CN102533704A

CN102533704A - Trypsin preparation prepared for immediate use

Info

Publication number: CN102533704A
Application number: CN2010106136810A
Authority: CN
Inventors: 李素霞; 赵致; 冯矗
Original assignee: SHANGHAI YAXIN BIOTECH CO Ltd
Current assignee: SHANGHAI YAXIN BIOTECH CO Ltd
Priority date: 2010-12-30
Filing date: 2010-12-30
Publication date: 2012-07-04

Abstract

The invention relates to a trypsin preparation prepared for immediate use. The trypsin preparation comprises a compound, trypsin freeze-drying powder and an aseptic solvent, wherein the trypsin freeze-drying powder is prepared by directly freeze-drying a trypsin solution or freeze-drying after adding mannitol or trehalose and carrying out aseptic filtration; and the aseptic solvent comprises a cell balance solution and low-concentration EDTA (Ethylene Diamine Tetraacetic Acid). According to the invention, the trypsin is freeze-dried into the aseptic solid powder which is stable in form and easy to transport and store, and the pollution risk is reduced.

Description

Instant trypsin preparation

Technical Field

The invention belongs to the field of biotechnology; more particularly, the present invention relates to ready-to-use formulations of trypsin.

Background

The trypsin solution is easily self-degradable, is generally prepared before use, and is used immediately after sterile filtration. However, since a trypsin solution is a commonly used reagent for cell digestion, a prepared trypsin cell digestion solution is now on the market.

The main component of the cell digestive juice is trypsin, the cell digestive juice in the current market is a solution, and transportation and storage are carried out under the condition of lower than 4 ℃, if the transportation and storage are improper, the cell digestive juice is very easy to inactivate, and the cell digestive juice loses the digestive ability after inactivation, so that the cell digestive effect is unstable when the cell digestive juice is used.

Further, the trypsin cell digestion solutions on the market at present are calculated by the weight of trypsin contained therein, not by its unit activity, and thus, when used for cell digestion, the unit activity is not clear, so that the correct amount cannot be accurately determined.

Therefore, there is an urgent need in the art to improve the current state of formulation and use of trypsin preparations and to find a more suitable and accurate method for formulation and use of trypsin preparations.

Disclosure of Invention

The invention aims to provide a trypsin preparation which is ready to use and is prepared immediately.

In a first aspect of the invention there is provided a trypsin formulation combination comprising:

(1) the trypsin freeze-dried powder is prepared by directly freeze-drying a trypsin liquid or freeze-drying after adding mannitol or trehalose; and

(2) a solvent, said solvent comprising: a cell balancing solution, and EDTA with a final concentration of 0.001-0.01% (w/v).

In another preferred embodiment, the cell balance fluid does not comprise: ca²⁺，Mg²⁺Ions.

In another preferred embodiment, the mannitol or trehalose is added at a final concentration of 0.1-10% (w/v) when preparing the trypsin lyophilized powder.

In another preferred embodiment, the mannitol or trehalose is added at a final concentration of 0.5-6% (w/v) when preparing the trypsin lyophilized powder.

In another preferred embodiment, the mannitol or trehalose is added at a final concentration of 1-5% (w/v) when preparing the trypsin lyophilized powder.

In another preferred embodiment, the solvent comprises: EDTA at a final concentration of 0.003-0.008% (w/v).

In another aspect of the invention, the use of the trypsin preparation combination is provided for preparing a kit for enzymatic digestion or digestion of cells.

In another aspect of the invention, the trypsin freeze-dried powder is prepared by adding mannitol or trehalose into a trypsin liquid and then freeze-drying.

In another aspect of the present invention, there is provided a solvent comprising: a cell balancing solution, and EDTA with a final concentration of 0.001-0.01% (w/v).

In another aspect of the invention, a solution for enzymolysis or digestion of cells is provided, which is formed by mixing the trypsin lyophilized powder and the solvent.

In another preferred embodiment, the enzymatic activity of trypsin in said solution is 500-15000 units (U); or

In another preferred embodiment, the enzymatic activity of the trypsin solution is 1000-12000 units (U).

In another preferred embodiment, the enzymatic activity of the trypsin solution is 1000-10000 units (U).

In another preferred embodiment, the enzymatic activity of the trypsin solution is 1000-6000 units (U).

In another preferred embodiment, the enzyme activity of the trypsin solution is 1500-3000 units (U).

In another preferred embodiment, the final concentration of EDTA in the solution is 0.001-0.01% (w/v).

In another aspect of the present invention, there is provided a method of digesting or digesting cells, the method comprising: and treating the cells needing enzymolysis or digestion by using the solution for enzymolysis or digestion of the cells.

In another aspect of the invention, a kit is provided comprising the combination of reagents.

