CN104342420B - A kind of recombinant long-acting people hyaluronidase, its encoding gene, production method and application - Google Patents
A kind of recombinant long-acting people hyaluronidase, its encoding gene, production method and application Download PDFInfo
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- CN104342420B CN104342420B CN201310325056.XA CN201310325056A CN104342420B CN 104342420 B CN104342420 B CN 104342420B CN 201310325056 A CN201310325056 A CN 201310325056A CN 104342420 B CN104342420 B CN 104342420B
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- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C—CHEMISTRY; METALLURGY
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- C12Y—ENZYMES
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- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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Abstract
It is the extracellular parts of people's hyaluronidase PH20 and people's source protein the invention discloses a kind of recombinant long-acting people hyaluronidase（Human albumin HSA fragments or human immunoglobulin(HIg) IgG2Fc fragments）Fusion protein PH20 HSA or PH20 IgFc, its amino acid sequence has the advantages that internal safe, long half time respectively as shown in SEQ ID No.4 or SEQ ID No.5；The invention also discloses encoding gene, expression vector, host cell, production method and the stabilization formulations of above-mentioned recombinant long-acting people hyaluronidase, using rich in the high expression system expression of GC and China's ground hamster cell（CHO）Production can reach industrialized level and obtain the active product that can be purified；The invention also discloses application of the above-mentioned recombinant long-acting people hyaluronidase in administration and production micromolecule hyaluronic acid is coordinated.
Description
Technical field
The invention mainly relates to biological technical field, more particularly to a kind of recombinant long-acting people hyaluronidase, its coding
Gene, production method and application.
Background technology
Hyaluronic acid is main mucopolysaccharide form in corium, is disaccharide unit D-Glucose aldehydic acid and N- by repeating
The linear polysaccharide of acetylglucosamine composition.Hyaluronic acid can remain above the moisture of 500 times of own wt, be formed very sticky
Solution, be filled within extracellular matrix collagen fabric skeleton, influence extracellular matrix mass transfer characteristics.Extracellular matrix is to be permitted
Drug carries out hypodermic major obstacle.The solid-state pedestal that collagen fabric is constituted and embedded sticking rich in hyaluronic acid
Property gel, causes certain resistance to mass tranfer, limits pharmacokinetics and can be subcutaneously injected from same site
Liquid volume, and reach blood vessel speed and quantity.
Hyaluronidase is the enzyme family of degraded hyaluronic acid, by being catalyzed the hydrolysis of hyaluronic acid, hyaluronidase drop
The viscosity of low hyaluronic acid, reversibly changes the structure of corium, thus improves the permeability of tissue.Hyaluronic acid half life of enzyme
15-20h, renewal time is short in vivo, and skin can be repaired quickly.Based on hyaluronidase get through extracellular matrix barrier to
Medicine is a kind of new and effective administration platform.According to the mechanism of action and the difference of catabolite, hyaluronidase can be divided into three classes,
The first kind includes β-D- acetyl-D- hexosaminidases, regard HMW degradation of substrates as main end-product into tetrose.Testis
Ball hyaluronidase is the prototype of this species, and it can be catalyzed transglycosylation, therefore, during hyaluronic acid is hydrolyzed
Also hexose, disaccharides and eight carbon sugar can be formed.Equations of The Second Kind, using leech hyaluronidase as the hyaluronidase of representative.3rd
Class, bacterial hyaluronidase is a kind of lyases.
Since 1940s, the invasin containing hyaluronidase activity extracted in animal tissue is as rush diffusant
For clinical administration, but its application is defined in emergency always, is primarily due to the not exclusively clear and definite enzyme of composition
The misgivings of other impurities composition in preparation.Animal tissue extracts active ingredient in hyaluronidase preparation and is less than 1%, the overwhelming majority
For gous impurities albumen.After gender bender's hyaluronidase gene discovery, the recombination human source hyaluronidase originated using DNA
(rHuPH20) hyaluronidase of substitution animal sources purifying, reduces immunogenicity, improves security, application also with
Expansion, be mainly used in hypodermoclysis, periocular surgery anesthesia, chemical drug and biological medicament promote to ooze, in urography contrast agent suction
Receive.
In many solid tumors (including prostate cancer, breast cancer, cancer of pancreas, colon cancer and non-small cell lung cancer) cell table
There is the enrichment of hyaluronic acid in face, the cell micro-environment rich in hyaluronic acid often between tumor development exist associate there is provided
The condition of malignant growth diffusion.Using the tissue or organ or processing tumour cell table of hyaluronic acid ferment treatment tumour growth
The hyaluronic acid integument in face simultaneously combines anti-tumor medicine there is provided a kind of anticancer new strategy.
