TWI480066B - Preparation of platelet dry powder - Google Patents

Description

Method for producing platelet dry powder

The present invention relates to a method for producing a dry powder of platelets, and more particularly to a method for producing a dry powder of platelets protected by adenosine diphosphate (ADP).

The dry powder of platelets is a dry powder of platelets containing intact platelet cells by a specific method using blood or blood preparations such as platelet-rich plasma (PRP). The platelet dry powder can be used for various purposes, such as treatment of baldness (see TW-201215402), beauty (see TW-201215402), and the like. The technology for making platelet dry powder, although known in the art, for example, see Taiwan No. I300806, I270375 Announcement Patent Case, Taiwan No. 201004659, 200526680 Patent Patent Case, US 7,659,052, 6,060,233, 5,736,313, 5,589,462 Announcement Patent Case, but such methods, Or platelet cells are unprotected, so most of the platelet cells in the process have ruptured, causing the growth factors in the cells to be easily deteriorated, for example, the US 5,589,462 announcement patent; or the protective effect is not good, the platelet function is significantly better than Low, for example, the US 6,060,233, 5,736,313 announcement patent case; or the protection agent is not easy to obtain, the dosage is not easy to control, the cost is extremely high, and the process control is not easy or the cost is too high, such as I300806, I270375 announcement patent case, Taiwan 201004659, 200526680 The public patent case, the US 7,659,052 announcement patent case, taking the process disclosed in the TW-I270375 announcement patent case as an example, which uses thrombin (thrombin) protects platelet cells, but thrombin often varies greatly depending on the source (human, bovine, pig, etc.), and the activity varies greatly. Take Sigma-Aldrich's product as an example. See http://www.sigmaaldrich.com/ Life-science/metabolomics/enzyme-explorer/analytical-enzymes/thrombins.html#References, human thrombin: T1063 is >2800 NIH units/mg (hereinafter referred to as units/mg), T7572 is 50~300 NIH units/mg, bovine thrombin: T9549 is 1500~2500 NIH units/mg, T6200 is 40~300 NIH units/mg, so the process control is not easy; further, its stability is not high, so it must be Cryopreservation, such as T9326, is stored at -70 °C, see http://www.sigmaaldrich.com/catalog/ProductDetail.do? N4=T9326|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC.

The invention provides a method for producing a dry powder of platelets.

The present invention provides a method for producing a platelet dry powder which protects platelet cells with ADP.

The method for producing a dry platelet powder of the present invention comprises: adding 0.1 to 10 mM of an ADP solution to a mammalian PRP and uniformly stirring to protect the platelet, wherein the amount of the ADP is calculated per 1 million platelet cells in the PRP. 0.04 to 4 Nemo ADP; the protected PRP is removed from the plasma layer to obtain a protected platelet layer; After freezing the platelet layer to a suitable low temperature, it is freeze-dried to form a platelet dry powder.

The above-mentioned mammal refers to a mammal that is accepted by a medical unit, such as cattle, pigs, sheep, humans, and the like.

The PRP obtained from the above-mentioned mammals may be heterologous PRP, homologous PRP, or autologous PRP. Based on the psychological considerations of the user, the PRP is preferably a heterologous PRP or an autologous PRP. It is better to use autologous PRP. However, based on the factors of easy and mass production, the PRP is preferably a heterologous PRP or a heterogeneous PRP, and a heterogeneous PRP is more preferred.

The above-mentioned PRP obtained from mammals can be obtained by any conventional method, for example, using a blood cell separator to separate PRP from whole blood, and of course, directly or indirectly from various blood donation centers or the like. Acquired.

The above ADP solution has a concentration of 0.1 to 10 mM ADP solution, and the concentration is less than 0.1 mM, which is not infeasible, but it will cause difficulty in centrifugation (relatively large volume); concentration higher than 10 mM is not infeasible, but will Causes difficulty in control (sometimes clotting). In general, 0.2 to 5 mM is preferred, and 0.5 to 2 mM is more preferred. The solvent of the above ADP solution is water, but in order to form an isotonic solution, or close to an isotonic solution, a physiological saline solution (forming an isotonic solution) or a mixed solution of reverse osmosis water and physiological saline (forming a near isotonic solution) is used. It is preferred to prepare the ADP solution instead of the reverse osmosis water, and it is more preferable to prepare the ADP solution by using the physiological saline instead of the reverse osmosis water.

