[go: up one dir, main page]

WO2022035334A2 - Procédé d'obtention d'aprotinine et produit obtenu à partir de celui-ci - Google Patents

Procédé d'obtention d'aprotinine et produit obtenu à partir de celui-ci Download PDF

Info

Publication number
WO2022035334A2
WO2022035334A2 PCT/PL2021/050058 PL2021050058W WO2022035334A2 WO 2022035334 A2 WO2022035334 A2 WO 2022035334A2 PL 2021050058 W PL2021050058 W PL 2021050058W WO 2022035334 A2 WO2022035334 A2 WO 2022035334A2
Authority
WO
WIPO (PCT)
Prior art keywords
obtaining
aprotinin
carried out
concentration
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/PL2021/050058
Other languages
English (en)
Other versions
WO2022035334A3 (fr
Inventor
Grzegorz CIURA
Magdalena MATWIEJCZYK
Monika BESMAN
Filip Porzucek
Konrad BABIJ
Michał LOBOCKI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharmfactor Sp Z OO
Original Assignee
Pharmfactor Sp Z OO
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmfactor Sp Z OO filed Critical Pharmfactor Sp Z OO
Publication of WO2022035334A2 publication Critical patent/WO2022035334A2/fr
Publication of WO2022035334A3 publication Critical patent/WO2022035334A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)