In another preferred embodiment, the kit further comprises an instruction for use.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 shows the results of cell digestion with the enzyme protein preparation of the present invention. Wherein,

I. digesting for 1-2 min, increasing cell gaps, deforming cells, adding a culture medium, and stopping digestion.

And II, the cells are basically in a single cell state after being suspended, and no agglomeration phenomenon exists.

And III, after the cells grow for 2d by adherence, the cells grow basically singly without agglomeration.

Detailed Description

Aiming at the problem that the existing preparation and use technologies of trypsin lag behind, the inventor develops a method suitable for preparing a trypsin lyophilized powder preparation and an optimized solvent for dissolving the lyophilized powder preparation and simultaneously serving as a cell balance solution through intensive research. The freeze-dried powder and the solvent are stored separately and can be conveniently mixed and used before use.

As used herein, the terms "comprising," having, "or" including "include" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

Freeze-dried powder

Since trypsin has self-digestion ability and poor stability, there are many problems in storage and use. Typically, they are formulated prior to use and immediately after formulation, which adds complexity to the testing procedure for the tester. The inventor has long devoted efforts to develop a method for long-term storage and convenient use of trypsin, and found that the conventional method for preparing trypsin lyophilized powder may cause activity loss during lyophilization, a specific protective agent needs to be added, and not all lyophilized protective agents are suitable. Therefore, the inventor screens and optimizes the lyoprotectant and the concentration thereof, and finally finds out the lyoprotectant used and the concentration thereof suitable for use.

Therefore, as a preferred mode of the invention, the trypsin freeze-dried powder is prepared by adding mannitol or trehalose into a trypsin liquid and then freeze-drying. Preferably, when preparing the trypsin lyophilized powder, the mannitol or trehalose is added at a final concentration of 0.5-6% (w/v); more preferably, the mannitol or trehalose is added at a final concentration of 1-5% (w/v). After mannitol or trehalose with the concentration is added into the enzyme solution, the trypsin freeze-dried powder has good forming property and solubility, and the enzyme activity is not lost at all, so that the mannitol or trehalose can effectively improve the stability of the trypsin freeze-drying process and can be used as an excellent freeze-drying protective agent for trypsin.

Other methods of lyophilizing trypsin may employ lyophilization techniques that are conventional in the art. In a preferred embodiment of the present invention, a freeze vacuum drying method is used.

The trypsin suitable for use in the present invention may be a recombinant protein, a natural protein, a synthetic protein, preferably a recombinant protein. The proteins of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells). The trypsin of the invention can be extracted from pig pancreas and bovine pancreas, and can also be recombinant trypsin. The trypsin of the invention is preferably a recombinant protein which ensures the absence of viral contamination of animal origin.

Mannitol or trehalose suitable for use in the present invention are all known products known in the art and are readily available in a commercially available manner, for example, from Shanghai Bioengineering, Inc.

Solvent(s)

The trypsin cell digestion solution (solution for digesting or digesting cells) is used for digesting cells, and is not only required to ensure that the cells themselves have good and stable enzyme activity, but also required to provide good environment for the cells so that the cells after digestion have a good state. Therefore, optimization of the solvent used to formulate the trypsin cell digest is also important. The inventor aims to find a solvent with a proper formula, and after extensive research, the inventor finds that the addition of EDTA with a low concentration in a cell balance solution can effectively assist the digestion effect of trypsin on cells, so that the trypsin achieves the same effect as a high-activity unit under the condition of a low-activity unit. However, since EDTA at a high concentration has a cell killing effect when used in combination with trypsin, digestion is carried out using EDTA at a concentration as low as possible in combination with trypsin.

Accordingly, the present invention provides a solvent comprising: cell balance solution and EDTA with final concentration of 0.001-0.01% (w/v); preferably, the solvent comprises: EDTA at a final concentration of 0.003-0.008% (w/v).

The cell-balancing solution of the present invention is not particularly limited, and may be any basic balancing solution as long as it can provide a desired balanced pH and osmotic pressure environment for cell growth. For example, the cell balancing fluid is selected from: ringer, PBS, earle, hanks, dulbecco or d-hanks. The formulation and formulation of these cell balancing solutions are known to those skilled in the art. In one embodiment of the present invention, the cell-balancing solution is PBS.

The solvent is typically prepared in sterile form, for example by aseptic filling, to avoid contamination of the cells.

Trypsin cell digestive juice

The trypsin cell digestive juice can be prepared by the trypsin freeze-dried powder and the solvent of the cells and used for enzymolysis or cell digestion. Therefore, the invention provides a solution for enzymolysis or digestion of cells, which is formed by mixing the trypsin freeze-dried powder and the solvent.

