CN105582883A

CN105582883A - Activated diatomaceous earth and application of diatomaceous earth in production of blood products

Info

Publication number: CN105582883A
Application number: CN201510985707.7A
Authority: CN
Inventors: 石清东; 莫丽影
Original assignee: BEIHAI KAIYUAN BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: Beijing Haomei Cell Gene Biotechnology Co ltd
Priority date: 2015-12-25
Filing date: 2015-12-25
Publication date: 2016-05-18
Anticipated expiration: 2035-12-25
Also published as: CN105582883B

Abstract

The invention discloses activated diatomaceous earth and an application of diatomaceous earth in production of blood products. In production of the blood products, the diatomaceous earth is required to be used in a common low-temperature ethanol filter press separation method and acts as a filter aid for solid-liquid separation or an adsorbent, but the diatomaceous earth can produce dust in the use process and blocks an air conditioning unit and a room air filter; secondly, the diatomaceous earth easily enters a filter press plate to block filter holes, thereby causing high filter pressure and prolonging of filter time; thirdly, although the diatomaceous earth is treated before the diatomaceous earth leaves the factory, metal ions in the diatomaceous earth affect the ion strength of a product solution, and abnormal yield and stability of different batches are caused. The activated diatomaceous earth is obtained through steps as follows: dry roasting, acid pickling, water washing and ethanol washing and is applied to production of the blood products. The metal ions or oxides are removed, the diatomaceous earth is filtered by a 600-mesh stainless steel filter net, fine particles gradually pass through the filter net during washing in each step, and the purpose of separation of the fine particles from the diatomaceous earth is achieved.

Description

A kind of activate diatomite and blood product produce in application

Technical field

What the present invention relates to is a kind of activation diatomite, also relates to the application of described activation diatomite in blood product is produced, and belongs to field of biological pharmacy.

Background technology

Diatomite mainly by ancient times silicon fall and the siliceous remains of other microorganisms form, its 80%～90%, even more than 90% chemical composition is SiO₂, also have a small amount of Al₂O₃、Fe₂O₃, CaO, MgO etc. Diatomite stable performance, have acidproof, pore volume is large, aperture is large, specific area is large, adsorptivity is strong, can adsorb the liquid of 1.5～4 times of sole masses, the wet goods character of 11～15 times of sole masses of absorption. Be widely applied as filter aid, adsorbent, filler, catalyst carrier, abrasive material reinforcing agent and animal feed replenishers etc. in many industries such as environment, chemical industry, oil, building materials, medicine manufactures.

In the production technology of blood product, at present the most frequently used is exactly cold ethanol press filtration partition method, the diatomite all can additional proportion in each step separation process not waiting, and its effect has two kinds: a kind of is the filter aid of Separation of Solid and Liquid, and the 2nd, as adsorbent. But in use usually there is following several problem: first, diatomite is added in goods with dry powder form conventionally, how slow the speed no matter adding have, how attention control, but because diatomite density is little, in the process adding, all can produce airborne dust, stop up in the course of time air-conditioning unit and space air filter. Secondly, filter press plate is a kind of principle of in-depth filtration, diatomite particle heterogeneity, and first tiny particle easily enters in filter press plate, stops up filter opening, causes in filter process pressure large, and filtration time extends and increases pollution risk impact and produce. The 3rd, although before diatomite dispatches from the factory, to do some and processed, the existence of metal ion (such as some metal ions or its oxide), more or less affects goods solution ion strength, and between causing batch, yield, stability is abnormal.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of activate diatomite and the application in blood product is produced, and solves diatomite as the generation airborne dust occurring in filter aid use procedure, stops up filter press plate filter opening, the problem that contains impurity metal ion.

For the problems referred to above, adopt following technique measures, a kind of activation diatomite, is activated by following method:

1. dry roasting: weigh pending diatomaceous weight, be placed in stainless steel cask and put into 180 DEG C of hot air drying ovens, heating 120min, leaves standstill and naturally cool to room temperature;

2. pickling: the diatomite after dry baking is placed in to the pyrogen-free diatomite container handling of pretreatment, add the water for injection of 2 times of diatomite weight, adding glacial acetic acid to make ultimate density is 0.20 ~ 0.30mol/L, agitator treating 1 hour, discharge washing raffinate, repeat said process 1-2 time;

3. washing: in diatomite container handling, add the water for injection of 2 times of diatomite weight, slowly agitator treating 30min, discharges washing raffinate after washing, repeat said process 1-2 time, is 5-7 until washing raffinate is measured pH value;

4. alcohol wash: according to the ethanolic solution of the concentration of alcohol preparation same concentrations of cold ethanol method separating blood product producing process separating step, in diatomite container handling, add the ethanolic solution of the described concentration weighing with diatomite etc., stir after 10～20 minutes, discharge cleaning solution, so operation repeats once, obtains activation diatomite.