Definitions

  • the object of the invention is a method of obtaining aprotinin and a product obtained by the method.
  • the invention will find application in pharmacy, medicine, biotechnology, biochemistry and molecular biology, e.g., for the protection of proteins against the action of serine-threonine proteases.
  • many methods comprise steps of enzymatic digestion of tissues (e.g., with proteases), which can affect the efficiency and safety of the process.
  • An example of such a document is, e.g., DE2615436A1.
  • isolation and purification techniques often used are complex and ineffective in terms of cost on a large scale.
  • many processes use affinity chromatography in at least one step of the preparation method. In this way, some methods, despite their high yield, may in practice not be industrially applicable due to cost and complexity.
  • the present invention is directed to a method of obtaining aprotinin that does not use strong proteolytic enzymes or complicated and unprofitable techniques (in particular the affinity chromatography), while maintaining a sufficiently high efficiency and safety of the product (meets the pharmacopoeial standards and requirements).
  • the object of the invention is a method of obtaining aprotinin, in particular from bovine lungs, comprising sequentially the steps of: a. Comminution b. Extraction c. Centrifugation d. Microfiltration e. Chromatography I f. Concentration I. g. Salting out h. Filtration i. Ultrafiltration I j. Chromatography II k. Ultrafiltration II l. Concentration II m. Ultrafiltration III n. Freeze-drying o. Washing
  • the method of obtaining aprotinin according to the invention is characterized in that the tissue comminution step is carried out by grinding the tissue in an industrial electrical meat grinder.
  • the method of obtaining aprotinin according to the invention is characterized in that the extraction step is carried out in purified water having a pH of 4-5 and a temperature of 45-60°C for a period of 35-55 minutes, and heating up the mixture to a temperature of 65-75°C.
  • the method of obtaining aprotinin according to the invention is characterized in that the centrifugation step is carried out by gravity filtration on a filter cloth for at least 3 hours and centrifugation in basket centrifuges for 30 minutes with a rotation frequency of 1000 rpm.
  • the method of obtaining aprotinin according to the invention is characterized in that the microfiltration step is carried out on ceramic filters with a pore diameter of 0.45 micrometers.
  • the method of obtaining aprotinin according to the invention is characterized in that the chromatography step I is carried out by means of liquid chromatography with a strong cation exchanger bed, selectively concentrating the aprotinin, using a gradient elution system with increasing sodium chloride concentration (0-1 M NaCI), separating the fractions with the highest aprotinin content.
  • the method of obtaining aprotinin according to the invention is characterized in that the concentration step I is carried out on a vacuum evaporator at a pressure of 10 mbar and a temperature of 26°C, preferably to about 20% of the starting volume, preferably to a protein concentration of about 35 mg/mL.
  • the method of obtaining aprotinin according to the invention is characterized in that the step of salting out is carried out using calcium chloride to a final concentration of 50 mM and sodium chloride to a concentration of 30%, then adjusting the pH with hydrochloric acid to 2.5-4.5, preferably to pH 3.5, leaving the solution in a refrigerator, preferably at a temperature of about 8°C, preferably for 24 hours, then gravitationally filtering on a Nutsche filter lined with filter cloth, preferably BT-17, and then reducing the pressure on the filtrate side, preferably with a vacuum pump, draining the precipitate to dryness.
  • the step of salting out is carried out using calcium chloride to a final concentration of 50 mM and sodium chloride to a concentration of 30%, then adjusting the pH with hydrochloric acid to 2.5-4.5, preferably to pH 3.5, leaving the solution in a refrigerator, preferably at a temperature of about 8°C, preferably for 24 hours, then gravitationally filtering on a Nutsche filter
  • the method of obtaining aprotinin according to the invention is characterized in that the filtration step is carried out by dissolving 1 kg of the chloride precipitate in about 25 L of purified water, adding about 2 L of a regenerated strong anion exchanger bed to the solution, while stirring continuously, measuring the pH of the solution after about 20 minutes, and in the case of pH being below 8, adding about 200 mL of the regenerated anion exchanger and measuring the pH again until the pH is above 8, and after stabilizing the pH of the solution, pouring it on a column with a polyamide mesh membrane with a pore size of 100 pm, passing the filtrate to the further purification steps, the strong anion exchanger bed being first prepared by adding a 2 M sodium hydroxide solution in a volume ratio of 1 to 3 to the strong anion exchanger bed and stirring for at least about 4 hours, then separating it from the solution on a column with a polyamide mesh membrane with a pore size of 100 pm, finally washing the bed on the column with purified water to the
  • the use of the anion exchanger reduces the volume of solution necessary to dissolve the chloride precipitate (so that the conductivity is below 5 mS).
  • the need of adding sodium hydroxide directly to the product is eliminated, and the purity and specific activity of aprotinin are increased.
  • the method of obtaining aprotinin according to the invention is characterized in that the ultrafiltration step I is carried out by filtering the solution obtained in the previous step on tubular filters with a MWCO of 300 kDa to 1000 kDa to a 5-L volume of retentate, then performing diafiltration of the obtained retentate in an intermittent-operation system by 5-times washing with 10 L of purified water.
  • the ultrafiltration step I is carried out by filtering the solution obtained in the previous step on tubular filters with a MWCO of 300 kDa to 1000 kDa to a 5-L volume of retentate, then performing diafiltration of the obtained retentate in an intermittent-operation system by 5-times washing with 10 L of purified water.
  • the method of obtaining aprotinin according to the invention is characterized in that the chromatography step II is carried out on a chromatography column with a weak cation exchanger using a gradient elution system with increasing concentration of ammonium acetate buffer having pH 8.8-9.2.