But in the rHuPH20 bodies do not transformed in blood half-life short in 1 minute, it is impossible to input and realize through blood and control in vivo
The effect for the treatment of, someone using polyethylene glycol chemistry coupling rHuPH20, produce long-acting people's hyaluronidase, and have been used for people
Tumour is treated in class clinical trial.
On the other hand, hyaluronidase can degrade the restructuring macromolecule hyaluronic acid manually produced, produce small point
Son amount hyaluronic acid.Small-molecular-weight hyaluronic acid, including the small-molecular-weight hyaluronic acid oligosaccharide containing 3-10 dissacharide units
(oHA) endothelial cell surface CD44 can be activated and promote endothelial cell proliferation, moreover it is possible to VEGF (VEGF) phase
Collaboration, promotes endothelial cell angiogenesis, is the form of hyaluronic acid for having biological effect.
Therefore, small-molecular-weight hyaluronic acid is widely used in cosmetic field, because its molecular weight is small, can penetrate into dermis of skin
Layer corium, with stronger biological effect.And degraded at present more than industrial production small-molecular-weight hyaluronic acid using soda acid or super
Sound is degraded.
The content of the invention
It is an object of the invention to provide a kind of safe, long half time recombinant long-acting people's hyaluronidase in vivo.
To achieve the above object, the present invention is adopted the following technical scheme that：A kind of recombinant long-acting people hyaluronidase, is that people is saturating
The sour extracellular parts of enzyme PH20 of bright matter and the fusion protein of people's source protein, people's source protein is human albumin HSA fragments or people
Immunoglobulin IgG2 Fc fragments；The extracellular parts of people's hyaluronidase PH20 and the fusion protein of human albumin HSA fragments are
PH20-HSA, its amino acid sequence is as shown in SEQ ID No.4；Ball is immunized with people in the extracellular parts of people's hyaluronidase PH20
The fusion protein of protein I gG2 Fc fragments is PH20-IgFc, and its amino acid sequence is as shown in SEQ ID No.5.
Second object of the present invention is to provide a kind of encoding gene of above-mentioned recombinant long-acting people hyaluronidase, using such as
Lower technical scheme：Its gene order is as shown in SEQ ID No.6 or SEQ ID No.7.
Third object of the present invention is to provide a kind of comprising gene order shown in SEQ ID No.6 or SEQ ID No.7
Expression vector and host cell.
Fourth object of the present invention is to provide the production method of above-mentioned people's hyaluronidase, adopts the following technical scheme that：
Using animal cell expression vectors pMH3, pMH4 or pMH5 rich in GC in Chinese ground hamster cell CHO expressing gene sequence
SEQ ID No.6 or SEQ ID No.7, then isolate and purify producing；Described expression vector pMH3, pMH4 or pMH5 base
Because sequence is respectively as shown in SEQ ID No.8,9,10.
Further, the PH20-HSA is isolated and purified using Protein A affinity purification methods；It is described
PH20-IgFc is isolated and purified using the blue glue series connection ion method of the hydrophobic series connection of ion series connection.
The 5th purpose of the present invention is to provide the preparation that a kind of storage-stable has above-mentioned people's hyaluronidase, using as follows
Technical scheme：The preparation includes 150-1500IU PH20-HSA or PH20-IgFc, 10mM Na2HPO4, 0.03%
CaCl2, 0.1%EDTA-Na2, 145mM NaCl, 0.1%HSA, and pH value is 5.5-7.5.
The 6th purpose of the present invention is to provide a kind of compound preparation, using following technical side's scheme：Compound preparation includes
The preparation and particular treatment medicine of above-mentioned storage-stable someone's hyaluronidase.
The 7th purpose of the present invention is to provide application of the above-mentioned recombinant long-acting people hyaluronidase in administration is coordinated, and adopts
With following technical side's scheme：By recombinant long-acting people's hyaluronidase activity be delivered in the case where not being degraded human albumin or
The tissue of human immunoglobulin(HIg) combination, organ, position；Or by the tissue where particular treatment drug delivery to disease, organ or
Position.
The 8th purpose of the present invention is to provide above-mentioned recombinant long-acting hyaluronidase application, IgFc and HSA pieces therein
Section provides non-functional site and comes chemical coupling diagnosticum, developer, medicine, treatment toxin, gene into diseased tissue organ.
It is transparent in industrial production small molecule that the 9th purpose of the present invention is to provide above-mentioned recombinant long-acting people hyaluronidase
Application in matter acid, is adopted the following technical scheme that：The recombinant long-acting people hyaluronidase is saturating as toolenzyme degraded macromolecular
Bright matter acid produces micromolecule hyaluronic acid.