Adopt the present invention of said method activation, the advantage of its activation method is the washing through several steps, remove corresponding metal ion or oxide, reduced the impact of cold ethanol intermediate ion intensity on separating effect, product quality and yield are further ensured. And through the filtration of 600 order stainless steel filtering nets, little fine grained, in the washing of each step, sees through filter screen gradually, reach the object that separates diatomite fine particle. And in whole process, mixing speed is unhappy, can not bring injury to diatomite. Main diatomite adds in hygrometric state mode, avoids the obstruction of airborne dust to cleaning shop air filtering system.

In actual application by adding after the present invention, no matter also can be reflected qualitatively from filter pressure, yield and final products. The present invention's filtering velocity in press filtration is fast, has greatly shortened filtration time, and the pollution because of artificial participation that filtration difficulty brings is reduced to minimum. Simultaneously in diatomite, contained metal ion or oxide be after treatment, its content is little, the impact that cold ethanol method in blood product is separated to each component reduces greatly, differences between batches reduce, product quality is further ensured, effective low obstruction that reduces air conditioner filter, product stability, yield also improve greatly.

Brief description of the drawings

Fig. 1 is the structural representation of diatomite container handling.

Detailed description of the invention

Embodiment 1

Diatomaceous activation

1. dry roasting: weigh pending diatomite 50kg and be placed in stainless steel cask, put into when hot air drying oven temperature reaches 180 DEG C and start timing, heating 120min, powered-down, leaves standstill that nature is cooling treats that temperature is reduced to room temperature;

2. pickling: rinse the diatomite processor of precoating alkali with water for injection, pour cooled diatomite into after qualified to pyrogen testing, add water for injection 100Kg, adding glacial acetic acid 1500ml under slowly stirring, agitator treating 1h, discharges washing raffinate; So repeat once;

3. washing: in diatomite container handling, add 100Kg water for injection, stirring at low speed washing 30min, discharges washing raffinate after washing; So repeating 1-2 time, is 5-7 until washing raffinate is measured pH value;

4. alcohol wash: according to the ethanolic solution of the concentration of alcohol preparation same concentrations of cold ethanol method separating blood product producing process separating step, in diatomite container handling, add 50 kilograms of the ethanolic solutions of same concentrations, stir after 10～20 minutes, discharge cleaning solution, so operation repeats once, obtains activation diatomite; The concentration of alcohol of described cold ethanol method separating blood product producing process separating step refers in existing cold ethanol method technique, as component I in human serum albumin separates corresponding concentration of alcohol 8%-10%(V/V), when component I I+III separates, corresponding concentration of alcohol is 20%-25%(V/V), it is 40%-42%(V/V that component I V, component V separate corresponding concentration of alcohol), human immunoglobulin(HIg) based article component III separates corresponding concentration of alcohol 14-17%, and component I I separates corresponding concentration of alcohol 23-27%(v/v).

Described diatomite container handling as shown in Figure 1, comprises container body 4, is provided with motor 1 at container body 4 tops, and motor 1 drives paddle 3 to rotate, and is provided with 600 order stainless steel filtering nets 2 in the bottom of container body 4. When use, diatomite and cleaning solution wash in diatomite container handling, and motor 1 drives paddle 3 rotations that diatomite is fully washed, and 600 order stainless steel screen packs 2 of container bottom can separate small particles.

Application Example 2

The present invention applies in quiet note human immunoglobulin(HIg)

Step 1F I supernatant is made

Blood plasma temperature after merging is controlled to 0 DEG C, regulating protein concentration with 0 DEG C of physiological saline is 5.1%, with pH4.0HAc-NaAc buffer solution adjusting pH to 7.10 ± 0.05, adding-15 DEG C of following ethanol to make ethanol ultimate density is 8% (v/v), limit adds the cooling of ethanol limit, final temperature is controlled at-2.5 DEG C, stirring reaction 1 hour. To the diatomite 4 ~ 8Kg (alcohol wash step concentration of alcohol is 8% (v/v)) that adds activation in every kilolitre solution, stir press filtration after 40 minutes and separate, isolate F I precipitation, collect F I supernatant.