  • the method of obtaining aprotinin according to the invention is characterized in that the ultrafiltration step II is carried out by filtering on a 10-kDa-MWCO filter in an intermittent-operation system, running the process until there remains about 10 L of the solution being concentrated, and then washing with three 20-L portions of purified water until the volume equals 10 L.
  • the method of obtaining aprotinin according to the invention is characterized in that the concentration step II is carried out on a vacuum evaporator at a pressure of 10 mbar and a temperature of 26°C, preferably to about 20% of the starting volume, preferably to a protein concentration of about 50 mg/mL.
  • the method of obtaining aprotinin according to the invention is characterized in that the ultrafiltration step III is carried out by filtering the solution on a 30- kDa-MWCO tubular filter to obtain a 1-L volume of retentate, then performing diafiltration of the obtained retentate in an intermittent-operation system by washing 5 times with 1 L of purified water.
  • the method of obtaining aprotinin according to the invention is characterized in that the freeze-drying step is carried out by freeze-drying the retentate for the first 24 hours at a condenser temperature of -45°C, a shelf temperature of -30°C and atmospheric pressure, then the pressure is reduced to 0.05 mbar and the shelf temperature is gradually increased to 24°C for 72 h, and the last 24 h it is carried out at a shelf temperature of 24°C and a vacuum pressure of 0.001 mbar.
  • the method of obtaining aprotinin according to the invention is characterized in that the washing step is carried out by first subjecting the lyophilisate to a milling process, preferably in a ball mill, preferably for at least 1 hour, and then subjecting the powder to at least one, preferably at least two cycles of suspending in anhydrous acetone at a ratio of 1 :4, stirring at room temperature for at least 1 hour, separating acetone from the powder on a Schott funnel, and resuspending in acetone at 1 :4 ratio.
  • a milling process preferably in a ball mill, preferably for at least 1 hour
  • the powder to at least one, preferably at least two cycles of suspending in anhydrous acetone at a ratio of 1 :4, stirring at room temperature for at least 1 hour, separating acetone from the powder on a Schott funnel, and resuspending in acetone at 1 :4 ratio.
  • the method of obtaining aprotinin according to the invention is characterized in that it further comprises at least one of the steps selected from the group including in-process control, sterilization, terminal sterilization, quality control, which steps may be repeated, occur before the first step, in the middle and at the end of the method of obtaining.
  • the subject of the invention is also the product obtained by the method of obtaining according to the invention.
  • the method of obtaining aprotinin according to the invention does not use strong proteolytic enzymes or technologically complicated and unprofitable techniques (such as, e.g., affinity chromatography), and at the same time gives a product with sufficiently high efficiency and safety (meeting pharmacopoeial standards and requirements).
  • the applied step of salting out unexpectedly precipitates 90% of the aprotinin as a precipitate, and about 60% of the remaining proteins stay in the supernatant, which significantly increases the degree of purification.
  • the need of adding sodium hydroxide directly to the product has been eliminated, and the consumption of water used to dissolve the precipitate has been reduced.
  • step o it was unexpectedly possible to normalize the content of aprotinin dimer from the final product test.
  • the invention will find application in pharmacy, medicine, biotechnology, biochemistry and molecular biology, by being used in the protection of proteins against the action of serine-threonine proteases.
  • Fig. 1 - shows a block diagram of an embodiment of a method of obtaining aprotinin according to the invention
  • Fig. 2 - shows the result of a chromatographic separation of a freeze-dried product not treated with acetone
  • Fig. 3 - shows the result of a chromatographic separation of the freeze-dried product, which has been washed with acetone
  • Fig. 4 - shows the product obtained by the method of obtaining according to the invention
  • Bovine lungs comminuted by grinding in an industrial electrical meat grinder were used as the raw material for the process.
  • the term bovine lungs means in particular cow lungs, which have been used in this embodiment, but does not restrict the scope of the invention as far as the age (e.g., calf, heifer, cow, bull), sex (e.g., male, female), or other representatives of cattle and wild cattle are concerned.
  • the term bovine lungs also means lung mixtures of the foregoing. Extraction was carried out in purified water, the pH of which was adjusted to 4-5 and then it was heated to 45-60°C for a period of 35-55 minutes.
  • the mixture was then heated up to 65-75°C.
  • the pH adjustment, heating, and heating up were performed using standard laboratory techniques.
  • the mixture was gravity- filtered on filter cloth in basket centrifuges. After about 3 hours of gravity filtration, the basket centrifuges were additionally turned on to fully centrifuge the extract.
  • the extract was centrifuged for 30 minutes at a rotation frequency of 1000 rpm, but a person skilled in the art will appreciate the possibility of modifying the process parameters as needed (e.g., the amount and the degree of processing of the starting material).
  • the extract thus obtained was subjected to microfiltration on ceramic filters with a pore diameter of 0.45 micrometers. Subsequently, the aprotinin was selectively concentrated by liquid chromatography with a strong cation exchanger bed.
  • the next step was elution with a gradient of sodium chloride (0- 1 M NaCI), separating fractions with the highest content of aprotinin.
  • the resulting eluate was concentrated on a vacuum evaporator to about 20% of the starting volume at a pressure of 10 mbar and a temperature of 26°C.
  • concentration to a total protein concentration of 35 mg/mL
  • the solution was salted out with calcium chloride to a final concentration of 50 mM and sodium chloride to a concentration of 30%.
  • the pH was then adjusted with hydrochloric acid to a pH of 3.5.
  • the solution was allowed to stand in a refrigerator (at about 8°C) for about 24 hours, for at least 8 hours.
  • the chloride precipitate formed after this time was poured onto a Nutsche filter lined with filter cloth (BT-17), it was then filtered by gravity for at least 2 hours, and then the pressure on the filtrate side was lowered (using a vacuum pump) and it was filtered to dryness. The filtrate was sent for disposal and the precipitate was passed on to further purification steps.
