Recombinant protein a lyophilized powder and method of preparation

Abstract

The present application relates to the field of protein purification and immunoadsorption, in particular to a recombinant protein A freeze-dried powder and a preparation method thereof. The present application provides a recombinant protein A freeze-dried powder and a preparation method thereof, wherein the recombinant protein A freeze-dried powder comprises recombinant protein A and two protein protective agents (sodium chloride and TCEP), compared with existing products, the composition is simple, after reconstitution of the freeze-dried protein, the freeze-drying protective agent does not need to be removed, and subsequent coupling reaction can be directly carried out, the preparation steps are simplified, the influence on protein activity is small, the processing cost is low, and the present application is suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein purification and immunoadsorption, in particular to a lyophilized powder of recombinant protein A and a preparation method thereof. BACKGROUND

[0002] Protein A is a cell wall protein isolated from Staphylococcus aureus type A, which can bind to the Fc segment of human IgG and is commonly used for immunological analysis and IgG purification. However, the structure of natural protein A is stable and not resistant to acid and alkali. Recombinant protein A is modified on the basis of natural protein A, which can significantly enhance the specificity of binding to the Fc segment, and improve the stability and alkali resistance of the protein.

[0003] Effective use of recombinant protein A requires long-term maintenance of protein activity. Whether it is storage, transportation or subsequent coupling with solid separation medium, protein denaturation and activity loss should be avoided. Vacuum freeze-drying technology can protect the biological activity of proteins with less damage to protein structure, and can be effectively applied in the field of biological products.

[0004] At present, a freeze-drying protective agent is added to the protein solution before freeze-drying to protect the protein structure and activity from the influence of drying stress, freezing stress and low temperature stress. Common freeze-drying protective agents include phosphate, polyhydroxy compound, amino acid, surfactant, sugar and the like. For example, phosphate is a common buffer in freeze-dried protein formulations to avoid the influence of environmental pH on protein stability. Polyhydroxy compounds and amino acids are often used as fillers to prevent protein product collapse and improve formulation solubility. Sugars are common excipients for freeze-dried proteins, which provide stability by replacing hydrogen bonds between proteins and water. In order to ensure good use effect, the freeze-dried protective agent needs to be removed before subsequent use after the freeze-dried protein is reconstituted. This will prolong the production time and increase the production cost.

[0005] In addition, when recombinant protein A is applied to chromatographic fillers or immunoadsorbents, the adsorption performance is related to the coupling method of recombinant protein A and solid microspheres. The single-point coupling method can improve the adsorption performance. Single-point coupling is usually achieved by the thiol group of cysteine in recombinant protein A. The thiol group of cysteine in recombinant protein A is oxidized into a disulfide bond. In the preparation of chromatographic fillers or immunoadsorbents, recombinant protein A needs to be reduced to thiol group first, and then the next coupling reaction can be carried out, which makes the preparation process relatively complex. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide a lyophilized powder of recombinant protein A and a preparation process thereof.

[0007] The application provides a recombinant protein A freeze-dried powder, raw materials for preparing the recombinant protein A freeze-dried powder include recombinant protein A, sodium chloride and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) ; the recombinant protein A is staphylococcus aureus cell wall protein, and can recognize and combine immunoglobulin IgG.

[0008] Further, in the recombinant protein A freeze-dried powder, the mass ratio of sodium chloride, tris (2-carboxyethyl) phosphine hydrochloride and recombinant protein A is (0.03-1) :(0.009-0.3):1.

[0009] Further, in the recombinant protein A freeze-dried powder, the mass ratio of sodium chloride, tris (2-carboxyethyl) phosphine hydrochloride and recombinant protein A is (0.03-1) :(0.009-0.3):1.