The trypsin cell digest is formulated to have comparable enzymatic activity, which is useful to one skilled in the art in determining the amount needed. As a preferred embodiment of the present invention, the enzyme activity of trypsin in the solution is 500-15000 units (U); more preferably, the trypsin solution has an enzymatic activity of 1000-12000 units (U); more preferably, the trypsin solution has an enzymatic activity of 1000-10000 units (U); more preferably, the enzymatic activity of the trypsin solution is 1000-6000 units (U); more preferably, the trypsin solution has an enzymatic activity of 1500-.

The trypsin cell digest was also formulated as a solution with an appropriate concentration of EDTA, since EDTA was able to work in concert with trypsin to digest the cells. As a preferred mode of the invention, the final concentration of EDTA in the solution is 0.001-0.01%; more preferably, the concentration is 0.003-0.008%.

The trypsin cell digest may be adapted for digestion or enzymatic digestion of any cell or cell culture, as determined by the nature of the cells it digests. Such as, but not limited to: cells needing digestive dispersion or adherent culture, such as interstitial neuroma cells, various epithelial cells, cancer cells, fibroblasts, various primary cells and the like.

Reagent combination and kit

Based on the above improvement, the present invention also provides a reagent combination, comprising: the trypsin freeze-dried powder is prepared by adding mannitol or trehalose into a trypsin liquid and then freeze-drying; and a solvent, said solvent comprising: cell balance liquid, and EDTA with final concentration of 0.001-0.01%.

The invention also provides a kit, which comprises the reagent combination.

Of course, the kit may also include other reagents or instructions, including, for example, instructions for formulation or use; or for example, cell culture media, cell lysates, etc.

The reagent combination or the kit can be convenient for the preparation and the use of the trypsin cell digestive juice by the technicians in the field, has high stability and is convenient for long-term storage or transportation.

The main advantages of the invention are:

(1) the trypsin cell digestive juice is prepared and used, and is lyophilized into solid powder after being aseptically filled, so that the solid form is stable and easy to transport and store. And the trypsin is packaged in a solid state, so that the pollution risk is reduced.

(2) The trypsin preparation of the invention has high activity, can be digested at room temperature (25 ℃) and does not need the condition of 37 ℃. And the purity is high, so that the influence of impurities on the cell growth is avoided.

(3) The trypsin cell digestion solution of the present invention does not contain Ca²⁺，Mg²⁺Ions, avoid Ca²⁺，Mg²⁺The influence of ions on the growth of cells, etc. Overcomes the defect that Ca must be added into the trypsinase cell digestive juice which is conventionally considered by the people in the field²⁺，Mg²⁺The technical prejudice of ion to protect the enzyme activity.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1 preparation of equilibrium solution

The basic equilibrium solution was formulated according to the formulation shown in table 1 below, and provided the equilibrium pH and osmotic pressure environment required for cell growth. And sequentially adding the reagents into 800mL of sterile water, dissolving the reagents under magnetic stirring, and fixing the volume to 1000mL of double distilled water. Autoclaving at 121 deg.C for 15 min.

TABLE 1 different methods for preparing cell-balancing solutions

g/L	ringer	PBS	earle	hanks	dulbecco	d-hanks
							NaCl	9	8	6.8	8	8	8
KCl	0.42	0.2	0.4	0.4	0.2	0.4
							CaCl₂	0.25		0.2	0.14	0.1
MgCl₂·6H₂O					0.1
							MgSO₄·7H₂O			0.2	0.2
Na₂HPO₄·H₂O		1.56		0.06		0.06
							Na₂HPO₄·2H₂O			1.14		1.42
KH₂PO₄		0.2		0.06	0.2	0.06
							NaHCO₃			2.2	0.35		0.35
Glucose			1	1
							Phenol Red			0.02	0.02	0.02	0.02

The balance liquid can provide the balance pH and osmotic pressure environment required by cell growth.

Example 2 Activity content of Trypsin in digestive juice

The passaged murine T3 cells (purchased from Shanghai cell Bank of Chinese academy of sciences) were passaged in 24-well plates and cultured in complete cell culture medium. After the cells grow adherent to the wall and are basically full of the cells, the culture solution is sucked out, and 1ml of cell digestive juice with different formulas is added. The morphological change of the cells is recorded by observing under a microscope, and the cells are photographed and the time is recorded. Comparing the cell morphology change and comparing the time difference.

Cell digests (using equilibrium PBS) containing recombinant human trypsin 2(hT2, wild type, GenBank accession No. NM — 002770) of different activities were prepared and EDTA was added at different final concentrations to observe their cell digests, the results are shown in table 2. The cell digestion solution containing trypsin with different activities has different digestion capacities on cells, the low-concentration EDTA and the trypsin have synergistic effect on cell digestion, the same effect as the high-concentration trypsin is achieved, and the dosage of the trypsin can be reduced under the action of the low-concentration EDTA. However, since EDTA at a high concentration has a cell killing effect when used in combination with trypsin, digestion is carried out using EDTA at a concentration as low as possible in combination with trypsin.