  • Table 1 the effect of salting out aprotinin increased the degree of purification almost 3-fold.
  • the invention relies on, among others, the salting out of aprotinin from the concentrated solution of the main fractions obtained after ion exchange chromatography. Unexpectedly, more than 90% of the aprotinin precipitates and falls out of the solution as a precipitate, and about 60% of the remaining proteins stay in the supernatant, which significantly increases the degree of purification.
  • the anion exchanger was prepared as follows. A 2 M sodium hydroxide solution at a 1 to 3 volume ratio was added to the strong anion exchanger bed and stirred for at least about 4 hours. The bed was separated from the solution on a column with a polyamide mesh membrane with a pore size of 100 pm. The bed was washed on the column with purified water until the pH of the effluent was below pH 9. Thus prepared anion exchanger was used for the filtration of the aprotinin solution. Thereafter, the obtained 1 kg of chloride precipitate was dissolved in 25 L of purified water.
  • the invention additionally consists in, among others, the use of filtration of dissolved chloride precipitate of aprotinin on the ion exchanger, the anion exchanger, which as a result made it possible to reduce the volume of water used to dissolve the precipitate.
  • the use of anion exchanger reduces the volume of solution necessary to dissolve the chloride precipitate (so that the conductivity is below 5 mS). The need of adding sodium hydroxide directly to the product has been eliminated, and the purity and specific activity of aprotinin have been increased.
  • the solution was filtered on tubular filters with MWCO from 300 kDa to 1000 kDa (using polymer filters) until a 5-L volume of retentate was obtained, then performing diafiltration of the obtained retentate in an intermittent-operation system by washing 5 times with 10 L of purified water.
  • the collected filtrate was adsorbed on a chromatography column with a weak cation exchanger and eluted using a gradient elution system with increasing concentration of ammonium acetate buffer having pH 8.8-9.2. The fraction with the highest purity and content of aprotinin was isolated.
  • filtration was carried out on a 10-kDa- MWCO filter in an intermittent-operation system, proceeding with the process until about 10 L of solution remained in the retentate.
  • the retentate was washed with three 20-L portions of purified water until the volume of the retentate was equal to 10 L.
  • the retentate after 10-kDa ultrafiltration was sent for disposal, and the filtrate was concentrated on a vacuum evaporator to about 20% of the starting volume at a pressure of 10 mbar and a temperature of 26°C (until protein concentration of 50 mg/mL was obtained).
  • the solution was filtered on a 30-kDa-MWCO tube filter until a 1 -L volume of retentate was obtained, then performing diafiltration of the obtained retentate in an intermittent-operation system by washing 5 times with 1 L of purified water, wherein the product was on the filtrate side.
  • the filtrate was packed in a sterile container and passed on for freeze-drying. The freeze-drying process was carried out for the first 24 hours at a condenser temperature of -45°C, a shelf temperature of -30°C and atmospheric pressure.
  • the pressure was reduced to 0.05 mbar and the shelf temperature was gradually increased to 24°C over 72 h, and the last 24 h the process was run at a shelf temperature of 24°C and a vacuum pressure of 0.001 mbar.
  • the lyophilisate obtained was subjected to a milling process for a period of 1 hour. A ball mill was used for this purpose, but a skilled artisan will appreciate the wide selection of mills and milling techniques. 100 g of the lyophilisate after milling was used for analytical purposes. The powder was then suspended in anhydrous acetone at a ratio of 1 :4.
  • aprotinin obtained with the unmodified method after freeze-drying and carrying out the dimerization process (for the analytical test), did not form a dimer form.
  • the test result was qualified as non-compliant.
  • This fact is illustrated in Fig. 2 and Fig. 3 showing the chromatographic separation of aprotinin on molecular sieve columns.
  • the chromatogram in Fig. 2 shows the result of a chromatographic separation of the freeze- dried product not treated with acetone. Although the lyophilisate was subjected to dimerization (for the analytical test), no aprotinin dimer was formed.
  • FIG. 3 shows the result of a chromatographic separation of the freeze-dried product that has been washed with acetone.
  • the lyophilisate was subjected to the dimerization process (for the analytical test), which resulted in the formation of an aprotinin dimer (peak with a retention time of 31.5 min). Its content is within the standard imposed by the pharmacopoeial monograph, and therefore the product meets the pharmacopoeial requirements.
  • a person of skill in the art understands the need for quality assurance or control and will modify the above method of obtaining aprotinin as necessary, adding one or more steps required or indicated by local or international guidelines or regulations.
  • these may be one or more in-process control steps, one or more sterilization or terminal sterilization steps, one or more quality control steps.
  • These steps may occur at all stages of the method of obtaining aprotinin according to the invention, also before the first step and after the last step.
  • the additional steps may also follow one another.
  • the described technology allows obtaining 74 mg of aprotinin with a purity of over 98% and an activity of over 3 PhEur units/mg on a dry weight basis. Recalculated to kallikrein units, the obtained product has a specific activity of not less than 5850 KlU/mg and is characterized by a process yield of about 60%.
  • the method of obtaining according to the invention is characterized by, among others, a simplification of the process in terms of technological sophistication of the steps, exclusion of techniques that are counter-effective cost-wise and a reduction of equipment requirements, a reduction of water losses, hence and indirectly, a reduction of costs and contamination, exclusion of direct addition of sodium hydroxide to the product and proteolytic enzymes, which in total makes the method according to the invention competitive with the methods characterized by a higher yield.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Steroid Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Compounds Of Unknown Constitution (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)