[0010] In the application, the recombinant protein A includes protein A with original amino acid sequence, and can also include recombinant protein A, the recombinant protein A can be selected from at least one of the amino acid sequences shown in SEQ ID NO:1-SEQ ID NO:15 or a tandem of each of the amino acid sequences shown in SEQ ID NO:1-SEQ ID NO:15;

[0011] The number of the tandem is multiple, specifically, can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0012] More specifically, the amino acid sequence of the recombinant protein A is selected from at least one of the following sequences:

[0013] (SEQ ID NO: 1)1, (SEQ ID NO: 2)1, (SEQ ID NO: 3)1, (SEQ ID NO: 4)1, (SEQ ID NO: 5)1, (SEQ ID NO: 6)1, (SEQ ID NO: 7)1, (SEQ ID NO: 8)1, (SEQ ID NO: 9)1, (SEQ ID NO: 10)1, (SEQ ID NO: 11)1, (SEQ ID NO: 12)1, (SEQ ID NO: 13)1, (SEQ ID NO: 14)1, (SEQ ID NO: 15)1, (SEQ ID NO: 7)3, (SEQ ID NO: 7)4, (SEQ ID NO: 7)5, (SEQ ID NO: 7)6, (SEQ ID NO: 12)3, (SEQ ID NO: 12)4, (SEQ ID NO: 12)5, (SEQ ID NO: 12)6, (SEQ ID NO: 13)3, (SEQ ID NO: 13)4, (SEQ ID NO: 13)5, (SEQ ID NO: 13)6, (SEQ ID NO: 14)3, (SEQ ID NO: 14)4, (SEQ ID NO: 14)5, (SEQ ID NO: 14)6, (SEQ ID NO: 15)3, (SEQ ID NO: 15)4, (SEQ ID NO: 15)5 or (SEQ ID NO: 15)6; wherein the number outside the amino acid sequence is the number of tandem recombinant protein A of the indicated sequence;

[0014] The amino acid sequences of the recombinant protein A as shown in SEQ ID NO: 1- SEQ ID NO: 15 are as follows:

[0015] SEQ ID NO: 1:

[0016]

[0017]

[0018] SEQ ID NO: 2:

[0019]

[0020] SEQ ID NO: 3:

[0021]

[0022] SEQ ID NO: 4:

[0023]

[0024] SEQ ID NO:5:

[0025]

[0026] SEQ ID NO:6:

[0027]

[0028] SEQ ID NO:7:

[0029]

[0030] SEQ ID NO:8:

[0031]

[0032] SEQ ID NO:9:

[0033]

[0034] SEQ ID NO:10:

[0035]

[0036] SEQ ID NO:11:

[0037]

[0038]

[0039] SEQ ID NO:12:

[0040]

[0041] SEQ ID NO:13:

[0042]

[0043] SEQ ID NO:14:

[0044]

[0045] SEQ ID NO:15:

[0046]

[0047] The present application provides a preparation method of the recombinant Protein A freeze-dried powder, which comprises:

[0048] The method comprises the following steps: mixing recombinant protein A, tris (2-carboxyethyl) phosphine hydrochloride and sodium chloride to obtain a mixed solution, and then freeze-drying the mixed solution, wherein the freeze-drying comprises pre-freezing, gradient freeze-drying and vacuum sublimation;

[0049] The pre-freezing temperature is -48℃ to -46℃, and the pre-freezing time is 2 to 4 hours.

[0050] The gradient freeze-drying program comprises freeze-drying stage 1 and freeze-drying stage 2.

[0051] The starting temperature of the freeze-drying stage 1 is -45℃, the starting temperature is kept for 1 to 4 hours, the ending temperature is -36℃ to -38℃, the ending temperature is kept for 2 to 4 hours, and the temperature is increased by 2 to 5℃ at each time, and each time the temperature is kept for 1 to 4 hours.

[0052] The starting temperature of the freeze-drying stage 2 is -32℃, the starting temperature is kept for 1 to 4 hours, the ending temperature is 30℃, the ending temperature is kept for 1 to 4 hours, and the temperature is increased by 5 to 10℃ at each time, and each time the temperature is kept for 1 to 4 hours.

[0053] Further, in the specific embodiments of the present application, the gradient freeze-drying program comprises:

[0054] The gradient freeze-drying program comprises:

[0055] -45℃ is kept for 1 to 4 hours;

[0056] The temperature is increased to -40℃ and kept for 1 to 4 hours;

[0057] The temperature is increased to -36℃ to -38℃ and kept for 2 to 4 hours;

[0058] The temperature is increased to -32℃ and kept for 1 to 4 hours;

[0059] The temperature is increased to -28℃ and kept for 1 to 4 hours;

[0060] The temperature is increased to -18℃ and kept for 1 to 4 hours;

[0061] The temperature is increased to -8℃ and kept for 1 to 4 hours;

[0062] The temperature is increased to 0℃ and kept for 1 to 4 hours;

[0063] The temperature is increased to 10℃ and kept for 1 to 4 hours;

[0064] The temperature is increased to 20℃ and kept for 1 to 4 hours;

[0065] The temperature is increased to 30℃ and kept for 1 to 4 hours.