The above ADP solution is used in an amount of 0.04 to 4 neMole of ADP per 1 million platelet cells in the PRP. Dose below 0.04 neMer may cause platelet cell insufficiency; doses above 4 nemore may cause excessive coagulation. In general, 0.1 to 2 nanomoles is preferred for each 1 million platelet cells in the PRP, and 0.2 to 1 nanomolar is preferred. The method for removing the plasma layer may be any plasma layer removal method of any known blood preparation, for example, membrane plasma separation, centrifugation, etc., preferably by centrifugation, that is, It is preferred to remove the plasma layer after centrifugation in a high speed centrifuge.

The platelet layer after removing the plasma layer (hereinafter referred to as PLT), the freezing temperature of the freezing program may be the freezing storage temperature of the general blood preparation or the lyophilization temperature, or the temperature between them, preferably from -10 to -50 ° C. It is better to use -30~-40°C.

The above freeze-drying method may employ a freezing method of a general blood preparation, for example, a method employed in the above-mentioned prior art, for example, -30 ° C / 20 Pa - 40 ° C / 10 Pa.

Before the above ADP solution is added, it is preferred to first determine the number of platelet cells in the PRP as a basis for adding the amount of the ADP solution, and the concentration and volume of the ADP solution are as described above.

After the above plasma layer is removed, if necessary, it may be diluted with water or an aqueous solution to facilitate subsequent operations, and it is preferred to add reverse osmosis water, physiological saline solution, or a mixture thereof, and reverse osmosis water is more preferable.

The above-mentioned PLT after removing the plasma layer can be directly subjected to freezing and/or freeze-drying procedures in situ (original centrifuge container); but if necessary (for example, a conversion container) Freezing and/or freeze-drying procedure), adding water or an aqueous solution (for example, physiological saline) to form a PLT diluent for operation (for example, a conversion container), preferably using a PLT diluent for freezing and/or freeze-drying procedures. . It is preferable to use reverse osmosis water, physiological saline, or a mixture thereof at the time of dilution, and it is more preferable to use reverse osmosis water. Wherein the PLT diluent may be added with any active ingredient having a positive effect, such as an anti-inflammatory component, an analgesic component, a nutrient component, and/or an ingredient which enhances absorption, or a component which has no negative effect or side effect, such as pharmacology, as needed. Acceptable fragrances, colorants, additives, etc. Based on the efficiency considerations of the freezing and lyophilization steps, the platelet content of the PLT diluent is preferably higher than 100 million per ml, preferably more than 1 billion per ml.

To further illustrate the teachings of the present invention, the preferred embodiment is as follows:

Example 1:

The patient was bled with a blood cell separator (MCS System 9000, http://www.haemonetics.com/) to obtain 36.7 mL of autologous PRP.

In a 1.5 mL microcentrifuge tube, add 0.2 mL of physiological saline and 0.2 mL of PRP, and mix well; use a fully automated blood analyzer (Sysmex Corporation KX-21 Automated Hematology Analyzer, see http://www.blockscientific.com) /) The number of platelet cells measured was 476 K/μL (476,000 platelets per microliter), that is, the number of platelets in the original PRP was 952 K/μL, which was confirmed to meet the quality requirements.

With the remaining 36.5 mL of PRP, add 22 mL of a physiological saline solution containing 1 mM ADP (ie 1 mM ADP per liter), ie ADP dosage 0.633 nmole/Mega (0.633 nm per 1 million platelet cells) Moore's ADP), after shaking for 30 minutes, centrifuge at 4000 rpm for 7 minutes, remove the plasma layer, add reverse osmosis water to 35 mL and mix well, and dispense into 35 bottles (1 mL per bottle) Platelet solution. 35 bottles of platelet solution were sent to a -40 ° C freezer (ultra-low temperature horizontal freezer, RS-CF 330LT, Ruey Shing Refrigeration Equipment Co., Ltd.), frozen for 24 hours, and then freeze-dried The machine (FD2-6P-M, Jinming Industrial Co., Ltd. - KINGMECH CO., LTD) was freeze-dried (-40 ° C, 10 Pa) for 36 hours to obtain 35 bottles of platelet dry powder, each containing about one billion platelets.