The separation of step 2F II+III precipitation

Metering F I supernatant volume, with pH4.0HAc-NaAc buffer solution adjusting F I supernatant pH value to 6.95 ± 0.15, adding-15 DEG C of following ethanol to make ethanol ultimate density is 20% (v/v), limit adds the cooling of ethanol limit, solution final temperature-5.0 DEG C, stirring reaction 2 hours, to the diatomite 4 ~ 8Kg (alcohol wash step concentration of alcohol is 20% (v/v)) that adds activation in every kilolitre solution, stirring press filtration after 40 minutes separates, filtrate is F II+III supernatant, make and separate for F IV-1, collect F II+III precipitation.

The making of step 3F III supernatant

F II+III precipitation is dissolved with the sodium dihydrogen phosphate of 2～8 DEG C of 0.01M of 6 ~ 7 times of weight, adjust pH to 5.00 ~ 5.10 with 2mol/LHAc, temperature is controlled to-1.0 ~-3.0 DEG C, adding-15 DEG C of following ethanol to make ethanol ultimate density is 17% (v/v), stir after 3 ~ 4 hours, to the diatomite 4 ~ 8Kg (alcohol wash step concentration of alcohol is 17% (v/v)) that adds activation in every kilolitre solution, stirring reaction is press filtration separation after 1 hour, collecting filtrate is F III supernatant, and precipitation is abandoned.

The separation of step 4F II precipitation

Metering F III supernatant volume, adds sodium chloride by 5g/L. With 1mol/LNaOH solution adjusting F III supernatant pH value to 6.9 ~ 7.1, adding-15 DEG C of following ethanol to make ethanol ultimate density is 25% (v/v), limit adds the cooling of ethanol limit, solution is finally controlled at-5.5 DEG C ~--6.0 DEG C., stirring reaction 2 hours, leave standstill after 2 hours, to the diatomite 4 ~ 8Kg (alcohol wash step concentration of alcohol is 25% (v/v)) that adds activation in every kilolitre solution, stirring reaction is press filtration separation after 1 hour, is precipitated as F II precipitation, and supernatant is abandoned.

Dissolving and the ultrafiltration dialysis of step 5F II precipitation

By 4 ~ 5 times of F II precipitation weight, 2 ~ 8 DEG C of waters for injection dissolve F II precipitation, and 2mol/L glacial acetic acid regulates PH to 3.8 ~ 4.4, dissolves after 4 hours and leaves standstill and filter, and filtrate is F II solution. By F II solution through ultrafiltration concentration to protein concentration 4.0～8.0%, with the continuous equal-volumes dialysis of 6 times of 2 ~ 8 DEG C of waters for injection, and be concentrated into protein concentration 6.0～8.0%, stop concentrating. Protein concentration is controlled to 5.0%～6.0% with water for injection, adjusts pH to 4.70～5.30 with 2mol/LHAc, adding sorbitol concentration is 28.0%～35.0%.

Step 6 pasteurization

The solution preparing is put in pasteurization tank, at the temperature of 59.5 ~ 60.5 DEG C, heated and within 10 hours, carry out pasteurization.

Step 7 purification refine

Pasteurization is complete, carry out ultrafiltration dialysis for the second time, after removing sorbierite, protein concentration is controlled between 1.4～2.5%, control temperature-4.0～-5.0 DEG C of solution, adding-15 DEG C of following ethanol to make ethanol ultimate density is 17% (v/v), with 2mol/LHAc or 1mol/LNaOH rectification goods pH to 5.15～5.30. After making, stir half an hour add above diatomite (alcohol wash step ethanol content is 17%) (4Kg diatomite/1000L goods) stir again 1 hour leave standstill 4～8 hours, press filtration separate. Collect filtrate, precipitation is abandoned.

Step 8 ultrafiltration for the third time and semi-finished product preparation

Filtrate is carried out to ultrafiltration for the third time, remove ethanol, salt, and be concentrated into protein concentration 4.0～8.0%. With 0.5mol/LHCl tune pH to 3.80～4.40, interpolation maltose is to maltose content at 90～110g/L, and dilute solution is to protein content > 5.0%. Again through incubated at low pH, degerming packing, censorship, lamp inspection, packaging, warehouse-in.