[0066] The vacuum pressure plug is under the condition of 10 -3 ~10 -1 Pa.

[0067] In the specific embodiments of the present application, the co-crystallization temperature range is -36℃ to -38℃, different solution compositions correspond to specific co-crystallization temperatures, which can be adjusted according to the solution composition; in the specific embodiments of the present application, the co-crystallization temperature of the solution with a mass ratio of recombinant protein A, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and sodium chloride of 1:0.29:1 is -37.6℃.

[0068] The step of mixing the recombinant protein A, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and sodium chloride uniformly comprises:

[0069] Step 1, mixing the recombinant protein A solution with the TCEP reducing agent to obtain a mixed solution;

[0070] Step 2, replacing the solution system of the mixed solution with a sodium chloride solution system.

[0071] The concentration of the TCEP reducing agent is 1-10 mmol / L;

[0072] The concentration of the TCEP reducing agent is 1-10 mmol / L;

[0073] The concentration of the protein A is 10-30 mg / mL;

[0074] The concentration of the sodium chloride solution is 1-10 mg / mL;

[0075] The device for replacing the solution system of the mixed solution adopts an ultrafiltration system.

[0076] The present application optimizes the component ratio of the recombinant protein A freeze-dried powder protective agent, and the test results show that, compared with the recombinant protein A freeze-dried powder without the protective agent, the long-term storage activity of the recombinant protein A freeze-dried powder with the protective agent sodium chloride and TCEP does not decrease significantly, the adsorption effect of the prepared adsorption product is good, the adsorption performance decreases less, and the mass ratio of sodium chloride, tris (2-carboxyethyl) phosphine hydrochloride, recombinant protein A and recombinant protein A in the recombinant protein A freeze-dried powder is 1:0.29:1, the long-term storage activity of the recombinant protein A freeze-dried powder decreases the least, and the adsorption performance of the prepared adsorbent is closest to that of the recombinant protein A adsorbent obtained by direct purification; at the same time, the freeze-drying process is optimized, and the test results show that, under the condition that the component ratio of the freeze-drying protective agent is the same, the recombinant protein A freeze-dried powder prepared by using the freeze-drying process of the present application has the best stability, and the long-term storage activity decreases less.

[0077] The preparation method of the application has the advantages of simple preparation process, little influence on protein activity, low processing cost, and suitability for large-scale production; the long-term stored recombinant protein A lyophilized powder can be directly reconstituted for coupling with a carrier without pre-removal of the lyophilized protein protective agent and pre-reduction of protein A, thus simplifying the preparation process of the carrier.

[0078] Compared with other existing protein protective agents or other component protein protective agents, the recombinant protein A lyophilized powder prepared by using the neutral salt sodium chloride and TCEP as protective agents has more stable performance and less influence on protein activity; in the protective agent system of the application, sodium chloride increases the hydration degree of protein, forms a hydration film on the surface of protein, protects the spatial chain of protein from direct exposure to the environment, thus maintaining the protein activity and reducing the loss of lyophilized activity; TCEP reacts with cysteine in protein to generate thiol (-SH) and phosphine oxide, and the phosphine oxide can prevent the thiol from being oxidized during lyophilization, thus playing a protective role in lyophilization and reducing the loss of lyophilized protein activity; the lyophilized protective agent only contains sodium chloride and TCEP, and the composition of the protective agent is simple; compared with existing recombinant protein A lyophilized products, the reconstituted product can directly undergo subsequent coupling reaction without removal of the lyophilized protective agent, thus simplifying the operation steps, shortening the production time, and reducing the production cost.

[0079] Meanwhile, the preparation method of the application is also applicable to the lyophilization of other protein products, including but not limited to protein A and other proteins.