One of the bottles (Sample 1) was immediately rehydrated according to the following method, and the platelet count and PDGF titer were determined; the remaining 34 bottles were immediately vacuum-packed and externally sterilized. Stored at room temperature for one week (sample 2), two weeks later (sample 3), four weeks later (sample 4), eight weeks later (sample 5), twelve weeks later (sample 6), after sixteen weeks (sample 7) Take one bottle of sterilized dry powder of platelets, rehydrate according to the following method, and measure the PDGF titer.

Rehydration procedure: Re-watering samples 1-7 with 4 mL reverse osmosis water.

Determination of PDGF titer: 1 mL of sample 1~7 of the reconstituted solution was diluted 40 times, and then colorimetrically analyzed by Enzyme-linked immunoassay, using a filter analyzer (Thermo Fisher Scientific Multiskan FC Filter-based microplate photometer, see http://www.ninolab.se/fileadmin/Ninolab/pdf/thermolabsystems/MultiskanFC_Brochure_0308-01_LR.pdf) Analysis of PDGF titer, the resulting titer multiplied by 40 times, respectively, 5016 (Sample 1), 4985 (Sample 2), 4968 (Sample 3), 4902 (Sample 4), 4803 (Sample 5), 4852 (Sample 6), 4785 (Sample 7) pg/mL.

The experimental data showed that the PDGF titer in the platelet dry powder was quite stable, and the PDGF titer at the 16th week was 95.4% (4785/5016) of the original PDGF titer.

Example 2:

According to the method of Example 1, the number of platelet cells of PRP taken from the blood donation center was measured to be 1386 K/μL (693 K/μL x 2), and it was confirmed that the quality requirements were met. 14.5 mL of this PRP was taken, and the ADP protection procedure of Example 1 was carried out with the ADP concentration and amount of Table 2. After shaking for 30 minutes, the plasma layer was centrifuged and removed to obtain PLT. Add reverse osmosis water to 20 mL and mix well to form a platelet solution. Dispense the platelet solution into 10 bottles (2 mL per bottle), then transfer to a -40 ° C freezer, freeze for 24 hours, then freeze. The dryer was freeze-dried (-40 ° C, 10 Pa) for 36 hours to obtain 10 bottles of platelet dry powder, each containing about 2 billion platelets.

Take a bottle (sample 1) of platelet dry powder, immediately rehydrate according to the method described in Example 1, take 0.5 mL of rehydration solution, dilute 80 times, and then color by enzyme immunoassay, and then analyze the PDGF effect by filter analyzer. Price The titer was multiplied by 80 times and the results are shown in Table 3. The remaining 9 bottles were immediately vacuum-packed and sub-sterilized.

After storage for four weeks at room temperature, take a bottle of sterilized dry powder of platelets (Sample 2), rehydrate according to the method described in Example 1, take 0.5 mL of rehydration solution, dilute 80 times, and then color by enzyme immunoassay. The PDGF titer was analyzed by a filter analyzer, and the obtained titer was multiplied by 80 times. The results are shown in Table 3.

Examples 3-8:

According to the method of Example 1, the number of platelet cells of PRP taken from the blood donation center was 1,476, 1182, 1340, 1296, 1164, and 1278 K/μL, respectively. 16.0, 17.0, 15.0, 17.5, and 16.0 mL of each of the PRPs were taken, and the platelet dry powder was produced in the same manner as in Example 2 except that the ADP concentration and amount were different, see Table 2. Then, the PDGF titers of the platelets of Examples 3 to 8 were measured in the same manner as in Example 2, and the test results are shown in Table 3.

From the ratio of the PDGF titer of the sample 2 to the PDGF titer of the sample 1, it can be seen that the dry powder stability of Examples 3, 4, 5, and 6 is better, and the dry powder stability of Examples 2, 7, and 8 is acceptable.

Example 9

Similarly to Example 2, 14.5 mL of PRP taken from a blood donation center was taken, and the ADP protection procedure of Example 1 was carried out in accordance with the ADP concentration and amount of Table 4. After shaking for 30 minutes, the plasma layer was centrifuged and removed to obtain PLT. Add reverse osmosis water to 20 mL and mix well to form a platelet solution. Dispense the platelet solution into 5 bottles (4 mL per bottle), then transfer to a -40 ° C freezer, freeze for 24 hours, then freeze. The dryer was freeze-dried (-40 ° C, 10 Pa) for 36 hours to obtain 5 bottles of platelet dry powder, each containing approximately 4 billion platelets.

One bottle (Sample 1) of platelet dry powder was taken, and immediately rehydrated according to the method described in Example 1, and the number of platelets was measured. The results are shown in Table 5. The remaining 4 bottles were immediately vacuum-packed and sub-sterilized.