Due to using above-mentioned technical proposal, the present invention at least has advantages below：
(1) after human immunoglobulin(HIg) IgG2 Fc fragments and human albumin HSA segment composition people's hyaluronidases, extend
Half-life period in the blood of people's hyaluronidase, disease location, extension of validity can be transported to through blood or other method.
(2) human immunoglobulin(HIg) IgG2 Fc fragments and human albumin HSA fragments can be with specific acceptor and knots in human body
Hop protein is combined, and people's hyaluronidase of fusion can be carried into these positions.
(3) compared with people's hyaluronidase rHuPH20 of polyethylene glycol chemistry coupling, people's hyaluronidase people's immune globulin
White IgG2 Fc fragment fusion proteins (PH20-IgFc) and people's hyaluronidase human albumin fusion protein (PH20-HSA) are
Full people's source protein matter, in vivo using safer.
(4) although compared with hyaluronidase rHuPH20, people's hyaluronidase human albumin fusion protein (PH20-
) and people hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragment fusion proteins (PH20-IgFc) external activity (specific activity) HSA
Reduction, but their activity in vivo is more superior.
(5) after PH20-HSA and PH20-IgFc mammalian cells of the invention production, can using purifying HSA and
IgFc specific method purifying, better than polyethylene glycol chemistry coupling rHuPH20 production length in terms of production method and production prices
The method for imitating people's hyaluronidase.
(6) can the long-acting fusion protein of industrialized production people's hyaluronidase be that a technology is chosen to Microbial Expression Systems
War, even if being extremely difficult to industrialized level using mammalian cell liquid and obtaining the active product that can be purified.The present invention is adopted
Active energy purifying is produced with the animal cell expression vectors rich in GC and China's ground hamster cell (CHO) successful commercialization
People's hyaluronidase human albumin fusion protein (PH20-HSA) and people's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragments
Fusion protein (PH20-IgFc).
(7) people's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragment fusion proteins (PH20-IgFc) and people are saturating
The sour enzyme human albumin fusion protein (PH20-HSA) of bright matter can be used to do long-acting not degradable toolenzyme, and degraded macromolecular is saturating
Bright matter acid, industrialized production micromolecule hyaluronic acid.
(8) people's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragment fusion proteins (PH20-IgFc) and people are saturating
IgFc the and HSA fragments of the sour enzyme human albumin fusion protein (PH20-HSA) of bright matter can be with the non-functional site of offer come chemical idol
Join diagnosticum, developer, medicine, treatment toxin, gene and enter diseased tissue organ.
Brief description of the drawings
Above-mentioned is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, below
With reference to accompanying drawing, the present invention is described in further detail with embodiment.
Fig. 1 is 37 DEG C of stability test result schematic diagrams of fusion protein PH20-IgFc of the present invention.
Fig. 2 is 37 DEG C of stability test result schematic diagrams of fusion protein PH20-HSA of the present invention.
Fig. 3 is the fusion protein PH20-IgFc of the present invention and compares the specific activity of people's hyaluronidase in big white mouse body
Experimental result schematic diagram.
Fig. 4 is intravenous injection PH20-IgFc animals (A), intravenous injection PH20-HSA animals (B), intravenous injection PH20
Animal (C) subcutaneous injection site absorption 99mTc-sestamibi radioactivity absorbs distribution situation schematic diagram.
Fig. 5 is the people's hyaluronidase PH20 and people's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragments of 99mTc marks
Blood clearance situations of the fusion protein PH20-IgFc in rat body.
Fig. 6 is the people's hyaluronidase PH20 and people's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragments of 99mTc marks
Distribution situations of the fusion protein PH20-IgFc in rat body.
Embodiment
The preferred embodiments of the present invention are illustrated below in conjunction with accompanying drawing, it should be understood that described herein to be preferable to carry out
Example is merely to illustrate and explain the present invention, and is not intended to limit the present invention.
The present invention is mainly used rich in GC animal cell expressions technology (WO/2008/091276) and zooblast reaction
Device (WO2006/138143 and WO2007/142664) technology, produces rapidly non-polyethylene glycol chemistry coupling rHuPH20 length
People's hyaluronidase is imitated, including people's hyaluronidase human albumin fusion protein PH20-HSA and people's hyaluronidase people are immunized
Globulin IgG2 Fc fragment fusion proteins PH20-IgFc.People hyaluronidase PH20, its extracellular partial amino-acid series is such as
Shown in SEQ ID No.1；The amino acid sequence of human albumin HSA fragments is as shown in SEQ ID No.2；Human immunoglobulin(HIg) IgG2
The amino acid sequence of Fc fragments is as shown in SEQ ID No.3.