[0080] The application provides the use of any one of the following I) to II) in the preparation of a product for protein purification and / or immunoadsorption:

[0081] I) the recombinant protein A lyophilized powder according to the application;

[0082] II) the recombinant protein A lyophilized powder prepared by the preparation method according to the application.

[0083] The application provides a product for protein purification and / or immunoadsorption, which comprises at least one of the recombinant protein A lyophilized powder according to the application or the recombinant protein A lyophilized powder prepared by the preparation method according to the application and a coupling carrier.

[0084] Further, the coupling carrier comprises agarose gel.

[0085] The application provides a preparation method of the product, which comprises the following steps:

[0086] The recombinant protein A lyophilized powder according to the application is reconstituted with water to obtain a recombinant protein A lyophilized powder solution, and the recombinant protein A lyophilized powder solution is reacted with an agarose solution to obtain the product.

[0087] In the specific embodiments of the present application, the freeze-dried powder solution of recombinant protein A is reacted with the agarose solution, and then a capping step is further included, which comprises reacting the conjugate obtained by reacting the freeze-dried powder solution of recombinant protein A with the agarose solution after washing.

[0088] The reaction condition of the freeze-dried powder solution of recombinant protein A with the agarose solution is 35℃ oscillation reaction for 1h;

[0089] The concentration volume ratio of the freeze-dried powder of recombinant protein A to agarose is 1:(1-3);

[0090] The washing reagent is pure water;

[0091] The concentration of the ethanolamine is 1M, and the volume ratio of the conjugate to the ethanolamine is 1:2;

[0092] The reaction condition with the ethanolamine is 35℃, 180rpm reaction for 8-12h.

[0093] In the present application, one of the capping methods is used in the embodiments of the present application, and the capping further includes other methods, which are not limited in the present application.

[0094] The present application provides a method for protein purification and / or immunoadsorption, which is carried out by using the product of the present application for protein purification or immunoadsorption.

[0095] The present application provides a freeze-dried powder of recombinant protein A and a preparation method thereof, which comprises recombinant protein A and two protein protective agents (sodium chloride and TCEP). Compared with the existing products, the freeze-dried powder of recombinant protein A has simple composition, small influence on protein activity, low processing cost, and is suitable for large-scale production. After reconstitution of the freeze-dried protein, the freeze-dried protective agent does not need to be removed, and the subsequent coupling reaction can be directly carried out, thereby simplifying the preparation steps of the adsorbent. DETAILED DESCRIPTION

[0096] The present application provides a freeze-dried powder of recombinant protein A and a preparation process, and those skilled in the art can refer to the content herein and appropriately improve the process parameters for implementation. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0097] The reagents and consumables used in the present application are ordinary market products, which can be purchased in the market.

[0098] The present application will be further described below in combination with embodiments:

[0099] Example 1: Preparation of Recombinant Protein A Lyophilization Protectant Solution

[0100] I. Preparation of Ultrafiltration System

[0101] 1. First, flush out the storage solution in the ultrafiltration system with 1 kg of purified water.

[0102] 2. Circulate and clean the ultrafiltration system with 3 kg of 0.5 M NaOH solution for more than 30 minutes, then rinse the ultrafiltration system with more than 1 kg of purified water cooled by the condenser until the pH of the effluent from the reflux end and the permeate end is neutral, and drain the liquid from the ultrafiltration system.

[0103] II. Solution Preparation

[0104] 1. Prepare recombinant protein A to obtain a recombinant protein A solution with a purity of over 95%, and concentrate the solution to a concentration between 10 and 30 mg / mL.

[0105] 2. Add TCEP (tris(2-carboxyethyl)phosphine hydrochloride) to the recombinant protein A concentrate and adjust the final concentrations to 1, 5, and 10 mM, respectively.

[0106] 3. Replace the solution system with sodium chloride solution (concentration of 1, 5, or 10 mg / mL) at least 5 times the volume of the concentrated solution. After replacement, drain the solution from the pipeline (inlet pressure ≤ 1.5 bar during concentration and replacement).

[0107] Orthogonal experiments were designed using Minitab software. According to Table 1, control groups 1-3 and experimental groups 1-9 were prepared.