After storing at room temperature for four weeks, take a bottle of sterilized dry powder of platelets (Sample 2), rehydrate according to the method described in Example 1, and measure the number of platelets. The results are as follows. table 5.

Examples 10-15:

The procedure is the same as in Example 9, but the amount of ADP is different, see Table 4. The number of platelets was determined in the same manner as in Example 9, and the test results are shown in Table 5.

From the ratio of the number of platelets in sample 2 to the number of platelets in sample 1, it can be seen that the dry powder protection effects of Examples 10 to 13 are better (the number of platelets in sample 1 is higher), and the stability is also better (sample 2 and sample 1) The platelet ratio was higher. The dry powder protection effects and stability of Examples 9 and 14 were relatively poor.

Test Example 1:

From a blood cell separator, 620 mL of heterogeneous (porcine) PRP was isolated from approximately 7 liters of pig blood. In a 1.5 mL microcentrifuge tube, 0.8 mL of physiological saline and 0.2 mL of PRP were added and mixed thoroughly. The platelet count was 574 K/μL, which is the number of platelets of the original PRP. 2.87 M/μL (2.77 million platelets per microliter).

Example 16

Take 21 mL of pig PRP and add 30 mL of physiological saline solution containing 1 mM ADP (equivalent to 0.50 nmole ADP/Mega platelets). After shaking for 30 minutes, the plasma layer was centrifuged and removed to obtain PLT. Add reverse osmosis water to 20 ml, mix quickly and form a platelet solution, and dispense into 20 bottles of platelet solution (1 mL per bottle). The platelet solution was sent to a -40 ° C freezer, frozen for 24 hours, and then freeze-dried (-40 ° C, 10 Pa) in a freeze dryer for 36 hours to obtain 20 bottles of platelet dry powder, each bottle containing about 3 billion platelets .

One bottle (Sample 1) of platelet dry powder was taken, and immediately rehydrated according to the method described in Example 1, and the number of platelets was measured. The results are shown in Table 6. The remaining bottles were immediately vacuum-packed and sub-sterilized.

After storing at room temperature for four weeks, a bottle of sterilized dry powder of platelets (Sample 2) was taken, rehydrated as described in Example 1, and the number of platelets was measured. The results are shown in Table 6.

Example 17

The procedure was the same as in Example 16, except that 30 mL of a physiological saline solution containing 1 mM of ADP was used instead of 30 mL of a reverse osmosis aqueous solution containing 1 mM of ADP.

The number of platelets was determined in the same manner as in Example 16 and the test results are shown in Table 6.

Example 18

The procedure was the same as in Example 16, except that 30 mL of a physiological saline solution containing 1 mM of ADP was used instead of a mixed solution of 15 mL of a physiological saline solution containing 1 mM of ADP and 15 mL of a reverse osmosis aqueous solution containing 1 mM of ADP.

The number of platelets was determined in the same manner as in Example 16 and the test results are shown in Table 6.

Example 19

The procedure was the same as in Example 16, except that the PLT of the plasma layer was centrifuged and removed, and diluted to 20 ml with physiological saline.

The number of platelets was determined in the same manner as in Example 16 and the test results are shown in Table 6.

Example 20

The procedure was the same as in Example 16, except that the PLT of the plasma layer was centrifuged and removed, and 10 mL of physiological saline was added, and then diluted with reverse osmosis water to 20 ml.

The number of platelets was determined in the same manner as in Example 16 and the test results are shown in Table 6.

Example 21:

The procedure was the same as in Example 17, except that the PLT of the plasma layer was centrifuged and removed, and diluted to 20 ml with physiological saline.

The number of platelets was determined in the same manner as in Example 16 and the test results are shown in Table 6.

Example 22

The procedure was the same as in Example 17, except that the PLT of the plasma layer was centrifuged and removed, and 10 mL of physiological saline was added, and then diluted with reverse osmosis water to 20 ml.

The number of platelets was determined in the same manner as in Example 16 and the test results are shown in Table 6.

Example 23

The procedure was the same as in Example 18, except that the PLT of the plasma layer was centrifuged and removed, and diluted to 20 ml with physiological saline.

The number of platelets was determined in the same manner as in Example 16 and the test results are shown in Table 6.