Embodiment one
Purpose：The gene constructed of fusion protein PH20-HSA or PH20-IgFc, expression, immunology detection, Activity determination
With reactor production.
Method：By PCR method by structural gene PH20-HSA (SEQ ID No.6) or PH20-IgFc (SEQ ID
No.7) it is building up in animal cell expression vectors pMH3,4,5 rich in GC (SEQ ID No.8,9,10).It is several that this is prepared in a small amount
Plant after expression plasmid, in the Chinese hamster ovary celI for being stably transfected into serum free suspension culture.Screened by G418, with the manual picking of pipette tips
Stable clone is into 96 orifice plates.When cell confluency degree is more than 50%, serum-free fresh culture is changed；After 3-6 hours, receive
Collect media samples, expression quantity is detected by the Fc antibody dot blot of anti-igg 2 or ELISA method, expression quantity highest gram is selected
It is grand multiple, continue to carry out free serum culture in the small shaking flask in sharp bottom and do mitotic stability research；Select the stable clone of passage
Expanded in 5L rip current type reactors.
As a result：(1) fusion protein PH20-IgFc or the PH20-HSA successful expression in Chinese hamster ovary celI, dot blot and
Western blot results show to screen produced by the stable high-expression clone of fusion protein PH20-IgFc or PH20-HSA
Band molecular size range is correct；(2) high-expression clone carries out free serum culture in the small shaking flask in sharp bottom and does passage stabilization；(3)
Stable high-expression clone carries out hyaluronic acid enzyme activity in stream plus culture amplification, each batch good harvest liquid in 5L rip current type reactors
Property reaches 600-1000 active units/milliliter, has reached commercial production level.
Conclusion：Use animal cell expression vectors pMH3,4,5 rich in GC, fusion protein PH20-IgFc or PH20-
Hyaluronidase activity reaches 600-1000 active units/milli in HSA successful expressions in Chinese hamster ovary celI, each batch good harvest liquid
Rise, industrialized production can be met.
Embodiment two
Purpose：Fusion protein PH20-HSA's or PH20-IgFc isolates and purifies.
Method：The separation and purification of protein strategy that the present embodiment is used is (1) PH20-IgFc mainly using Protein A parents
And purifying；(2) PH20-HSA uses the purifying process of the hydrophobic blue glue of ion series connection.
(1) PH20-IgFc receives liquid purifying.
Harvest after nutrient solution, 6min is centrifuged using 5000rpm, takes centrifuged supernatant to be filtered, three metafiltration membrane apertures point
Wei not be 0.8 μm, 0.45 μm, 0.22 μm.Loading is to equilibrium liquid A liquid (20mM Tris+100mM NaCl, pH 7.4) balance
Good Mabselect pillars, elution to baseline it is constant after, using eluent B liquid (100mM Gly+50mM Arg+0.01%
Tween 80, pH 3.5) eluted.Situation is isolated and purified by SDS-PAGE detection products.
(2) PH20-HSA receives liquid purifying
PH20-HSA cell culture fluids are received after liquid, are centrifuged 6min using 5000rpm, are taken centrifuged supernatant to be filtered, and three
Metafiltration membrane aperture is respectively 0.8 μm, 0.45 μm, 0.22 μm.The supernatant after filtering is carried out using Millipore30kDa films bag
Liquid is changed in concentration, and displacement liquid is anion column Q SFF level pads.
Concentration is changed into protein liquid loading after liquid to the Q for using A liquid (20mM Tris+50mM NaCl, pH 7.4) to balance
SFF posts, elution to baseline it is constant after, baseline is washed till using C liquid (20mM Tris+100mM NaCl, pH 7.0) in advance constant, uses E
Liquid (20mM Tris+450mM NaCl, pH 7.0) is eluted, and collects eluting peak.
Q SFF eluting peaks are added into (NH4)2SO4To 0.5M, CaCl is added2To 0.1mM, pH to 7.0 is adjusted, with 2ml/
Min flow velocitys loading is to A liquid (20mM Tris+0.5M (NH4)2SO4+0.1mM CaCl2, pH 7.0) and the Phenyl that has balanced
Post, elution to baseline it is constant after, E liquid (20mM Tris+0.25M (NH4)2SO4+0.1mM CaCl2, pH 7.0) and prewashing is carried out,
With B liquid (20mM Tris+0.1mM CaCl2, pH 7.0) eluted.
