claim 1, which is characterized in that the actrapid monotard includes: organic synthesis
Actrapid monotard or rh-insulin.
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| CN112341535A (en) * | 2019-08-07 | 2021-02-09 | 中国科学院大连化学物理研究所 | Method for preparing insulin by separation and purification through ion exchange chromatography |
| CN111778231A (en) * | 2020-07-29 | 2020-10-16 | 珠海冀百康生物科技有限公司 | Purification method of lysyl endopeptidase |
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| CN1313866A (en) * | 1998-08-24 | 2001-09-19 | 阿文蒂斯药物德国有限公司 | Method for chromatographically purifying insulins |
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