Example 24:

The procedure was the same as in Example 18, except that the PLT of the plasma layer was centrifuged and removed, and 10 mL of physiological saline was added, and then diluted with reverse osmosis water to 20 ml.

The number of platelets was determined in the same manner as in Example 16 and the test results are shown in Table 6.

From the ratio of the number of platelets of the sample 2 to the number of platelets of the sample 1, it can be seen that the dry powders of Examples 16 to 24 are all excellent in stability.

The number of platelets of sample 1 of Examples 16, 17, 18 (635 K / μL ± 1.7%), no significant difference; the number of platelets of sample 1 of Examples 19, 21, 23 (602 K / μL ± 2.0%), no significant Difference; the number of platelets of sample 1 of Examples 20, 22, and 24 (615 K/μL ± 4.2%) showed no significant difference; in the protective step, the ADP solution was treated with physiological saline solution, reverse osmosis water, or a mixed solution thereof. The protective effect is not much different, but based on the requirement of the isotonic solution, it is preferred to use a physiological saline solution, or a mixed solution of reverse osmosis water and physiological saline, and it is more preferable to use physiological saline.

The number of platelets of sample 1 of Examples 16, 19, 20 (627 K / μL ± 2.1%), no significant difference; the number of platelets of sample 1 of Examples 17, 21, 22 (621 K / μL ± 4.0%), no significant Difference; the number of platelets of sample 1 in Examples 18, 23, and 24 (604 K / μL ± 4.5%), no significant difference; showing the dilution step, the dilution solution is physiological saline, reverse osmosis water, or a mixed solution thereof, The protection effect is not much different, but based on the requirement of dry powder purity, it is mixed with reverse osmosis water or reverse osmosis water and physiological saline. The liquid is preferably used to preferably use reverse osmosis water.

The results of Examples 9-24 show that the number of platelet cells in heterogeneous PRP is higher than the number of platelets in allogeneic and autologous PRP in terms of platelet cell number; allogeneic PRP (source: blood donation center, taken from healthy The number of platelet cells in a donor is usually higher than the number of platelets in the autologous (patient) PRP. Accordingly, in terms of the number of platelet cells in the PRP and the difficulty in obtaining the source, it is preferable to use the allogeneic PRP or the heterogeneous PRP, and the heterogeneous PRP is preferable; but considering the patient's psychology, the autologous PRP or the allogeneic PRP is used. Preferably, autologous PRP is preferred.

Claims (12)

A method for producing a dry powder of a platelet comprising: adding a 0.1 to 10 mM adenosine diphosphate (ADP) solution to a platelet-rich plasma (PRP) of a mammal and stirring it evenly To protect platelets, wherein the ADP is used in an amount of 0.04 to 4 nanomolar ADP per 1 million platelet cells in the PRP; the protected PRP is removed from the plasma layer to obtain a protected platelet layer; The protected platelet layer is chilled to a suitable low temperature and lyophilized to form a platelet dry powder. The manufacturing method according to claim 1, wherein after removing the plasma layer, the diluent is further diluted, wherein the diluent is reverse osmosis water, physiological saline, or a mixture thereof, and diluted after dilution. The platelet content of the liquid is higher than 100 million per ml. The manufacturing method according to claim 2, wherein the diluted diluent has a platelet content of more than 1 billion per ml. The manufacturing method according to claim 1, wherein the PRP is a heterologous PRP, an allogeneic PRP, or an autologous PRP obtained from cattle, pigs, and sheep. The manufacturing method according to claim 1, wherein the ADP solution has a concentration of 0.2 to 5.0 mM. The manufacturing method according to claim 5, wherein the ADP solution has a concentration of 0.5 to 2.0 mM. The manufacturing method according to claim 1, wherein the ADP solution is used in an amount of 0.1 to 2.0 nanomolar ADP per 1 million platelet cells in the PRP. The manufacturing method according to claim 7, wherein the ADP solution is used in an amount of 0.2 to 1.0 nanomolar ADP per 1 million platelet cells in the PRP. The manufacturing method according to claim 1, 2 or 3, wherein the freezing temperature is -10 to -50 °C. The manufacturing method according to claim 9, wherein the freezing temperature is -30 to -40 °C. The manufacturing method according to claim 1, wherein the operating condition of the freeze-drying method is -30 ° C / 20 Pa - 40 ° C / 10 Pa. The manufacturing method according to claim 1, wherein the plasma layer is removed by centrifugation, and the protected PRP is centrifuged at a high speed to remove the plasma layer.