CN102552177B - Freeze-dried recombinant human brain natriuretic peptide (rhBNP) preparation and preparation method thereof - Google Patents

Abstract

The invention discloses a freeze-dried recombinant human brain natriuretic peptide (rhBNP) preparation. The preparation is prepared from the following Chinese herbal medicines and auxiliary materials in part by weight: 0.5 part of rhBNP, 0.2 to 20 parts of buffer solution system, 1 to 20 parts of excipient and 1 to 20 parts of NaC1. The invention also discloses a preparation method for the freeze-dried rhBNP preparation. By optimization of the auxiliary materials, rhBNP has high stability, and effectiveness and safety of medicines are ensured.

Description

Lyophilized formulations of rhBNP and preparation method thereof

Technical field

The present invention relates to lyophilized formulations of rhBNP and preparation method thereof, belong to drug world.

Background technology

RhBNP (rhBNP) is succeeded in developing by the U.S., and be used for the treatment of clinically the medicine of new generation of congestive heart failure, mechanism of its treatment heart failure be it to a certain extent can the antagonism epinephrine, the activity of Re-A-A element and endothelin system, relax vascular smooth muscle and expand the peripheral artery vein blood vessel, increase the drainage of sodium and sharp sodium, diuresis, alleviate the front and back load of heart, be of value to the alleviation of the heart failure state of an illness.

The lyophilizing recombinant human brain natriuretic peptide of domestic clinical practice, trade name are newly lived plain, every 0.5mg.For acute heart failure patient's treatment, detailed directions: the plain 0.5mg that newly lives is dissolved in 100ml liquid, gives loading dose 1.5 μ g/kg, intravenous injection, and the time of injecting must not be less than 90 seconds, the remaining vena axillaris 0.01 μ g (kgmin) that instils ^-1, persistent instillation, SM 48～72 hours.For example, be the patient of 60kg for body weight, will extract the quiet notes of 18～20ml in the 100ml liquid, remaining liquid 7～8ml/h continuous intravenous infusion.For the treatment of chronic heart failure, detailed directions is: give (0.0075～0.01) μ g (kgmin) ^-1Intravenous drip, 1～2 outpatient service vein treatment weekly, be 12 weeks the course for the treatment of.Yet, because rhBNP is polypeptide drug, it may stand the chemical degradation of Various Complex and physical change under the environment and inactivation in vivo and in vitro, therefore, the method that research improves the stability of this pharmaceutical preparation just seems particularly necessary, at present also not about improving the pertinent literature report of the plain lyophilized formulations stability of new work.

Summary of the invention

Technical scheme of the present invention has provided a kind of rhBNP lyophilized formulations of good stability.This bright preparation method that this lyophilized formulations also is provided.

The invention provides a kind of rhBNP lyophilized formulations, it is to be prepared from by the crude drug of following weight proportion and adjuvant:

0.5 part of rhBNP, buffer solution system 0.2-20 part, excipient 1-20 part, NaCl 1-20 part.

Wherein, described buffering liquid is phosphate, citric acid and acetate; Be preferably phosphate or citric acid.

Wherein, described buffering liquid is phosphate.

Wherein, described excipient is human serum albumin, mannitol, glycine, hetastarch; Be preferably human serum albumin or mannitol.

Wherein, described excipient is the human serum albumin.

RhBNP lyophilized formulations of the present invention is that crude drug and the adjuvant by following weight proportion is prepared from:

0.5 part of rhBNP, human serum albumin 1-20 part, Na ₂HPO ₄0.1-10 part, NaH ₂PO ₄0.1-10 part, NaCl 1-20 part.

Further preferably, it is that crude drug and adjuvant by following weight proportion is prepared from:

0.5 part of rhBNP, 10 parts of human serum albumins, Na ₂HPO ₄1.73 part, NaH ₂PO ₄0.93 part, 9 parts of NaCl.

The present invention also provides the preparation method of this rhBNP lyophilized formulations, and it comprises the steps:

(1) gets rhBNP, buffer solution system, excipient, the NaCl of recipe quantity, with water for injection dissolving, solution for standby; Or

(2) get buffer solution system, excipient, the NaCl of recipe quantity, be dissolved in the solution of the rhBNP that contains recipe quantity the gained solution for standby;

(3) behind the solution impurity removal and purification with step (1) or (2) gained, with the refined liquid packing, lyophilization gets product.

