CN106554949A

CN106554949A - The purification process of the uricase of PEGization

Info

Publication number: CN106554949A
Application number: CN201510638356.2A
Authority: CN
Inventors: 江筠; 黄琳秋; 楼觉人
Original assignee: SHANGHAI INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Current assignee: SHANGHAI INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Priority date: 2015-09-29
Filing date: 2015-09-29
Publication date: 2017-04-05
Anticipated expiration: 2035-09-29
Also published as: CN106554949B

Abstract

The invention discloses a kind of method that uricase to PEGization carries out purification, specifically, the invention discloses method comprise the steps：I () provides a raw material to be purified, wherein the raw material uricase for containing PEGization, the uricase and PEGylation that do not have the upper PEG of connection；(ii) raw material to be purified is splined on into cation exchange column so that the uricase of the PEGization is incorporated into described cation exchange column；(iii) eluting is carried out to the cation exchange column after loading with eluent, so as to obtain the eluent (Elution) of the uricase containing PEGization, the uricase of as purified PEGization.Purified with the method the PEGization for obtaining uricase have very high purity, therefore, be with a wide range of applications.

Description

The purification process of the uricase of PEGization

Technical field

The present invention relates to protein purification art, in particular it relates to a kind of purification process of the uricase of PEGization.

Background technology

PEG modifications method is referred on a molecular scale, in vitro the side chain special groups of pharmaceutical grade protein are covalently attached with activation PEG by manual method, to extend the protein half-life in vivo, reduce immunogenicity and antigenicity, it can in addition contain weaken the hydrolysis of protease, increase the solubility of protein molecular.Protein Jing after PEG modifications is sufficiently complex mixture, it is necessary to isolated and purified.

After connection PEG, the molecular weight of protein has different degrees of raising, generally use size exclusion chromatograph (SEC) to be separated, but the separating effect of the different modifying product close for molecular weight after modification is bad, and SEC method applied sample amounts low (being less than column volume 1%), elution time is long, the big cost format high throughput of energy consumption is little, is not suitable for expanding production.Ion exchange chromatography (IEC) is a kind of separation method that can process sample on a large scale, it is widely used in the separation of product after PEG is modified, conventionally employed DEAE is separated, but DEAE belongs to weak anionic displacement chromatography, the adsorption effect of sample is not good, and impurity can not be removed well.

PEGization albumen is separated at present and is most commonly used that strong anion displacement chromatography Q-sepharose, first afford separation product through multistep using Q-sepharose in the environment of highly basic, then carry out product purification with Sephacryl S-200.The method complex operation, multi step strategy cause sample loss amount big, the bad control of multiple close pH in elution requirement, and during have acid precipitation step, it is most likely that make uricase loss of activity.Sephacryl S-200 used in purification step fall within the old product of comparison in addition, process loaded down with trivial details, and elution efficiency is not high.

Therefore, this area has very high activity in the urgent need to developing a kind of purification process of the uricase of new PEGization, the uricase of the PEGization obtained with the method.

The content of the invention

It is an object of the invention to provide a kind of purification process of the uricase of new PEGization, the uricase of the PEGization obtained with the method is with very high activity.

First aspect present invention provides a kind of method that uricase to PEGization carries out purification, including step：

I () provides a raw material to be purified, wherein the raw material contains uricase, the uricase of non-PEGization and the free PEG of PEGization；

(ii) raw material to be purified is splined on into cation exchange column so that the uricase of the PEGization is incorporated into described cation exchange column；With

(iii) eluting is carried out to the cation exchange column after loading with eluent, so as to obtain the eluent (Elution) of the uricase containing PEGization, the uricase of as purified PEGization.

In another preference, described uricase includes wild type and saltant type uricase.

In another preference, the uricase of described PEGization includes the uricase of site directed pegylation.

In another preference, the uricase of described PEGization includes cysteine site by the uricase of PEGization.

In another preference, the uricase of described PEGization includes the saltant type uricase of PEGization.

In another preference, described saltant type uricase includes the cysteine residues introduced with restructuring, and described cysteine residues are located at the saltant type uricase of the non-active region of uricase.

In another preference, described saltant type uricase has wild type uricase (SEQ ID NO.:1) enzymatic activity of at least 70% (>=70%).

In another preference, described non-active region is the region being selected from the group of uricase：

The 103rd ± 3, the 148th ± 3, the 177th ± 3, the 202nd ± 3, the 228th ± 3, the 291st ± 3 of (a) uricase；

Before the N-terminal of (b) uricase；

After the C-terminal of (c) uricase；

The combination in any of (d) above-mentioned (a), (b) and (c)；

Wherein, described amino acid position is based on SEQ ID NO.:Uric acid enzyme sequence shown in 1.

In another preference, the quantity of the cysteine residues that described restructuring is introduced is 1-4, preferably 1-3, is more preferably 1-2, is most preferably 1.

In another preference, the incorporation way of the cysteine residues that described restructuring is introduced includes：Replace (displacement), insertion (when being located at non-end) and/or addition (when being located at N-terminal or C-terminal).