Table 1: effect contrast in quiet note human immunoglobulin(HIg)

Application Example 3

The application of the present invention in human serum albumin

The making of step 1F IV-1 supernatant

With step 2 gained F II+III supernatant pH5.25 in pH4.0HAc-NaAc buffer solution adjusting embodiment 2, control solution temperature-5.0 DEG C, drip ethanol to concentration 40% (v/v), to the diatomite 4～6kg (alcoholysis step concentration of alcohol is 40% (v/v)) that adds activation in every kilolitre solution, stirring reaction 40 minutes, press filtration, filtrate is F IV-1 supernatant.

The making of step 2F IV-4 supernatant

Metering F IV-1 supernatant volume, with 1mol/L sodium bicarbonate solution adjusting supernatant PH to 5.9 ± 0.05, F IV-1, temperature is-5.0 ± 0.05 DEG C, adding-15 DEG C of following ethanol to make ethanol ultimate density is 40% (v/v),, to the diatomite 5～7kg (alcohol wash step concentration of alcohol is 40% (v/v)) that adds activation in every kilolitre solution, stirring reaction 2 hours, press filtration, filtrate is F IV-4 supernatants.

Step 3F V precipitate and separate

Metering F IV-4 supernatant volume, with 2mol/LHAc-40% ethanolic solution adjusting supernatant pH to 4.8 ± 0.05, F IV-4, temperature is-7.5 ± 0.05 DEG C, drip ethanol to concentration 40% (v/v), to the diatomite 3～6kg (alcohol wash step concentration of alcohol is 40% (v/v)) that adds activation in every kilolitre solution, stirring reaction 1 hour, press filtration, is precipitated as F V precipitation.

Step 4F V deposition and purification

8% ethanolic solution (0 DEG C～5 DEG C) stirring and dissolving of 5 times of volumes for F V precipitation, cooling while stirring, until precipitation is dissolved completely. With pH4.0HAc-NaAc buffer solution tune pH to 4.55 ± 0.05. Continue to stir more than 1 hour, the control of goods final temperature is-2.5 DEG C ± 0.5 DEG C, leaves standstill more than 2 hours press filtration and separates.

Get filtrate and carry out ultrafiltration, be concentrated into protein concentration 10%～15%, first use 5 times of isopyknic 0.9% normal saline dialysis, then dialyse with 3 times of isopyknic waters for injection, after dialysis finishes, gained is albumin stoste. Regulate pH value to should be 6.60～7.20, the dosage of Sodium Caprylate is pressed 0.160mmol/g protein, with water for injection, goods being diluted to protein content is 195～205g/L, bath temperature is controlled at 60.0 DEG C, after constant temperature 10 hours, be down to room temperature, aseptic filtration, censorship, lamp inspection and visible foreign matters inspection, the qualified rear packaging of lamp inspection, warehouse-in.

Table 2: effect contrast in human serum albumin

Claims

1. an activation diatomite, is characterized in that described activation diatomite is activated by following method:

(1) dry roasting: weigh pending diatomaceous weight, be placed in stainless steel cask and put into 180 DEG C of hot air drying ovens, heating 120min, leaves standstill and naturally cool to room temperature;

(2) pickling: the diatomite after dry baking is placed in to the pyrogen-free diatomite container handling of pretreatment, add the water for injection of 2 times of diatomite weight, adding glacial acetic acid to make ultimate density is 0.20 ~ 0.30mol/L, agitator treating 1 hour, discharge washing raffinate, repeat said process 1-2 time;

(3) washing: in diatomite container handling, add the water for injection of 2 times of diatomite weight, slowly agitator treating 30min, discharges washing raffinate after washing, repeat said process 1-2 time, is 5-7 until washing raffinate is measured pH value;

(4) alcohol wash: the concentration of alcohol of the separating step according to diatomite in quiet note human immunoglobulin(HIg) and human serum albumin application, the ethanolic solution of preparation same concentrations, in diatomite container handling, adds the ethanolic solution of the described concentration weighing with diatomite etc.,

After agitator treating 10-20min, discharge ethanolic solution, so repeat said process 2 times, obtain activation diatomite.

2. activate diatomite application as filter aid in blood product is produced.