Preferred by to adjuvant of the present invention so that rhBNP has good stability, guaranteed effectiveness and the safety of medicine.

The specific embodiment of form is described in further detail foregoing of the present invention again by the following examples.But should not be understood as limiting the scope of the invention, all based on above-mentioned technological thought, the modification, replacement, the change that utilize ordinary skill knowledge and customary means to make all belong to scope of the present invention.

Description of drawings

Fig. 1 example schematic

The specific embodiment

The selection of embodiment 1 rhBNP bottling dosage

The bibliographical information of the clinical trial that external rhBNP is relevant: external clinical present recommended dose is that intravenous drip is 0.015ug/kg.min and 0.03ug/kg.min, continuous drip 6 hours, be 80kg by American-European average man body weight, therefore every dosage specification should be 432ug and 864ug.We, tentatively determine with 0.015 μ g/kg.min as dosage in conjunction with the document of abroad having delivered by the pharmacological effect test to animal; By Chinese's average weight in 60kg, continuously quiet 6 hours, then every dosage should be 324 μ g, so the bottling amount is increased to 500 μ g/ dosage.

Embodiment 2 rhBNP 0.5mg

Na ₂HPO ₄ 1.73mg

NaH ₂PO ₄ 0.93mg

Human serum albumin 10mg

NaCl9mg

Method for making: after the dissolving, add proper amount of active carbon, mixing, after suitably leaving standstill, through coarse filtration, fine straining, get refined liquid, prop up packing by 1ml/ after, lid is rolled in lyophilization, gets product, and contains rhBNP 500RU (0.5mg) in every finished product.

Embodiment 3 rhBNP 0.5mg

Na ₂HPO ₄ 1.73mg

NaH ₂PO ₄ 0.93mg

Mannitol 8mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 4 rhBNP 0.5mg

Na ₂HPO ₄ 1.73mg

NaH ₂PO ₄ 0.93mg

Glycine 6mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 5 rhBNP 0.5mg

Na ₂HPO ₄ 1.73mg

NaH ₂PO ₄ 0.93mg

Hetastarch 6mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 6 rhBNP 0.5mg

Sodium citrate 5.9mg

Citric acid 0.11mg

Human serum albumin 10mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 7 rhBNP 0.5mg

Sodium citrate 5.9mg

Citric acid 0.11mg

Mannitol 8mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 8 rhBNP 0.5mg

Sodium citrate 5.9mg

Citric acid 0.11mg

Glycine 6mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 9 rhBNP 0.5mg

Sodium citrate 5.9mg

Citric acid 0.11mg

Hetastarch 6mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 10 rhBNP 0.5mg

Sodium acetate 1.64mg

Acetic acid 0.002mg

Human serum albumin 10mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 11 rhBNP 0.5mg

Sodium acetate 1.64mg

Acetic acid 0.002mg

Mannitol 8mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 12 rhBNP 0.5mg

Sodium acetate 1.64mg

Acetic acid 0.002mg

Glycine 6mg

NaCl 9mg

Method for making is with embodiment 2

Embodiment 13 rhBNP 0.5mg

Sodium acetate 1.64mg

Acetic acid 0.002mg

Hetastarch 6mg

NaCl 9mg

Method for making is with embodiment 2

Below be long-term stable experiment and the activity test of prescription screening test and preparation.

Test example 1 rhBNP lyophilized formulations prescription optimization test

One. test method:

The preparation prescription optimization test is mainly determined by the stability that detects rhBNP under the different pharmaceutical formulation conditions, is specially with the RP-HPLC method content of the rhBNP in the finished product is measured.

Chromatographic column: Source 5RPC 4.6/150 prepacked column (Pharmacia Biotech)

Instrument: AKTA explorer100

The RP-HPLC condition is: mobile phase A is the water that contains 0.1%TFA, 5%Acetonitrile; Mobile phase B is the Acetonitrile that contains 0.1%TFA, 10% water; Gradient is: 0% → 20%B, 2CV, and 20% → 30%B, 3CV, 30% → 80%B, 1CV, applied sample amount are 500 μ l, flow velocity is 0.5ml/min, detects wavelength 215nm.