In another preference, described non-active region is the region being selected from the group of uricase：The 103rd ± 1, the 148th ± 1, the 177th ± 1, the 202nd ± 1, the 228th ± 1, the 291st ± 1 of uricase；More preferably, it is the 103rd, the 148th, the 177th, the 202nd, the 228th, the 291st of uricase.

In another preference, the incorporation way of the cysteine residues that described restructuring is introduced is replacement (or displacement).

In another preference, the original acid being replaced is selected from the group：K, N, Q, G or its combination.

In another preference, the original acid being replaced is selected from the group：103rd lysine, the 148th agedoite, the 177th glutamine, the 202nd glycine, the 228th lysine, the 291st lysine or its combination.

In another preference, the cysteine residues that described restructuring is introduced are located at N-terminal.

In another preference, the cysteine residues that described restructuring is introduced are located at C-terminal.

In another preference, the cysteine residues that described restructuring is introduced are selected from the group：K103C, N148C, G202C, K228C, K291C or its combination.

In another preference, described " non-active region " refers to that the activity of uricase does not have the region of substantial effect, after i.e. described non-active region is undergone mutation, the saltant type uricase for being formed still remains at least 70% (such as 70-200%, preferably 80-150%, more preferably 90-140%) wild type uricase activity.

On the other hand, present invention also offers the derivative polypeptide of above-mentioned saltant type uricase, these derivative polypeptides are on the basis of above-mentioned saltant type uricase, can also have the replacement of one or several amino acid residues (preferred 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residue), disappearance or add, and the derivative polypeptide uricase activity.

In another preference, activity preservation rate >=70% of the saltant type uricase, preferably 80-200% is more preferably 100-150%.

In another preference, enzyme average specific work >=10U/mg, the preferably 12-30U/mg of the saltant type uricase, are more preferably 15-25U/mg.

In another preference, the saltant type uricase is SEQ ID NO.:The mutated rear formation of uricase shown in 1.

In another preference, in SEQ ID NO.:The cysteine residues of introducing of recombinating in aminoacid sequence shown in 1 are selected from the group：K103C, N148C, G202C, K228C, K291C or its combination.

In another preference, the cation exchange column described in step (ii) is selected from the group：EzFsat Diamond SP Mustang、SP Bestarose FF、Bestarose Diamond MMC、SOURCE15S、HiTrap CM FF、MonoS5/50GL.

In another preference, comprise the following steps in step (ii)：

A () balances：Phosphate buffer of the balance solution used for concentration 0.05-0.2M (such as 0.1M)；

(b) loading：Sample solution cumulative volume is 0.1-10 with medium volume ratio：0.1-10 is (it is preferred that 1-5：1-5, more preferably, 1：1)；

Loading sample solution pH value is adjusted to 4-8 (preferably 5-7, more preferably 5.5-6)；Sample flow rate is 0.5-5ml/min (preferably 1-3ml/ml, more preferably 1.5-2ml/min).

In another preference, following steps are included in step (iii)：

C () washs：With the phosphate buffer drip washing of 0.05-0.2M (such as 0.1M), 1-20 column volume of abundant drip washing (preferably 2-10 column volume, more preferably 5-8 column volume)；

(d) eluting：With the CBS buffer stages eluting of 0.1-1M (such as 0.1M) and collect the eluent of the uricase containing PEGization, the uricase of as purified PEGization.

In another preference, described raw material to be purified is prepared with the method for comprising the following steps：

(1) provide a uricase raw material；

(2) described uricase raw material and PEGization reagent are reacted, so as to form the reactant mixture of the uricase containing PEGization, raw material as to be purified.

In another preference, described uricase raw material is purified uricase raw material.

In another preference, the described purified uricase raw material purification process preparation for comprising the following steps：

(a0) tunning is obtained into the precipitate containing uricase Jing after ammonium sulfate precipitation process；And/or

(b0) precipitate containing uricase is carried out into purification by anion exchange, so as to obtain purified uricase raw material.

In another preference, the ammonium sulfate precipitation is carried out under the following conditions：

The precipitating concentration of ammonium sulfate is 5%-35%, it is preferred that 10%-25%, more preferably, 10%-15%.

In another preference, in step (b0), described anion exchange is carried out under the conditions of one or more being selected from the group：

I () exchange media or filler are selected from the group：Bestarose Diamond MMA, DEAE, Source 15Q, Capto Q or its combination；

(ii) solution used by balance liquid：With the CBS buffer of concentration 0.1-1M (such as 0.1M)；Loading sample pH 7-11 (preferably 8-10.5, more preferably 9.5-10)；

(iii) solution used by eluent：With the CBS buffer of the 0.1-1M (such as 0.1M) of concentration 0.05-2M (such as 0.1M) NaCl.

In another preference, in described purified uricase raw material, purity >=80% of uricase.

In another preference, described method also includes：

(iv) eluent of the uricase containing PEGization obtained to previous step, carries out refinement treatment, so as to obtain the uric acid enzyme product of the PEGization of purification.