[0108] Table 1. Combinations of lyophilization protectant solutions of different concentrations of recombinant protein A

[0109]

[0110] Example 2: Preparation of Experimental Samples

[0111] The preparation of adsorption samples from freeze-dried samples of experimental groups 1–9 (composition as shown in Table 1) is as follows:

[0112] I. Freeze-drying steps

[0113] 1. Determination of the eutectic point:

[0114] The temperature probe of the eutectic point detector was inserted into the obtained recombinant protein A lyophilized protein protectant solution, and the eutectic point temperature was measured to be -36℃ to -38℃.

[0115] 2. Pre-freezing stage

[0116] Take 10 mL of the freeze-dried protective agent solution of recombinant protein A in a vial, and keep it at the pre-freezing temperature for about 4 h after reaching the pre-freezing temperature, and freeze it completely.

[0117] 3. Drying sublimation stage

[0118] 1) After the pre-freezing is completed, start the vacuum pump (set the vacuum degree to 10 -3 ~ 10 -1 Pa), and heat to -45℃ and keep for 4 h;

[0119] 2) Heat to -40℃ and keep for 4 h;

[0120] 3) Heat to the eutectic point and keep for 4 h;

[0121] 4) Heat to -32℃ and keep for 3 h;

[0122] 5) Heat to -28℃ and keep for 3 h;

[0123] 6) Heat to -18℃ and keep for 3 h;

[0124] 7) Heat to -8℃ and keep for 1 h;

[0125] 8) Heat to 0℃ and keep for 1 h;

[0126] 9) Heat to 10℃ and keep for 1 h;

[0127] 10) Heat to 20℃ and keep for 1 h;

[0128] 11) Heat to 30℃ for 1 h;

[0129] 4. Vacuum plugging stage

[0130] After the freeze-drying is completed, slowly press the cover plate of the freeze dryer in a vacuum environment to perform plugging treatment, so that the inside of the freeze-dried powder is in a vacuum state, and the sterile environment of the freeze-dried powder is ensured, and the freeze-dried powder of recombinant protein A is obtained, which has a fluffy and delicate texture, a smooth surface, and uniform color. The prepared freeze-dried powder of recombinant protein A is stored in a sealed state at room temperature.

[0131] II. Preparation of adsorbed samples

[0132] 1. Preparation of recombinant protein A solution: Take the freeze-dried powder of recombinant protein A prepared above and placed for one year, add purified water to each vial to dissolve, and prepare a 20 mg / mL protein A solution (the concentration of protein A is calculated according to Table 1), and each vial is made up to 20 mL.

[0133] 2. Recombinant Protein A Coupling Medium: Take 20 mL of pre-activated agarose microspheres (Bestarose 6FF), add 40 mL of PB buffer solution to obtain a microsphere solution, mix the microsphere solution with the protein solution at a volume ratio of 3:1, and shake at 35°C for 1 h. After the reaction is completed, wash with purified water until clean.

[0134] 3. End-capping: add 2 times the volume of the adsorbent of 1M ethanolamine to the coupled agarose, react at 35°C, 180 rpm overnight, after the reaction is completed, rinse with a large amount of purified water, and obtain the recombinant protein A adsorption sample, i.e., test samples 1-9, which are stored in 20% ethanol for standby.

[0135] Example 3 Preparation of Control Samples

[0136] Including attempts to see if there is a protective liquid component, without freeze-drying and freeze-drying steps

[0137] I. Control samples 1-3, ingredients as shown in Table 1 Control Group 1-3, without protective agent, after freeze-drying, the preparation of the adsorption sample, the steps are as follows:

[0138] 1. The freeze-drying step of control samples 1-3 is as in Example 2.

[0139] 2. The preparation steps of the adsorption sample of control samples 1-3 are as follows:

[0140] 1) Preparation of recombinant protein A solution: Take the above-mentioned recombinant protein A freeze-dried powder placed for one year, add purified water to each bottle to reconstitute, configure 20 mg / mL of protein A solution, and add 10 mmol / L TCEP to each bottle to react overnight to obtain an active protein solution.

[0141] 2) Recombinant Protein A Coupling Medium: Take 20 mL of agarose microspheres, add 40 mL of PB buffer solution to obtain a microsphere solution, mix the microsphere solution with the protein solution at a volume ratio of 3:1, and shake at 35°C for 1 h. After the reaction is completed, wash with purified water until clean.