Two. the pharmaceutical formulation adjuvant

Table 1: pharmaceutical formulation adjuvant

Adjuvant	Manufacturer
		Na 2HPO 4	The Chengdu chemical reagent factory
NaH 2PO 4	The Chengdu chemical reagent factory
		Citric acid	The Chengdu chemical reagent factory
Sodium citrate	The Chengdu chemical reagent factory
		Acetic acid	The Chengdu chemical reagent factory
Sodium acetate	The Chengdu chemical reagent factory
		NaCl	The Chengdu chemical reagent factory
Mannitol	The Chengdu chemical reagent factory
		Glycine	The Chengdu chemical reagent factory
Hetastarch	The Chengdu chemical reagent factory
		The human serum albumin	Chengdu Inst. of Biological Products

Three. the preparation prescription optimization test

Biological product will keep its biological activity as much as possible, and dosage form must be stablized, and is easy and simple to handle, is easy to repetition, and the management that can strictly regulate.Lyophilized formulations is a kind of dosage form that present biological product extensively adopt.

1. the selection of dosage

The bibliographical information of the clinical trial that external rhBNP is relevant: external clinical present recommended dose is that intravenous drip is 0.015ug/kg.min and 0.03ug/kg.min, continuous drip 6 hours, be 80kg by American-European average man body weight, therefore every dosage specification should be 432ug and 864ug.We, tentatively determine with 0.015 μ g/kg.min as dosage in conjunction with the document of abroad having delivered by the pharmacological effect test to animal; By Chinese's average weight in 60kg, continuously quiet 6 hours, then every dosage should be 324 μ g, considers the report that there is no at present Chinese's clinical trial, so the bottling amount is increased to 500 μ g/ dosage.

2. the selection of buffer system

500 μ grhBNP are dissolved in the 1ml buffer, and buffer system adopts respectively phosphate, citric acid and acetate, and concentration does not wait from 5mM-100mM, PH remains between the 5-8, and temperature is 2-8 ℃, and the time is one month, investigate the stability of sample, result of the test following (μ g):

Table 2: buffer system and PH are on the impact (concentration) of rhBNP stability

Buffer PH	5	5.5	6	6.5	7	7.5	8
								Phosphate	387	396	413	422	468	486	416
Citric acid	346	398	394	415	435	457	392
								Acetate	322	372	388	398	416	421	386

It is as follows to be converted into percentage result:

Table 3: buffer system and PH are on the impact (percentage ratio) of rhBNP stability

Buffer PH	5	5.5	6	6.5	7	7.5	8
								Phosphate	77.4	79.2	82.6	84.4	93.6	97.2	83.2
Citric acid	69.2	79.6	78.8	83.0	87.0	91.4	78.4
								Acetate	64.4	74.4	77.6	79.6	83.2	84.2	77.2

The result shows that buffer system adopts phosphate 20mM, PH7.5 (corresponding Na ₂HPO ₄1.7mg, NaH ₂PO ₄0.93mg), sample stability is best.

3. the selection of excipient

500 μ grhBNP are dissolved in phosphate buffer (20mM, PH7.5), difference end user serum albumin, mannitol, glycine, hetastarch excipient, and temperature is 2-8 ℃, the time is one month, investigates the stability of sample.

(1) human serum albumin

Table 4: the different amounts human serum albumin is on the impact of rhBNP stability

The human serum albumin	0	2.5mg	5mg	10mg	20mg	40mg
							RhBNP content	481	489	497	499	493	486
Percentage ratio	96.2	97.8	99.4	99.8	98.6	97.2

(2) mannitol

Table 5: different amounts mannitol is on the impact of rhBNP stability

Mannitol	0	2mg	4mg	8mg	16mg	32mg
							RhBNP content	483	487	497	498	489	487
Percentage ratio	96.6	97.4	99.4	99.6	97.8	97.4

(3) glycine

Table 6: the different amounts glycine is on the impact of rhBNP stability

Glycine	0	2mg	4mg	8mg	16mg	32mg
							RhBNP content	482	486	493	489	487	487
Percentage ratio	96.4	97.2	98.6	97.8	97.4	97.4

(4) hetastarch

Table 7: the different amounts hetastarch is on the impact of rhBNP stability

Hetastarch	0	2mg	4mg	8mg	16mg	32mg
							RhBNP content	483	487	494	491	488	486
Percentage ratio	96.6	97.4	98.8	98.2	97.6	97.2

The result shows human serum albumin or mannitol better effects if, and the human serum albumin has better effect.In the biological product manufacture process, adding the human serum albumin has good protective effect to the stability of albumen or polypeptide usually, thereby is often used as excipient.