In another preference, described refinement treatment is selected from the group：HPLC, Sephacryl S-200, Sephardex G200, EzFast Phenyl FF or its combination.

In another preference, described HPLC is RP-HPLC (reverse high performance liquid chromatography).

In another preference, described RP-HPLC is carried out under the conditions of one or more being selected from the group：

(e1) loading volume and medium volume be than 0.001-0.01 (it is preferred that 0.002-0.004, more preferably, 0.002)；

(e2) chromatographic column of high performance liquid chromatograph is C4 reversed-phase column XBridgeTM BEH300.

In another preference, purity >=85% of the uricase of PEGization in the uricase of described purified PEGization, preferably >=90%, more preferably >=92%, most preferably >=95%.

In another preference, the uricase of PEGization has one or more features being selected from the group：

(f1) there is enzymatic activity (the preferably enzymatic activity under the pH for highest enzymatic activity at least 50%) in pH6-10；

(f2) there is enzymatic activity (the preferably enzymatic activity at the temperature for highest enzymatic activity at least 30%) at 5 DEG C -45 DEG C.

In another preference, the method that purification is carried out to the uricase of PEGization includes step：

I () provides raw material to be purified, the raw material is saltant type uricase；

(ii) it is optional, the saltant type uricase is isolated and purified；

(iii) the saltant type uricase for isolating and purifying is carried out into PEG modifications；

(iv) the saltant type uricase after described PEG modifications is splined on into ion exchange column so that the saltant type uricase after PEG modifications is incorporated into described ion exchange column；With

V () carries out eluting to the ion exchange column after loading with elution buffer, flow through liquid containing the saltant type uricase (that is, the saltant type uricase of PEGization) after PEG modifications so as to obtain.

In another preference, in step (iii), including one or more conditions being selected from the group：

(a1) PEG and the mol ratio of saltant type uricase are 0.1-10：0.1-10, it is preferred that 1-8：1-8, more preferably, 1:1.

(a2) time of PEG modifications is 1-8 hours, it is preferred that 2-5 hours, more preferably, 2 hours.

(a3) pH of PEG modifications is 6-10, it is preferred that 7-9, more preferably, 8.

(a4) protein concentration of the saltant type uricase modified by PEG is 0.1-10mg/ml, it is preferred that 0.5-5mg/ml, more preferably, 1mg/ml.

In another preference, in step (iv), including one or more conditions being selected from the group：

(b1) sample solution cumulative volume and medium volume ratio are 0.1-10：0.1-10 is (it is preferred that 1-5：1-5, more preferably, 1：1)；

(b2) Ion Exchange Medium is EzFsat Diamond SP Mustang.

(b3) balance the phosphate buffer of solution used for concentration 0.05-0.2M (such as 0.1M).

In another preference, in step (v), after loading is finished, with the phosphate buffer drip washing of 0.05-0.2M (such as 0.1M), again with the CBS buffer stages eluting collection of 0.1-1M (such as 0.1M) containing the saltant type uricase after PEG modifications, i.e. the saltant type uricase of PEGization.

In another preference, methods described also includes step (vi)：To the eluent obtained in step (v), high performance liquid chromatograph is splined on, and is chromatographed, so as to obtain the uricase of the PEGization of purification.

In another preference, in step (vi), high performance liquid chromatograph is reverse high performance liquid chromatograph (RP-HPLC).

In another preference, in step (vi), the chromatography condition of high performance liquid chromatograph is：

(c1) loading volume and medium volume be than 0.001-0.01 (it is preferred that 0.002-0.004, more preferably, 0.002)；

(c2) chromatographic column of high performance liquid chromatograph is C4 reversed-phase column XBridgeTM BEH300.

In another preference, the saltant type uricase of the PEGization obtained from high performance liquid chromatograph, its purity >=90%, preferably >=92%, more preferably >=95%.

Second aspect present invention provides a kind of uricase of the PEGization of purification, method described in the uricase first aspect present invention of the PEGization of described purification is obtained, and described uricase has the cysteine residues that restructuring is introduced, and described cysteine residues are located at the non-active region of uricase.

It should be understood that within the scope of the present invention, can be combined with each other between above-mentioned each technical characteristic of the present invention and each technical characteristic for specifically describing in below (eg embodiment), so as to constitute new or preferred technical scheme.As space is limited, here is no longer tired out one by one and is stated.

Description of the drawings

Fig. 1 shows the impact that the ammonium sulfate precipitation of variable concentrations is distributed to mutant uricase K103C；

Wherein, M is Marker, and 1-6 is the supernatant after 10%-35% ammonium sulfate precipitations, and 7-12 is 0.1M sodium carbonate buffers redissolution after 10%-35% ammonium sulfate precipitations.

Fig. 2 shows the result that uricase (such as K103C) anion-exchange chromatography MMA is processed.

Fig. 3 shows separating effects of the PEG-K103C Jing after SP cation-exchange chromatographies.