Phenyl eluting peak components are diluted with water to conductance for 20mS/cm, and pH is adjusted extremely with 1M HCl/1M NaOH
7.0.Loading is to I liquid (10mM Tris+150mM KCl, pH 7.0) balance Blue posts, loading, with F liquid (10mM Tris+
600mM KCl, pH 8.0) eluted, carried out using H liquid (10mM Tris+1.5M KCl+50% ethylene glycol, pH 8.0)
Strip, flow velocity is 1ml/min, collects eluting peak.Situation is isolated and purified by SDS-PAGE detection products.
As a result：Isolate and purify strategy using above-mentioned, fusion protein PH20-IgFc or PH20-HSA purity 95% with
On, yield is more than 30%.
Conclusion：Fusion protein PH20-IgFc and PH20-HSA can be using the specific purification process of IgFc and HSA come pure
Change, purification yield meets the requirement further done and medicine is subcutaneously injected.
Embodiment three
Purpose：Fusion protein PH20-IgFc or PH20-HSA preparation and In vitro and in vivo activity research
Method：The pharmaceutical formulation of the fusion protein PH20-IgFc parenteral solutions of hyaluronidase is as follows：
PH20-IgFc+10mM Na2HPO4+ 0.03%CaCl2+ 0.1%EDTA-Na2+ 145mM NaCl+0.1%
HSA, pH 5.5-7.5
Solution after 500U/ml PH20-IgFc are filtered is dispensed into 2ml amperes of bottles, and 25 bottles of solution are placed in into 37 DEG C,
Humidity is in 75 ± 5% accelerated test incubator.Respectively the 0th, sampling in 7,14,21,28,35,42 days is according to 2010 editions medicines
Allusion quotation annex hyaluronidase Activity determination, stability test result (table 1, Fig. 1) display, since the 28th day, hyaluronidase
There is conspicuousness and declines (about 15%) in activity.
Table 1 PH20-IgFc, 37 DEG C of stability test results
| Time (d) | 1 | 2 | 3 | Average value (U/ml) | Deviation | CV |
| 0 | 468 | 512 | 491 | 490.33 | 22.01 | 4.49 |
| 7 | 544 | 420 | 520 | 494.67 | 65.77 | 13.30 |
| 14 | 496 | 447 | 507 | 483.33 | 31.94 | 6.61 |
| 21 | 575 | 489 | 443 | 502.33 | 67.00 | 13.34 |
| 28 | 426 | 444 | 435 | 435.00 | 9.00 | 2.07 |
| 35 | 485 | 449 | 437 | 457.00 | 24.98 | 5.47 |
| 42 | 447 | 361 | 448 | 418.67 | 49.94 | 11.93 |
The pharmaceutical formulation of the fusion protein PH20-HSA parenteral solutions of hyaluronidase is as follows：
150-1500IU PH20-HSA+10mM Na2HPO4+ 0.03%CaCl2+ 0.1EDTA-Na2+145mM NaCl
+ 0.1%HSA, pH5.5-7.5
Stability test：Solution after 500U/ml PH20-HSA are filtered is dispensed into 2ml amperes of bottles, molten by 25 bottles
Liquid is placed in 37 DEG C, and humidity is in 75 ± 5% accelerated test incubator.Respectively the 0th, sampling in 7,14,21,28,35,42 days presses
According to 2010 editions pharmacopeia annex hyaluronidase Activity determinations, stability test result (table 2, Fig. 2) is shown, since the 28th day, thoroughly
There is conspicuousness and declines (about 15%) in the activity of the sour enzyme of bright matter.
Table 2 PH20-HSA, 37 DEG C of stability test results
| Time | 1 | 2 | 3 | Average value (U/ml) | Deviation | CV |
| 1 | 468 | 513 | 481 | 487.17 | 22.88 | 4.70 |
| 7 | 420 | 520 | 534 | 491.33 | 62.17 | 12.65 |
| 14 | 496 | 547 | 507 | 516.67 | 26.84 | 5.19 |
| 21 | 443 | 489 | 502 | 478.00 | 31.00 | 6.49 |
| 28 | 486 | 434 | 450 | 456.67 | 26.63 | 5.83 |
| 35 | 485 | 459 | 437 | 460.33 | 24.03 | 5.22 |
| 42 | 447 | 390 | 448 | 428.33 | 33.20 | 7.75 |
Fusion protein PH20-IgFc and PH20-HSA specific activity and control people's hyaluronidase have done external contrast and ground
Study carefully, by setting up trypan blue diffusion model in young mouse body, determine PH20-IgFc or PH20-HSA activity in vivo, also use
Determined in half-life period in blood plasma.