Select the human serum albumin as excipient on the basis of test in front, and select the human serum albumin of 1% and 0.5% concentration under different temperatures, to carry out the study on the stability of sample.The result shows that the human serum albumin of two kinds of concentration all has good protective effect to rhBNP, but it is better to add 1% human serum albumin (being that human albumin's consumption is 10mg) molding effect.Therefore select 1% human serum albumin as formulation concentrations.

Table 8: variable concentrations human serum albumin condition of different temperatures is on the impact of rhBNP stability

The selection of NaCl

In the manufacturing process, contain NaCl in the rhBNP solution that obtains, consider that human body needs 150mM NaCl to keep etc. and oozes, so contain NaCl in the goods.And in lyophilizing, contain the goods of 150mM NaCl under the condition of identical lyophilizing, its moisture is lower, so more be conducive to the preservation of rhBNP lyophilized formulations.

The test of test example 2 lyophilized formulations prescription screenings

The difference of lyophilized formulations on outward appearance, dissolution time, clarity and stability that different formulations makes investigated in the prescription screening test.Outward appearance should be white shelly loosening body, and dissolubility will be got well (planted agent was dissolved fully in 5 minutes), should be clear liquid after the dissolving, and especially the stability of rhBNP will be got well.

The stability of rhBNP adopts the RP-HPLC method that the content of the rhBNP in the lyophilized formulations is measured to reflect.Concrete detection method is as follows:

Chromatographic column: Source 5RPC 4.6/150 prepacked column (Pharmacia Biotech)

Instrument: AKTA explorer100

The RP-HPLC condition is: mobile phase A is the water that contains 0.1%TFA, 5%Acetonitrile; Mobile phase B is the Acetonitrile that contains 0.1%TFA, 10% water; Gradient is: 0% → 20%B, 2CV, and 20% → 30%B, 3CV, 30% → 80%B, 1CV, applied sample amount are 500 l, flow velocity is 0.5ml/min, detects wavelength 215nm.

By the lyophilized formulations that above-mentioned prescription makes, temperature is 2-8 ℃, and the time is one month, comes the stability of judgement sample by the content of measuring rhBNP.

Lyophilized formulations prescription screening result of the test is as follows:

The test of table 9 lyophilized formulations prescription screening

Result of the test shows, adopts embodiment 2 (rhBNP 0.5mg, Na ₂HPO ₄1.73mg, NaH ₂PO ₄0.93mg, human serum albumin 10mg, NaCl 9mg) described preparation prescription the lyophilized formulations dissolubility, clarity, the optimal stability that obtain.

Long-term stable experiment and the activity test of test example 3 lyophilized formulations prescription

Investigate long-time stability and the activity of embodiment 2 described pharmaceutical formulations.The quality of the pharmaceutical preparations requires such as following table:

Table 10 formulation samples prescription

Index	Quality of the pharmaceutical preparations requirement
		Outward appearance	Should be white shelly loosening body.
Dissolution time	With the water for injection dissolving, the planted agent was dissolved fully in 5 minutes under the room temperature.
		Clarity	Little clear liquid with opalescence is not answered precipitation muddy, that contain foreign body or cannot not shake loosely.
PH value	6.5～8.0
		Active	500RU
Moisture	≤ 3.0% (g/g)

Content of the test:

1.2～8 ℃ of investigations that keep sample: this product is placed 2～8 ℃ of refrigerators, be set in respectively and March, June, JIUYUE, December, 18 months, 24 months, 30 months, 36 months philosophies measure above index.

2. the room temperature investigation that keeps sample: this product is placed 25 ℃ of calorstats, be set in respectively and March, June, JIUYUE, December, 18 months, 24 months, 30 months philosophies measure above index.