Fig. 4 shows the result of PEG-K103C each component electrophoresis after SP cation exchange layers；

Wherein, M:Marker；1:K103C；2,3:PEG-K103C；4,6:PEG-K103C crosses SP column chromatographies and flows through peak；5,7:PEG-K103C crosses SP column chromatography eluting peaks.

Fig. 5 show PEG-K103C it is purified after RP-HPLC analysis results；Wherein, upper figure is PEG-K103C tomographic maps, and figure below is mobile phase background.

Fig. 6 shows the separating effect of PEG-K103C Jing DEAE anion-exchange chromatographies.

Fig. 7 shows the separating effect of PEG-K103C Jing MMA anion-exchange chromatographies.

Fig. 8 shows vector plasmid pET3d and purpose fragment mutant PBC Jing NcoI and BamHI double digestion electrophoresis result figures.Wherein, PBC represents wild type uricase.

Fig. 9 a show the PCR qualification figures of multiple mutants that catastrophe point is located in sequence.Wherein, 1,2:K103C；3,4:N148C；5,6:Q177C；7,8:G202C；9,10:K228C；M:Marker (molecular weight standard)；11:Negative control (NC)；12,13:G274C；14,15:K291C.

Fig. 9 b show that catastrophe point is located at the PCR qualification figures of 2 mutants of sequence end.Wherein, 1-3:N-ter-C；4-6:C-ter-C；M:Marker (molecular weight standard).

Specific embodiment

The present inventor is through extensively in-depth study, by groping in a large number to production technology, it is surprised to find that first, based on specific cation chromatography media (such as SP), extremely efficient the saltant type uricase of PEGization can be separated with other impurities (including unnecessary PEG, the uricase of non-PEGization and other impurities), so as to carry out highly purified purification to the saltant type uricase of PEGization, and the purification process on the activity of uricase substantially free of impact.On this basis, the present inventor completes the present invention.

Specifically, the present inventor overturns tradition, employs cation-exchange chromatography to the uricase after PEG modifications so that the hanging column efficiency of uricase is improved, it is to avoid using extreme pH conditions, eluting is also more easy.

Additionally, uricase raw material of the present invention for non-PEGization, using ammonium sulfate precipitation and/or compound anion column (such as MMA) chromatography chromatography, such that it is able to obtain fast and efficiently preliminary purification to the uricase in tunning.

Term

As used herein, described " PEG " refers to Polyethylene Glycol, specifically, refers to the mixture of ethylene oxide condensate and water, by formula H (OCH₂CH₂)_nOH represents, wherein, n is at least 4.

As used herein, term " saltant type uricase ", " mutant uricase ", " uric acid enzyme mutant " are used interchangeably, the mutain for all referring to that the restructuring described in first aspect present invention introduces cysteine residues and retaining uricase activity.The restructuring of cysteine is introduced as fixed point and introduces, and specifically, the non-active region fixed point of N-terminal, C-terminal or uricase in uricase introduces cysteine.Introduce the activity of (such as 70-200%, preferably 80-150%, more preferably 90-140%) the wild type uricase that still remains at least 70% of the uricase (i.e. saltant type uricase) after cysteine.

Present invention also offers the derivative polypeptide of above-mentioned saltant type uricase, these derivative polypeptides are on the basis of above-mentioned saltant type uricase, can also have the replacement of one or several amino acid residues (preferred 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residue), disappearance or add, and the derivative polypeptide uricase activity.

As used herein, described " PEG " refers to Polyethylene Glycol, specifically, refers to the mixture of ethylene oxide condensate and water, by formula H (OCH₂CH₂)_nOH represents, wherein, n >=4.Generally, the molecular weight >=5KDa of PEG molecules, preferably preferably 10-40KDa, 15-30KDa.

Initial uricase

In the present invention, the uricase as initial uricase is not particularly limited, and can be the uricase in any source, and representational example includes (but being not limited to)：The uricase of mammal, the uricase of restructuring.Additionally, the uricase of initial uricase can be the uricase, or the saltant type uricase containing mutation of wild type.

A kind of preferred initial uricase is the chimeric protein comprising two or more mammalian amino acid sequences, such as includes the restructuring uricase of the section of pig uricase and baboon uricase.

In another preference, the N-terminal of the initial uricase derives from pig uricase, and its C-terminal derives from baboon uricase.More preferably, 225 aminoacid of the N-terminal of initial uricase from pig uricase, 79 aminoacid of its C-terminal from baboon uricase.

A kind of aminoacid sequence such as SEQ ID NO. of typical initial uricase:Shown in 1.

The saltant type uricase of PEGization

Saltant type uricase can pass through chemical bond covalent bond PEG using means known in the art.Generally, PEG can be combined with saltant type uricase by linking group, and wherein linking group can be selected from the group：Succinimido, amide groups, imide, carbamate groups, ester group, epoxy radicals, carboxyl, hydroxyl, carbohydrate group, L-Tyrosine group, cysteine residues, histidine group or its combination.In addition, saltant type uricase directly can also be coupled (i.e. no linking group) with PEG by amino, sulfydryl, hydroxyl or carboxyl.