As a result：Preparation stability in external test tube test result indicates that, fusion protein PH20-IgFc's and PH20-HSA
Activity 37 degree of stability in above preparation is both greater than 20 days, and loss of activity is less than 10%.
Experimental result is also shown that people's hyaluronic acid specific enzyme activity of control production is 110000 units/milli in external test tube
Gram albumen, fusion protein PH20-IgFc specific activity is 40000 units/milligram albumen, and PH20-HSA specific activity is
20000 units/milligram albumen, fusion protein PH20-IgFc specific activity is lower than people's hyaluronic acid specific enzyme activity of control production
40% or so；And PH20-HSA specific activities are only people's hyaluronidase 10% or so of control production.
Experimental result is also shown that people's hyaluronic acid half life of enzyme of control production is very short in big white mouse body vessel, is only
1-2min, fusion protein PH20-IgFc and PH20-HSA long half time reach 2.5h, fusion protein PH20-IgFc and
Half-life period is compare the people's hyaluronic acid specific enzyme activity produced 100 times or so in PH20-HSA blood.
Conclusion：Fusion protein PH20-IgFc and PH20-HSA activity 37 degree of stability in above preparation are more than
20 days, the ratio of fusion protein PH20-IgFc and PH20-HSA people hyaluronidase of the external specific activity less than control production
Activity, fusion protein PH20-IgFc and PH20-HSA people hyaluronic acid half life of enzyme of the half-life period much larger than control production.
The present invention determines internal the half of fusion protein using the method for hyaluronic acid degradation activity and Western blot
Decline the phase.Compared with PH20, the existence time (i.e. half-life period) in Mice Body is obviously prolonged PH20-IgFc or PH20-HSA, is pointed out
Its hyaluronic acid effect of degrading in vivo of PH20-IgFc or PH20-HSA should also extend compared with PH20, i.e., drug effect extends.PH20-
IgFc or PH20-HSA are the medicines of the degraded hyaluronic acid more more efficient than PH20.
Example IV
Purpose：Determine fusion protein PH20-IgFc and PH20-HSA 15 amino acid sequences of N-terminal
Method：Do not dyed after sample is separated with SDS-PAGE electrophoresis, its electricity gone into pvdf membrane with CAPS buffer solutions,
Coomassie brilliant blue staining, rinsing, decolouring are standby after natural drying to without blue background；Destination protein band is cut, ABI is used
PROCISETM494cLC instruments, N-terminal sequencing is carried out according to N-terminal sequencing standard method (SCI-S-006).
As a result：Sequencing result shows that N-terminal amino acid sequence is
Leu-Asn-Phe-Arg-Ala-Pro-Pro-Val-Ile-Pro-Asn-Val-Pro-
Phe-Leu is consistent with theoretical value.
Embodiment five
Purpose：People's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragment fusion proteins (PH20-IgFc) and people are transparent
Matter acid enzyme human albumin fusion protein (PH20-HSA) degraded macromolecular hyaluronic acid activity.
Method：
1) 3 250ml triangular flask is taken, 100ml purified waters are separately added into, labeled as 1,2, No. 3, into each triangular flask
1.17gNaCl is added, it is 0.2mol/L to make its concentration, regulation pH is 7.0.
2) each into every triangular flask to add 1-3g hyaluronic acid dry powder, it is 10-30g/L to make its concentration, and dissolving is stayed overnight.
3) after hyaluronic acid is completely dissolved, to 1, in 2, No. 3 bottles, adding enzyme amount in sample enzyme liquid, sample is respectively
6000U, 12000U, 18000U, enzyme concentration are respectively 60U/ml, 120U/ml, 180U/ml；It is placed in after stirring in shaking table,
40 DEG C of digestion 4h.
4) wait digestion that solution temperature is increased into 80 DEG C after terminating, 30min carries out enzyme-deactivating；
5) it is each into three samples to add the ethanol solutions of 300ml 95% precipitation, rear abandoning supernatant to be precipitated, with 95%
Suction filtration after ethanol dehydration.
6) 60 DEG C of vacuum drying after suction filtration, are detected with HPLC.
As a result：Sodium hyaluronate solution is gel liquid after dissolving, during digestion 2h, and solution starts to become in 3 triangular flasks
It is dilute, present watery；HPLC testing results are shown：The molecular weight of hyaluronic acid is relevant with the consumption of enzyme, and the consumption of enzyme gets over macromolecular
Amount is smaller.(500KDa during 60U/ml；300KDa during 120U/ml；180U/ml when 200KDa)
Conclusion：People's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragment fusion proteins (PH20-IgFc) and people
Hyaluronidase human albumin fusion protein (PH20-HSA) can be used to do long-acting not degradable toolenzyme, macromolecular of degrading
Hyaluronic acid, industrialized production micromolecule hyaluronic acid.