TABLE 2 Effect of EDTA content in formulations and equilibration solutions for different active trypsins on cell digestibility

In the table, ku represents 1000U; the percentage of EDTA is mass to volume (w/v).

The enzyme activity calculation method comprises the following steps: a BAEE unit was defined as an increase in absorbance at 253nm by 0.001 per minute by enzymatic digestion of N-benzoyl-L-arginine ethyl ester (BAEE) at 25 ℃ and pH7.6 in a reaction volume of 3.0ml (1em path).

By performing cell digestion experiments with different formulations of trypsin content and EDTA content, as shown in table 2, it was found that only 0.01% of EDTA was insufficient for the digestion capacity of the cells, and some cells could not be exfoliated at all. A certain amount of trypsin must be added to achieve the best effect.

The results of 2.0ku + 0.005% (w/v) EDTA digestion of the cells are shown in FIG. 1.

Example 3 equilibration of other Metal ions such as Ca²⁺，Mg²⁺Content of ion

Due to Ca²⁺，Mg²⁺The ion etc. has the enzyme activity protection function to the trypsin in the solution state, so the prior trypsin cell digestive juice contains Ca²⁺，Mg²⁺Ion, but due to Ca²⁺，Mg²⁺Since ions have an influence on cell growth and the like, the equilibrium liquid of the present invention does not contain Ca²⁺，Mg²⁺Ions.

Example 4 Trypsin lyophilization

The dissolved trypsin was sterile filtered and lyophilized to a solid powder.

Adding different kinds of protective agents (including mannitol, sorbitol, trehalose, sucrose, lactose, bovine serum albumin, NaCl and the like) with different concentrations into pure recombinant human trypsin 2(hT2) enzyme solution respectively for freeze vacuum drying. Respectively recording the appearance forms of the freeze-dried products, then adding 0.5ml of pure water for dissolving, recording the solubility of the freeze-dried products, and determining the enzyme activity of each freeze-dried product, wherein the initial enzyme activity before freeze-drying is 100%, and calculating the residual rate of the enzyme activity, thereby screening the protective agent capable of stably recombining hT2 in the freeze-drying process and keeping the biological activity of the target protein.

TABLE 3 Freeze-drying protective agent optimization table

Note: the shaping performance is excellent, and the freeze-dried product is fluffy and takes the shape of a cake; the shaping performance is better and the freeze-dried product is more fluffy, "+ + + + +"; "+" and the excipient is general, the freeze-dried product is compact; "-", the shaping property is poor, and the freeze-dried product is in a shrinkage state.

Example 5 Trypsin lyophilized powder stability

To the enzyme solution was added a lyophilized powder having a final concentration of 1% (w/v) mannitol or 1% (w/v) trehalose.

The verification proves that the stability of the freeze-dried powder is good, the recombinant trypsin freeze-dried powder is kept at 4 ℃, and the activity is not lost within 2 years.

Example 6 method of Using Ready-to-use Trypsin cell digest

The using method comprises the following steps:

1. adding 1ml of cold equilibrium solution into an EP tube containing trypsin freeze-dried powder;

2. fully dissolving trypsin;

3. all the materials are moved into a bottle filled with a balance liquid and used within 2 hours;

4. if the product is not used for one time, the product is prepared and then immediately packaged according to the use amount of each time, and the product is stored at the temperature of minus 20 ℃;

5. when in use, the product is taken out at the temperature of-20 ℃ and dissolved at room temperature for use without preheating at the temperature of 37 ℃.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A trypsin formulation combination comprising:

2. The trypsin formulation combination of claim 1, wherein said mannitol or trehalose is added at a final concentration of 0.1-10% (w/v) when said lyophilized trypsin powder is prepared.

3. Use of a combination of trypsin preparations according to any one of claims 1-2, for the preparation of a kit for enzymatic digestion or digestion of cells.

4. The trypsin freeze-dried powder is characterized in that the trypsin freeze-dried powder is prepared by adding mannitol or trehalose into a trypsin liquid and then freeze-drying the mixture.

5. A solvent, said solvent comprising: a cell balancing solution, and EDTA with a final concentration of 0.001-0.01% (w/v).

6. A solution for enzymatic digestion or digestion of cells, which is prepared by mixing the lyophilized trypsin powder of claim 5 and the solvent of claim 6.

7. The solution of claim 6, wherein the trypsin has an enzymatic activity of 500-15000 units (U).

8. The solution of claim 6, wherein the final concentration of EDTA in the solution is 0.001-0.01% (w/v).

9. A method of enzymatically digesting or digesting cells, said method comprising: treating cells in need of enzymolysis or digestion with the solution for digesting cells according to any one of claims 6 to 8.

10. A kit comprising the combination of reagents of claim 1.