The investigation 3.37 ℃ keep sample: this product is placed 37 ℃ of calorstats, be set in respectively and March, June, JIUYUE, December, 18 months, 24 months, 30 months philosophies measure above index.

3. biological activity test assay method (tremulous pulse bar method)

Material: the rhBNP sample, specification: 0.5mg/ props up,

Phenylephrine, Shanghai Hefeng Pharmaceutical Co., Ltd. produces, lot number: 981101

RhBNP work is with reference to product, and specification is that 0.1mg/ props up, and activity is 100U, is provided by this laboratory.

Principle: at first utilize Phenylephrine and its receptors bind on blood vessel, produce the contracting vascular effect, the resting tension of blood vessel is improved, and stable maintenance is at certain level, then utilize rhBNP to be combined with its natriuretic peptide receptor on blood vessel, generation by the variation of antiotasis, is measured the biological activity of rhBNP to the vasorelaxation of Rabbit Thoracic Aortic bar.

3.1 the compound method of solution:

The preparation of tissue fluid: the prescription of tyrode ' s liquid (with 1 liter of solution note):

NaCl 8g；KCl 0.2g；MgSO ₄.7H2O 0.26g；NaH ₂PO ₄ 0.065g；NaHCO ₃ 1g；Glucose 1g；CaCl ₂ 0.2g

First will be except CaCl ₂Other component in addition adds in the 600ml distilled water to fully dissolving, again with CaCl ₂Add in the 40ml distilled water to fully dissolving, again with CaCl ₂Solution slowly adds when stirring in the 600ml solution, and is at last that the solution dilution that mixes is for subsequent use to 1000ml.

With normal saline the concentration of Phenylephrine is formulated as 1.25 * 10 ^-2Mg/ml;

Respectively rhBNP work is dissolved in the 1ml normal saline with reference to product and testing sample first, and then is diluted to 0.1mg/ml concentration, press again 1: 10 dilution proportion standard sample and testing sample, and be numbered with manage number as follows:

The pipe number: No.1 No.2 No.3

RhBNP work is with reference to product---→ 0.1mg/ml---→ 0.01mg/ml---→ 0.001mg/ml

RhBNP testing sample---→ 0.1mg/ml---→ 0.01mg/ml---→ 0.001mg/ml

3.2 the preparation of tremulous pulse bar

Get one of 1.5-2.0kg new zealand rabbit, with pentobarbital sodium anesthesia, then put to death with dislocation method, get branches of descending thoracic aorta.The artery bar is cut into long, the wide tremulous pulse spiral bar of 2.5mm about 1.5cm, and hang in the Magnus' bath that contains 20ml tyrode's solution (37 ℃), logical oxygen, pH value remains on 7.4, blood vessel is added the 1g preload, stablized 2 hours, changed 1 time tyrode's solution therebetween in per 20 minutes, the monitor chart drive speed is adjusted to 2.5mm/min, sensitivity * 0.02.The tension variation of record blood vessel.

After baseline stability, the 0.05ml Phenylephrine that adding prepares is in Magnus' bath, and making its final concentration in Magnus' bath is 1.56 * 10 ^-4Mg/ml, curve rise to peak and stable after, begin to add the rhBNP standard sample by the cumulative concentration dose regimen, and the record tension variation.

3.3 accumulation dosage regimen: following steps, from work with reference to No. three of QC pipe, add respectively 6.25,6.25,12.5,25,50 in the Magnus' bath at every turn, 100ul, and then get second and manage, add respectively 20,40 at every turn, 80ul, get again the first pipe, add respectively 16,32, the 64ul sample at every turn.Each add sample after, wait until that tension curve is reduced to steady position after, enter into again next step dosing program, until tension curve is near baseline.

Step is as follows:

3.4 after finishing above-mentioned administration, eluting was also stablized 1 hour, changed tyrode's solution 1 time in per 20 minutes therebetween, after baseline stability, added Phenylephrine solution that 0.05ml prepares in Magnus' bath again, then its final concentration in Magnus' bath is 1.56 * 10 ^-4Mg/ml, curve rise to peak and stable after, begin to add the rhBNP testing sample by the cumulative concentration dose regimen, the accumulation medication with reference to product, and records tension variation with work.