In a preference of the present invention, PEG is coupled by the cysteine residues of introducing of recombinating on chemical bond (such as covalent bond) and saltant type uricase.

In a preference of the present invention, PEG is pointed decoration.Saltant type uricase can be monomer or the tetramer.The enzyme can be individual with covalent bond 1-4, it is preferred that 1-3, more preferably 1-2 PEG.These PEG can be linear chain, or branched chain.

A kind of molecular weight of the saltant type uricase (tetramer) of preferred PEGization is 160,000-220,000Da.

The cation exchange column of the present invention

As used herein, term " cation exchange column of the present invention " refers to the cation exchange resin that specifically the saltant type uricase of PEGization can be efficiently separated with unnecessary PEG and other impurities.

It is not particularly limited suitable for the cation exchange resin of the present invention, representational cation exchange resin includes (but being not limited to)：EzFsat Diamond SP Mustang、SP Bestarose FF、Bestarose Diamond MMC、SOURCE15S、HiTrap CM FF、MonoS5/50GL.

In the present invention, a kind of preferred cation exchange resin is EzFsat Diamond SP Mustang.Cation exchange column is balanced, phosphate buffer of the balance solution used for concentration 0.05-0.2M (such as 0.1M)；Then sample is splined on into ion column, sample solution cumulative volume is 0.1-10 with medium volume ratio：0.1-10 is (it is preferred that 1-5：1-5, more preferably, 1：1), loading sample solution pH value is adjusted to 4-8 (preferably 5-7, more preferably 5.5-6), sample flow rate 0.5-5ml/min (preferably 1-3ml/ml, more preferably 1.5-2ml/min)；Wash after loading afterwards：With the phosphate buffer drip washing of 0.05-0.2M (such as 0.1M), 1-20 column volume of abundant drip washing (preferably 2-10 column volume, more preferably 5-8 column volume)；Finally by target protein eluting:With the eluent of CBS buffer stages eluting collection containing target protein of 0.1-1M (such as 0.1M), that is, obtain purified target protein.

In the present invention, by screening a variety of separation methods and separating medium, it was found that the specific cation exchange resin of a class (including EzFsat Diamond SP Mustang) is higher compared with other media with the binding ability of the saltant type uricase of PEGization, and the medium is hardly combined if most of other impurities (such as unnecessary PEG)；As the saltant type uricase of PEGization uses the PBS of 0.1M-0.2M on ion exchange column, pH5.5-5.6 washes post, can remove a large amount of foreign proteins, further when using high concentration, the elution buffer of high pH, 0.1M-0.2M PBS i.e. containing 0.2-0.6M NaCl, the saltant type uricase of the very high PEGization of purity during pH7-7.5 eluting, can be obtained, which achieves that stock solution is a large amount of, quick, the purpose of effective purification.

Include the resin of EzFsat Diamond SP Mustang or similar character suitable for the cation exchange resin of the present invention.These resins can be prepared by known method or can be commercially available.

In the present invention, the consumption of chromatography media is not particularly limited, depending on the saltant type uricase quantity of PEGization that generally can be according to contained by raw material to be purified.

By taking the chromatography media of 100g or ml as an example, generally can primary sorption (or separate) the about PEGization of 500-1000mg saltant type uricase.Therefore, for the raw material of the uricase containing PEGization obtained after cell culture, generally for about 5L material liquids for, can use about 100-2000ml chromatography media, be preferably about 200-1000ml.

Purification process

The invention provides a kind of method that specific chromatography media above-mentioned based on the present invention carries out purification to the saltant type uricase of PEGization, including step：

I () provides a raw material to be purified, wherein the raw material uricase for containing PEGization, the uricase and PEGylation that do not have the upper PEG of connection；

(ii) raw material to be purified is splined on into cation exchange column so that the uricase of the PEGization is incorporated into described cation exchange column；

In a preference of the present invention, described method also includes：

In a preference of the present invention, described raw material to be purified is prepared with the method for comprising the following steps：

(1) provide a uricase raw material；

In a preference of the present invention, the method for the present invention is mainly included the following steps that：

(ii) it is optional, the saltant type uricase is isolated and purified；

V () carries out eluting to the ion exchange column after loading with elution buffer, so as to obtain the eluent containing the saltant type uricase (that is, the saltant type uricase of PEGization) after PEG modifications.

In another preference, the inventive method also includes corresponding pre-treatment, post processing, and/or PEGization step.

Pre-treatment：Ammonium sulfate, MMA purified uricase raw materials (such as saltant type uricase)；

Post processing：Refined (such as using HPLC, especially RP-HPLC is refined)；

PEGization step：Uricase raw material after pre-treatment and PEGization reagent are reacted, so as to form the reactant mixture of the uricase containing PEGization, raw material as to be purified.

In a preference of the present invention, in equilibrium step, buffer used is buffer A, phosphate buffer of its component for 0.1M-0.6M (it is preferred that 0.2M-0.4M), pH about 5.6 ± 0.1.