[0142] 3) End-capping: add 2 times the volume of the adsorbent of 1M ethanolamine to the coupled agarose, react at 35°C, 180 rpm overnight, after the reaction is completed, rinse with a large amount of purified water, and obtain the adsorption medium sample - control sample 1, control sample 2 and control sample 3, which are stored in 20% ethanol for standby.

[0143] II. Control sample 4, without freeze-drying, directly for the preparation of the adsorption sample, the steps are as follows:

[0144] 1. Take the recombinant protein A solution stored at -20°C, configure 20 mg / mL of protein A solution, and add 10 mmol / L TCEP to react overnight to obtain an active protein solution.

[0145] 2. Recombinant Protein A Coupling Medium: Take 20 mL of Sepharose microspheres, add 40 mL of PB buffer solution to obtain a microsphere solution, mix the microsphere solution with a protein solution at a volume ratio of 3:1, and shake at 35°C for 1 h. After the reaction is completed, wash with pure water until clean.

[0146] 3. End-capping: Add 2 times the volume of the adsorbent of 1 M ethanolamine to the coupled Sepharose and react at 35°C, 180 rpm overnight. After the reaction is completed, rinse with a large amount of purified water to obtain an adsorbent sample - control sample 4, and store in 20% ethanol for later use.

[0147] III. Control sample 5, ingredients as shown in Table 1, experimental group 3, after freeze-drying, prepare the adsorption sample, the steps are as follows:

[0148] 1. The freeze-drying steps of control sample 5 are as follows:

[0149] (1) Measurement of the eutectic point:

[0150] Insert the temperature probe of the eutectic point detector into the recombinant protein A freeze-dried protein protectant solution obtained according to the steps of Example 1, and measure the eutectic point temperature -37.6°C.

[0151] (2) Drying and sublimation stage:

[0152] 1) Cool the protein A solution to -48°C and maintain for 4 h;

[0153] 2) Warm up to -37.6°C and maintain for 4 h;

[0154] 3) Warm up to 0°C and maintain for 4 h;

[0155] 4) Warm up to 30°C for 4 h;

[0156] (3) Vacuum plugging stage

[0157] After freeze-drying is completed, slowly press the cover plate of the freeze dryer in a vacuum environment to perform plugging treatment, ensuring that the inside of the freeze-dried powder is in a vacuum state and ensuring the sterile environment of the freeze-dried powder. The recombinant protein A freeze-dried powder obtained has a fluffy and delicate texture, a smooth surface, and uniform color. The prepared recombinant protein A freeze-dried powder is properly stored.

[0158] 2. Control sample 5 adsorption sample preparation steps as in Example 2.

[0159] Example 4: Test of adsorption performance of experimental sample and control sample

[0160] Take the experimental sample and control sample respectively, add 80 mg IgG per milliliter of protein A filler, adsorb at 28℃, 120 rpm for 1 h. Collect the flow-through, use 20 times the column volume of 1×PBS to balance, add 8 times the column volume of 0.1M pH=3.0 acetic acid solution, elute at 28℃, 120 rpm for 15 min. Collect the eluent, dilute 20 times to measure OD 280 , the extinction coefficient is 1.38. The adsorption performance is calculated as follows:

[0161]

[0162] The results are shown in Table 2. From the test results, it can be seen that:

[0163] The adsorption performance of the adsorption sample prepared by reconstituting the freeze-dried powder of recombinant protein A in experimental samples 1-9 is slightly lower than that of the control sample 4 of non-frozen recombinant protein A, but is significantly higher than that of the freeze-dried powder without adding freeze-dried protein protectant in control samples 1-3. The freeze-dried protectant well protects the activity of the freeze-dried protein, meeting the needs of long-term storage.

[0164] The test sample 3 does not need to add TCEP reducing agent for reduction after reconstitution of the freeze-dried powder of recombinant protein A, and does not need to remove the freeze-dried protectant, and can directly proceed to the subsequent coupling. The adsorbent performance has no obvious change compared with the control sample 4 of non-frozen protein. However, the freeze-dried powder without adding freeze-dried protectant in control samples 1-3 still needs to add TCEP reducing agent for protein treatment after reconstitution. Therefore, the protein activity of the freeze-dried protein of the test sample 3 does not decrease obviously, the adsorption performance of the adsorbent after coupling is maintained at a high level, and the coupling process is simple, and the processing time and cost are obviously reduced.