3.5 the record of data and processing

The record format of according to the form below, data are recorded and process, final concentration value after wherein the Conc. representative sample adds in the Magnus' bath, LogC represents the common logarithm value of this final concentration, A representative in each adding work with reference to product and the tension force amplitude that behind tension stability, begins from baseline, OA represents the tension variation value that each adding work begins with reference to product and the stable tension platform from adding rising that Phenylephrine produces behind tension stability, OA% represents OA with respect to the relative value of the tension force of the stable tension platform that adds rising that Phenylephrine produces, B representative is in each testing sample and tension force amplitude that begins from baseline behind tension stability of adding, the tension variation value that OB representative each adding testing sample and the stable tension platform from adding rising that Phenylephrine produces behind tension stability begin, OB% represents OB with respect to the relative value of the tension force amplitude of the stable tension platform that adds rising that Phenylephrine produces.

Example schematic is seen Fig. 1: i.e. OA=K-A, OB=K-B, OA%=(K-A)/K, OB%=(K-B)/K

OA% and OB% have represented respectively the work of rhBNP with reference to the reaction percentage rate of product and vasodilator effect that testing sample produces, respectively LogC value and OA%, the input of OB% value in the software of the drug receptor Analytical Computer Program establishment that " Shanghai the first medical college journal " the 12nd volume the 5th phase 347-349 page or leaf is delivered, are obtained ED50 separately with reference to the Xu Duan full professor.

3.6 the calculating of testing sample activity

Active computing formula is:

Sample activity=standard substance activity * (standard substance ED ₅₀* diluted sample multiple)/(sample ED ₅₀* standard substance extension rate)

The result is as follows for the formulation samples stability test:

4～8 ℃ of investigation specification: 500RU that keep sample of table 11 recombinant human brain natriuretic peptide lyophilized injectable powder

The result: recombinant human brain natriuretic peptide lyophilized injectable powder (specification: 500RU/ props up) sample is at 2～8 ℃ of reserved sample observings, and sampling detects indices at the appointed time, and is relatively front, almost unchanged with test, activity stabilized (more than 97%), indices meets the requirements.

Table 12 recombinant human brain natriuretic peptide lyophilized injectable powder room temperature investigation specification: the 500RU that keeps sample

The result: recombinant human brain natriuretic peptide lyophilized injectable powder (specification: 500RU/ props up) sample is at the room temperature reserved sample observing, and sampling detects indices at the appointed time, and is relatively front, almost unchanged with test, activity stabilized (more than 93%), indices meets the requirements.

37 ℃ of investigation specification: 500RU that keep sample of table 13 recombinant human brain natriuretic peptide lyophilized injectable powder

The result: recombinant human brain natriuretic peptide lyophilized injectable powder (specification: 500RU/ props up) sample is at 37 ℃ of reserved sample observings, and sampling detects indices at the appointed time, and is relatively front, almost unchanged with test, activity stabilized (more than 85%), indices meet clinical front prescription.

The rhBNP lyophilized formulations was preserved 12 months under higher temperature, and its pharmaceutically active still can reach more than 85%, under nearly room temperature, preserved 12 months, its pharmaceutically active remains on more than 93%, stored refrigerated 12 months, and its pharmaceutically active remains on more than 97%.Preferred by to pharmaceutical formulation so that rhBNP has good stability, guaranteed effectiveness and the safety of medicine.

Claims (1)

1. rhBNP lyophilized formulations, it is characterized in that: it is to be prepared from by the crude drug of following weight proportion and adjuvant:

0.5 part of rhBNP, 10 parts of human serum albumins, Na ₂HPO ₄1.73 part, NaH ₂PO ₄0.93 part, 9 parts of NaCl.

2, the preparation method of rhBNP lyophilized formulations claimed in claim 1, it is characterized in that: it comprises the steps:

(1) gets rhBNP, buffer solution system, excipient, the NaCl of recipe quantity, with water for injection dissolving, solution for standby; Or

(2) get buffer solution system, excipient, the NaCl of recipe quantity, be dissolved in the solution of the rhBNP that contains recipe quantity the gained solution for standby;

(3) behind the solution impurity removal and purification with step (1) or (2) gained, with the refined liquid packing, lyophilization gets product.

Images