In step (iv), the raw material (liquid) of the saltant type uricase containing PEGization to be purified can be splined on cleaned and Balance Treatment ion exchange column, wherein, loading speed is not particularly limited, for chromatography media generally for about 500ml, flow velocity can be 10-100ml/min, preferably 20-60ml/min.

After loading, can be close with buffer property used in property and Balance Treatment or identical buffer is cleaned, so as to unconjugated impurity is washed.It is once purged, eluting process can be carried out with elution buffer, so that the saltant type uricase of the PEGization for combining is eluted out from the chromatography media.In this step, elution speed is not particularly limited, and for the chromatography media generally for about 500ml, flow velocity can be 10-100ml/min, preferably 20-60ml/min.

In elution process, the parameters such as the pH of effluent can be monitored, so as to the eluting peak for more accurately collecting the saltant type uricase containing PEGization.

In a preference of the present invention, in elution step, elution buffer used is buffer B, CBS buffer of its component for concentration 0.01M-0.1M, pH about 10.12 ± 0.1.

Preferably, the inventive method also includes further purification (refining) step, for example, carry out purification again with ion exchange column, or carry out purification with additive method.

One kind is preferably further purified (refine) and can be carried out by high performance liquid chromatograph, such as reverse high performance liquid chromatograph (RP-HPLC)；More preferably, the chromatographic column of described reverse high performance liquid chromatograph (RP-HPLC) is C4 reversed-phase column XBridgeTM BEH300.

Main advantages of the present invention include：

(1) purification process of the invention can be obtained the saltant type uricase of highly purified PEGization, its high purity more than 85%.

(2), when being isolated and purified with medium EzFsat Diamond SP Mustang, the destination protein for flowing through seldom, has reached the effect of unnecessary PEG and some other impurity after removal modification, and preferably, the response rate is high for eluting peak type.

(3) the purge process time is short, simple to operate, and loss of proteins is few, reduces cost, has saved the time, beneficial to follow-up extensive amplification production application.

In description (especially embodiment), the implication of part abbreviation is as follows：

CBS：Sodium bicarbonate-carbonate buffer

PBS：Phosphate buffer

MMA：Bestarose Diamond MMA

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are percentage by weight and parts by weight.

1 uricase mutation construction of embodiment and expression

The structure of 1.1 mutants

1.1.1 obtain mutant gene

The mutation of K291C, N-terminal and C-terminal is obtained with complete sequence synthesis mode, and K103C, N148C, Q177C, G202C, K228C are obtained with double PCR method, and primer sequence is as follows：

The design of primers of 1 different mutant nucleotide sequences of table

1.1.2 construction expression plasmid

PET3d plasmids and purpose fragment are carried out into double digestion (Fig. 8), is attached with T4 ligases after recovery, connection product is all added in the DH5 α competent cells of the firm defrosting of 50 μ L, flicks mixing；Ice bath 30min, 42 DEG C of heat shock 90s；After turning ice bath 2min immediately；500 μ L sterile LB mediums are added to mix, 37 DEG C of 200rpm cultivate 1h, make thalline recover；On flat board of the coating containing corresponding resistant, monoclonal bacterium colony, the successful bacterial strain of PCR identification conversions are selected.

1.1.3 build mutant strain

Plasmid is extracted, by above-mentioned sequencing correct plasmid transformation escherichia coli BL21 (DE3) pLysS, construction expression bacterial strain (mutant strain), PCR are identified (Fig. 9 a and Fig. 9 b) to mutant strain

The expression of 1.2 uric acid enzyme mutants

By taking K103C as an example (expression of other mutants is identical), take colibacillus engineering strain BL21 (DE3) pLysS-pET3d-K103C that can express uricase mutant protein for building to recover from -80 DEG C of glycerol stocks, cultivate to exponential phase (OD600=0.3) for 37 DEG C after switching, add IPTG to final concentration 0.4mM, 37 DEG C of abduction delivering 2h, 30 DEG C of overnight abduction deliverings, next day collects thalline, according to 1：10 ratios add bacterial cell disruption liquid, are put into -20 DEG C and stand overnight.Next day is dissolved in 25 DEG C of water temperatures, and ultrasonication 20kHz surpasses 5 seconds and stops 5 seconds, is crushed until completely.12000rpm centrifugation 10min separate broken supernatant precipitation.Precipitation is resuspended with isopyknic PBS, and after rinsing once, 12000rpm centrifugation 10min take precipitation, are dissolved with 0.1M pH10.12CBS (sodium carbonate buffer), and 12000rpm centrifugation 10min take supernatant, are enzyme extract.

After testing, in enzyme extract, the content of saltant type uricase is about 1-10mg/ml.