[0165] Compared with the control sample 5, the freeze-dried process of the test sample 3 is first pre-frozen, and the temperature is raised during the freeze-drying process. The adsorption performance of the adsorbent obtained by reconstituting the freeze-dried protein powder is higher than that of the product of rapid freeze-drying without pre-freezing, and the protein activity is maintained at a high level. Therefore, the design of the freeze-drying process of the present application is beneficial to the preservation of the activity of the freeze-dried protein.

[0166] Table 2. Adsorption performance of different recombinant protein A adsorbents

[0167] Sample Coupling adsorbent adsorption performance mg / mL Adsorption performance reduction rate % Control sample 1 28.87 28.86 Control sample 2 32.52 19.85 Control sample 3 30.93 23.77 Control sample 4 40.58 — Control sample 5 33.83 16.64 Test sample 1 37.19 8.35 Test sample 2 38.49 5.14 Test sample 3 40.31 0.66 Test sample 4 37.98 6.41 Test sample 5 40.06 1.27 Test sample 6 38.76 4.48 Test sample 7 39.01 3.87 Test sample 8 38.09 6.14 Test sample 9 39.80 1.93

[0168] Note: The adsorption performance decrease rate is controlled by the adsorption performance of the control sample 4, and the formula is: adsorption performance decrease rate%= (adsorption performance of control sample 4-adsorption performance of sample) / adsorption performance of control sample 4×100.

[0169] The above merely is the preferred embodiment of the present application, it should be pointed out that, for ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A lyophilized powder of recombinant Protein A, characterized in that, A mixture of recombinant protein A, tris (2-carboxyethyl) phosphine hydrochloride and sodium chloride is obtained by mixing and then freeze-drying, the freeze-drying comprising pre-freezing, gradient freeze-drying and vacuum plugging; The pre-freezing temperature is -48℃ to -46℃, and the pre-freezing time is 2 to 4 hours; The gradient freeze-drying procedure comprises freeze-drying stage 1 and freeze-drying stage 2; The starting temperature of the freeze-drying stage 1 is -45℃, the starting temperature is maintained for 1 to 4 hours, the temperature is increased by 2 to 5℃ at each gradient, each gradient is maintained for 1 to 4 hours, the ending temperature is -36℃ to -38℃, and the ending temperature is maintained for 2 to 4 hours; The starting temperature of the freeze-drying stage 2 is -32℃, the starting temperature is maintained for 1 to 4 hours, the temperature is increased by 5 to 10℃ at each gradient, each gradient is maintained for 1 to 4 hours, the ending temperature is 30℃, and the ending temperature is maintained for 1 to 4 hours; The recombinant protein A is a staphylococcus aureus cell wall protein; The mass ratio of sodium chloride, tris (2-carboxyethyl) phosphine hydrochloride and recombinant protein A is (0.03 to 1) : (0.009 to 0.3) :

1.

2. The recombinant protein A lyophilized powder according to claim 1, characterized in that, The mass ratio of sodium chloride, tris (2-carboxyethyl) phosphine hydrochloride and recombinant protein A is 1:0.29:

1.

3. The recombinant protein A freeze-dried powder of claim 1 or 2 for use in the preparation of a product for protein purification and / or immunosorption. The use does not include disease diagnosis and treatment methods.

4. A product of protein purification and / or immunoadsorption, characterized in that, The product comprises the recombinant protein A freeze-dried powder of claim 1 or 2 and a coupling carrier.

5. The product of claim 4, wherein, The coupling carrier comprises at least one of agarose gel, chitosan, dextran, resin and / or cellulose.

6. Process for the preparation of the product according to claim 4 or 5, characterized in that, The product comprises the following steps: The recombinant protein A freeze-dried powder solution is obtained by reconstituting the recombinant protein A freeze-dried powder of claim 1 or 2 with water, the recombinant protein A freeze-dried powder solution is reacted with an agarose solution, and the product is obtained.

Images