Embodiment six
Purpose：Using the biological penetrating agent people hyaluronidase of radioactive tracer imaging comparative studies, people hyaluronidase people
Immunoglobulin IgG2 Fc fragment fusion proteins (PH20-IgFc), people's hyaluronidase human albumin fusion protein (PH20-
HSA the effect of tissue drug absorption) is strengthened.
Method：People's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragment fusion proteins (PH20-IgFc), people's hyalomitome
It is small that sour enzyme human albumin fusion protein (PH20-HSA), people's hyaluronidase (PH20) are injected separately into four groups of health ICR through vein
Mouse, 2 hours after treatment, the tracer methoxy isobutyl isonitrile of 99mTc marks is subcutaneously injected at left or right thigh position
(99mTc-sestamibi), obtains live body mouse whole body imaging using the special γ cameras of high-resolution toy, observes radioactivity mark
Infiltration rate and body tissue's distribution after the drug simulated compound of note is subcutaneously injected.Note：99mTc-sestamibi is used as medicine
Thing substitute is used for tumor drug resistance evaluation for many years, using method detection hyaluronidase reinforcing drug absorption, with sensitive, fast
Fast, directly perceived, reliable the advantages of.
As a result：As shown in figure 4, intravenous injection PH20-IgFc animals (A), intravenous injection PH20-HSA animals (B), quiet
Arteries and veins injection PH20 animals (C) subcutaneous injection site absorption 99mTc-sestamibi radioactivity absorbs distribution situation and shown,
99mTc-sestamibi increased radioactivity and major organs radiation in PH20-IgFc and PH20-HSA treatment groups Mice Body
Level is apparently higher than PH20 animals, through image quantitative analysis, 2 hours after treatment, is subcutaneously inhaled promoting 99mTc-sestamibi
Receive in performance, PH20-IgFc and PH20-HSA are strong compared with PH20 2.3 times.
Conclusion：Due to the change of PH20-IgFc and PH20-HSA blood halflifes, PH20-IgFc and PH20-HSA
In terms of drug absorption is promoted, hence it is evident that better than PH20.
Embodiment seven
Purpose：Study the people's hyaluronidase (PH20) and people's hyaluronidase human immunoglobulin(HIg) IgG2 of 99mTc marks
The plasma half-life of Fc fragment fusion proteins (PH20-IgFc).
Method：People's hyaluronidase (PH20 and the people hyaluronidase human immunoglobulin(HIg) IgG2 Fc that 99mTc is marked
Fragment fusion protein (PH20-IgFc) is injected intravenously in Sprague Dawley rat bodies, and it is determined using living imaging instrument
Plasma half-life (Blood clearance (%peak) of99mTc-labeled PH20-Fc and PH20).
As a result：As shown in figure 5, people's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragment fusion proteins of 99mTc marks
(PH20-IgFc) plasma half-life is substantially longer than the plasma half-life of people hyaluronidase (PH20).
Conclusion：The blood plasma of people's hyaluronidase human immunoglobulin(HIg) IgG2 Fc fragment fusion proteins (PH20-IgFc) half
Decline the phase substantially it is longer than the plasma half-life of people hyaluronidase (PH20).
Embodiment eight
Purpose：Study the people's hyaluronidase (PH20) and people's hyaluronidase human immunoglobulin(HIg) IgG2 of 999mTc marks
Absorption distribution of the Fc fragment fusion proteins (PH20-IgFc) in rat body.
Method：People's hyaluronidase (PH20 and the people hyaluronidase human immunoglobulin(HIg) IgG2 Fc that 99mTc is marked
Fragment fusion protein (PH20-IgFc) is injected intravenously in Sprague Dawley rat bodies, after 3 hours, uses living imaging
Instrument quantitative determines them and is distributed in the absorption of Different Organs.
As a result：As shown in fig. 6, the people's hyaluronidase (PH20) and people's hyaluronidase people's immune globulin of 99mTc marks
Absorption distribution of the white IgG2 Fc fragment fusion proteins (PH20-IgFc) in rat body is different.
Conclusion：The people's hyaluronidase (PH20) and people's hyaluronidase human immunoglobulin(HIg) IgG2 Fc pieces of 99mTc marks
Absorption distribution of the section fusion protein (PH20-IgFc) in rat body is different.