Purification before the modification of 2 saltant type uricase of embodiment

2.1 ammonium sulfate precipitation purification

In this step, carry out modifying front purification with ammonium sulfate precipitation method.10%, 15%, 20%, 25%, 30%, 35% saturated ammonium sulfate solution is separately added in the enzyme extract prepared in above-described embodiment 1.2, room temperature places 30min, 12000rpm centrifugation 10min, wherein, precipitate and dissolved with 0.1M pH10.12CBS.SDS-PAGE electrophoretic analysiss are done to the precipitation of supernatant and dissolving respectively, to determine optimal ammonium sulfate precipitation concentration.

By taking uricase K103C as an example, as shown in figure 1, when ammonium sulfate concentrations are 10%, you can make most of restructuring uricase K103C precipitations, wherein in enzyme extract >=90% restructuring uricase defines precipitation.When ammonium sulfate concentrations are 15%, PBS to be washed and have an obvious band at visible 34kDa, illustrates that the precipitation of restructuring uricase K103C is incomplete.And from the beginning of being 15% from ammonium sulfate concentrations, with the increase of ammonium sulfate concentrations, the precipitation capacity of the uricase K103C that recombinates is remained unchanged substantially, and the foreign protein amount detected in PBS washs supernatant gradually increases, the separation purpose without reaching fractional precipitation.Therefore, select 10% ammonium sulfate concentrations that the preliminary purification of the first step is carried out to saltant type uricase K103C.

Purification is carried out to other saltant type uricases using 10% ammonium sulfate concentrations (such as N148C, Q177C, G202C, K228C).Jing is determined, saltant type uricase rate of deposition A1/A0 >=90% (85%-95%) of Jing precipitations, and wherein A1 is the uricase quality of Jing precipitations, and A0 is the quality of uricase in enzyme extract.

2.2 anion exchange column purifications

In this step, further purification is carried out with ion exchange, concrete grammar is as follows：

Take MMA fillers (purchased from Bestchrom (Shanghai) Biotechnology Co., Ltd.), load chromatographic column, pillar is rinsed with 0.5N NaOH, remove impurity, pillar is balanced with balance liquid 0.1M pH 10.12CBS, Jing ammonium sulfate precipitations sample after purification is pressed into 2mL/min flow velocitys loading until overload, drip washing is carried out with balance liquid, unconjugated impurity is washed away fully, drip washing is to conductance and uv absorption value stabilization, with the 0.1M pH10.12CBS eluting containing 0.2M NaCl, according to the strong and weak collection eluting peak of 280nm absorption values, SDS-PAGE detection absworption peak albumen.

As a result as shown in Fig. 2 as a result showing, MMA can be with active adsorption uricase protein, and the effect for removing most of impurity can be reached, eluting peak conditions of streaking is not obvious, but eluting peak is not single, illustrate MMA can not nearly as the uricase of different aggregate forms separate.

The PEG modifications of 3 mutant uricase (such as K103C) of embodiment

By the Jing ammonium sulfate precipitations obtained in embodiment 2.1 uricase (respectively K103C after purification, N148C, Q177C, G202C, K228C) 2mg/ml is configured to, the MAL-Ala-mPEG (Beijing Kaizheng Biotech Engineering Development Co., Ltd. with 1.14mg/ml, molecular weight 20kD) equal-volume mixing, reaction system pH=8, final concentration of protein 1mg/ml, albumen are 1 with PEG mol ratios:1, reaction is carried out 2 hours in room temperature (about 25 DEG C), is reached reaction balance, is obtained the product of the uricase containing PEGization.Product is carried out into SDS-PAGE electrophoresis detection PEG modification efficiency.

By taking K103C as an example, experimental result as shown in 2 in Fig. 4, No. 3 swimming lanes, K103C monomer molecules amount about 35kD, by PEG modify after, the apparent molecular weight of saltant type uricase K103C monomers is increased to about 70kD.The apparent molecular weight of uricase (N148C, Q177C, G202C, the K228C) monomer of other saltant types is also increased to about 65-75kD.

The saltant type uricase of 4 PEGization of embodiment is isolated and purified

In the present embodiment, for the product of the obtained uricase containing PEGization in embodiment 3, ion-exchange chromatography is carried out with specific chromatography media EzFsat Diamond SP Mustang and purified.Method is as follows：

EzFsat Diamond SP Mustang fillers are taken, loads chromatographic column, with buffer (NaAc-HAc) the balance pillar of balance liquid 0.02M pH5.5；The sample of the product of the uricase containing PEGization is pressed into 2mL/min flow velocity loadings, until overload, is balanced to conductance and uv absorption value stabilization with balance liquid (NaAc-HAc) after end of the sample；With the PBS eluting of the 0.02M pH7.4 containing 0.2M NaCl, according to the strong and weak collection eluting peak of 280nm absorption values, SDS-PAGE detection absworption peak albumen.

In experimental result such as Fig. 3, Fig. 4 shown in swimming lane 5 and 7.As a result show, after SP posts, most foreign proteins, the albumen and PEGylation for having neither part nor lot in modification all pass through the form that flows through and removes, and eluting peak PEG modified outcomes can be very good to separate, and peak shape is symmetrical, good separating effect, high income.The albumen of electrophoresis showed PEG modification is primarily present in eluting peak.

Wherein, the molecular weight of the uricase of purified PEGization is about 160-200kD, then carries out Purity with the HPLC of embodiment 5 and further refines.

Purity analysis of the saltant type uricase of 5 PEGization of embodiment and refined

For the eluent of the uricase that the PEGization containing purification is obtained in embodiment 4, purity detecting is carried out with reverse high performance liquid chromatograph (RP-HPLC) and/or is refined.Specific experiment method is as follows：

1. balance：Using highly effective liquid phase chromatographic system 1200, C4 reversed-phase column XBridgeTM BEH300 (3.5 μm, 4.6*150mm) is installed in system, with A liquid (A:0.1%TFA water) pillar is rinsed until baseline values undulating value is less than 0.1mAU/min；

2. loading eluting：The 20 μ l loadings of uricase of the PEGization that previous step purification is obtained, mobile phase：A:0.1%TFA water, B:0.1%TFA acetonitriles, in 30min, the ratio of B liquid changes to 95% by 0%, carries out eluting, flow velocity：0.5mL/min, ultraviolet detection wavelength 280nm；

3. post is washed：Eluting cleans 3 column volumes with 95% Mobile phase B after terminating, experiment is stored in 100% acetonitrile after terminating.

Experimental result

By taking uricase K103C as an example, its result is as shown in Figure 5.As a result show, in embodiment 4, the purity of the Jing SP cation seperation columns uricase (such as PEG-K103C) of PEGization after purification reaches more than 85%.

Additionally, corresponding retention time is the sample of 16-17 minutes in collecting RP-HPLC, the product of the pegloticase of purity >=95% is obtained.

Comparative example 1

Repeat embodiment 4, difference is：With the cation exchange medium EzFsat Diamond SP Mustang in commercially available Ion Exchange Medium DEAE alternative embodiments 4.

As a result it is as shown in Figure 6.As a result show, when being purified with medium DEAE, the uricase protein (such as K103C) of most of Jing PEGization is flowed out in the form of flowing through and can not be suspended on pillar, cause greatly waste, do not play the effect for isolating and purifying.

Comparative example 2

Repeat embodiment 4, difference is：With the cation exchange medium EzFsat Diamond SP Mustang in commercially available Ion Exchange Medium MMA alternative embodiments 4.

As a result it is as shown in Figure 7.As a result show, when being purified with MMA, the uricase protein for still there are the Jing PEGization of a greater part of (＞ 50%) is lost in the form of flowing through, and separating effect is bad.

The all documents referred in the present invention are all incorporated as reference in this application, are individually recited just as each document such as reference.In addition, it is to be understood that after the above-mentioned teachings for having read the present invention, those skilled in the art can be made various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims limited range.

Claims

1. a kind of method that uricase to PEGization carries out purification, it is characterised in that including step：

I () provides a raw material to be purified, wherein the raw material contains the uricase of PEGization, non-PEGization Uricase and free PEG；

(ii) raw material to be purified is splined on into cation exchange column so that the uricase knot of the PEGization Together in described cation exchange column；With

(iii) eluting is carried out to the cation exchange column after loading with eluent, so as to obtain the urine containing PEGization The eluent (Elution) of sour enzyme, the uricase of as purified PEGization.

2. the method for claim 1, it is characterised in that the uricase of described PEGization includes fixed point The uricase of PEGization.

3. the method for claim 1, it is characterised in that the uricase of described PEGization includes PEG The saltant type uricase of change.

4. the method for claim 1, it is characterised in that in step (ii), described cation exchange Post is selected from the group：EzFsat Diamond SP Mustang、SP Bestarose FF、Bestarose Diamond MMC、SOURCE15S、HiTrap CM FF、MonoS5/50GL。

5. the method for claim 1, it is characterised in that described raw material to be purified be with include with Prepared by the method for lower step：

(1) provide a uricase raw material；

(2) described uricase raw material and PEGization reagent are reacted, so as to form the uric acid containing PEGization The reactant mixture of enzyme, raw material as to be purified.

6. method as claimed in claim 5, it is characterised in that described uricase raw material is purified urine Sour proenzyme material.

7. method as claimed in claim 6, it is characterised in that described purified uricase raw material bag Include the purification process preparation of following steps：

(b0) precipitate containing uricase is carried out into purification by anion exchange, it is purified so as to obtain Uricase raw material.

8. the method for claim 1, it is characterised in that described method also includes：

(iv) eluent of the uricase containing PEGization obtained to previous step, carries out refinement treatment, so as to obtain Obtain the uric acid enzyme product of the PEGization of purification.

9. method as claimed in claim 8, it is characterised in that described refinement treatment is selected from the group：HPLC、 Sephacryl S-200, Sephardex G200, EzFast Phenyl FF or its combination.

10. a kind of uricase of the PEGization of purification, it is characterised in that the uric acid of the PEGization of described purification Enzyme is obtained with the method described in claim 1, and described uricase has the cysteine that restructuring is introduced residual Base, and described cysteine residues are located at the non-active region of uricase.