The present invention produces long-acting restructuring fusion people's hyalomitome of non-polyethylene glycol chemistry coupling using mammalian cell
Sour enzyme human albumin fragment (PH20-HSA) and hyaluronidase human immunoglobulin(HIg) Fc fragments (PH20-IgFc), can make
Blood halflife extends in rHuPH20 bodies, so that applied to oncotherapy.The PH20-HSA and PH20-IgFc of the present invention is fed
After newborn zooblast production, it can be purified using purifying HSA and IgFc specific method, it is excellent in terms of production method and production prices
The method for producing long-acting people's hyaluronidase in polyethylene glycol chemistry coupling rHuPH20.In addition, HSA and IgFc are people's source proteins,
Internal security is better than polyethylene glycol.
The above described is only a preferred embodiment of the present invention, any formal limitation not is made to the present invention, this
Art personnel make a little simple modification, equivalent variations or modification using the technology contents of the disclosure above, all fall within this hair
In bright protection domain.
Claims (7)
1. a kind of method for producing recombinant long-acting people's hyaluronidase, the recombinant long-acting people hyaluronidase behaviour hyaluronic acid
The extracellular parts of enzyme PH20 and the fusion protein of people's source protein, people's source protein are that ball is immunized in human albumin HSA fragments or people
Protein I gG2Fc fragments；The fusion protein of the extracellular parts of people's hyaluronidase PH20 and human albumin HSA fragments is PH20-
HSA, its amino acid sequence is as shown in SEQ ID No.4；The extracellular parts of people's hyaluronidase PH20 and human immunoglobulin(HIg)
The fusion protein of IgG2Fc fragments is PH20-IgFc, and its amino acid sequence is as shown in SEQ ID No.5, it is characterised in that described
Method includes：
Using animal cell expression vectors pMH3, pMH4 or pMH5 rich in GC in Chinese ground hamster cell CHO expressing gene
Sequence SEQ ID No.6 or SEQ ID No.7 to obtain described PH20-HSA fusion proteins or PH20-IgFc fusion proteins,
Then isolate and purify producing；Described expression vector pMH3, pMH4 or pMH5 gene order respectively as SEQ ID No.8,
9th, shown in 10.
2. a kind of method for producing recombinant long-acting people's hyaluronidase according to claim 1, it is characterised in that described
PH20-HSA is isolated and purified using Protein A affinity purification methods；The PH20-IgFc is using the hydrophobic indigo plant of ion series connection
Gluing method is isolated and purified.
3. a kind of preparation, it is characterised in that storage-stable has the recombinant long-acting using the method production described in claim 1 or 2
People's hyaluronidase, the preparation includes 150-1500IU PH20-HSA or PH20-IgFc, 10mM Na2HPO4, 0.03%
CaCl2, 0.1%EDTA-Na2, 145mM NaCl, 0.1%HSA, and pH value is 5.5-7.5.
4. a kind of compound preparation, it is characterised in that include the preparation of storage-stable someone's hyaluronidase described in claim 3
With particular treatment medicine.
5. the recombinant long-acting people hyaluronidase of the method production described in claim 1 or 2 prepares the purposes with the use of medicine,
Characterized in that, the medicine that uses cooperatively prepared by people's hyaluronidase is used to transmit in the case where its activity is not degraded
To human albumin or the tissue of human immunoglobulin(HIg) combination, organ, position；Or for by particular treatment drug delivery to disease institute
Tissue, organ or position.
6. recombinant long-acting people's hyaluronidase of the method production described in claim 1 or 2 prepares the purposes of chemical coupling agent, institute
Stating the chemical coupling agent of people's hyaluronidase preparation is used to carry by IgFc the and HSA fragments of the recombinant long-acting hyaluronidase
Come chemical coupling diagnosticum, developer, medicine, treatment toxin, gene into diseased tissue organ for non-functional site.
7. the recombinant long-acting people hyaluronidase of the method production described in claim 1 or 2 is in industrial production small molecule hyalomitome
Application in acid, it is characterised in that the recombinant long-acting people hyaluronidase is produced as toolenzyme degraded macromolecular hyaluronic acid
Raw micromolecule hyaluronic acid.
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| CN109536476A (en) * | 2018-05-21 | 2019-03-29 | 沣潮医药科技（上海）有限公司 | Targent fused protein, the Preparation method and use for having hyaluronidase activity |
| CN114573715A (en) * | 2022-03-14 | 2022-06-03 | 江苏雅酶医药科技有限公司 | Recombinant long-acting human hyaluronidase as well as production method and application thereof |
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