Abstract

L'invention a pour objet un procédé d'obtention d'aprotinine, en particulier à partir de poumons de bovins, comprenant séquentiellement les étapes de : broyage, extraction, centrifugation, microfiltration, chromatographie I, concentration I, relargage, filtration, ultrafiltration I, chromatographie II, ultrafiltration II, concentration II, ultrafiltration III, lyophilisation, lavage. L'invention a également pour objet le produit obtenu par le procédé.
PCT/PL2021/050058 2020-08-13 2021-08-11 Procédé d'obtention d'aprotinine et produit obtenu à partir de celui-ci Ceased WO2022035334A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PL434960A PL434960A1 (pl) 2020-08-13 2020-08-13 Sposób otrzymywania aprotyniny oraz produkt otrzymany tym sposobem
PLP.434960 2020-08-13

Publications (2)

Publication Number Publication Date
WO2022035334A2 true WO2022035334A2 (fr) 2022-02-17
WO2022035334A3 WO2022035334A3 (fr) 2022-03-31

Family

ID=77989964

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/PL2021/050058 Ceased WO2022035334A2 (fr) 2020-08-13 2021-08-11 Procédé d'obtention d'aprotinine et produit obtenu à partir de celui-ci

Country Status (2)

Country Link
PL (1) PL434960A1 (fr)
WO (1) WO2022035334A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115259367A (zh) * 2022-07-26 2022-11-01 上海净豚环保科技有限公司 一种食品废水用厌氧颗粒污泥的增殖培菌的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006042327A2 (fr) * 2004-10-12 2006-04-20 Large Scale Biology Corporation Aprotinine de recombinaison d'origine vegetale et variants d'aprotinine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115259367A (zh) * 2022-07-26 2022-11-01 上海净豚环保科技有限公司 一种食品废水用厌氧颗粒污泥的增殖培菌的方法
CN115259367B (zh) * 2022-07-26 2023-11-17 上海净豚环保科技有限公司 一种食品废水用厌氧颗粒污泥的增殖培菌的方法

Also Published As

Publication number Publication date
WO2022035334A3 (fr) 2022-03-31
PL434960A1 (pl) 2022-02-14

Similar Documents

Publication Publication Date Title
JP3094167B2 (ja) 免疫血清グロブリンの精製方法
US7750119B2 (en) Production of 2S canola protein involving ion exchange
JPH03502921A (ja) 乳清からのラクトペルオキシダーゼおよびラクトフェリンの純粋な画分の抽出法
CN104672328B (zh) 一种人抗凝血酶ⅲ的生产方法
JP6650861B2 (ja) 傷口の癒合、コラーゲン増殖、血管新生、免疫細胞の活性化を促進し、また傷口の感染を減少させることができるペプチド及びその応用
EP0291264B1 (fr) Procédé de préparation de glycomacropeptide de caséine-K
JP2974434B2 (ja) 分泌性コンポネント含有組成物
JPH083197A (ja) ウイルスの不活性化および活性蛋白質の精製
WO2022035334A2 (fr) Procédé d'obtention d'aprotinine et produit obtenu à partir de celui-ci
PL244350B1 (pl) Sposób otrzymywania hialuronidazy ze zwierzęcych jąder oraz produkt otrzymany tym sposobem
CN119529313B (zh) 一种皂苷佐剂纯化方法
US7002007B2 (en) Production of high molecular weight hyaluronates
JP2678653B2 (ja) ガングリオシドの抽出および濃縮方法
CN112625110A (zh) 一种牛乳中β-酪蛋白的纯化制备方法
CN106866812B (zh) 一种从妇女尿液中提取多种尿蛋白的方法
US2797184A (en) Process for the recovery of heparin
RU2749424C1 (ru) Способ получения лекарственного средства, обладающего антикоагулянтной активностью
WO2000011890A2 (fr) Procedes relatifs a l'isolation de beta-caseine de recombinaison
KR102531611B1 (ko) 고순도 dna 단편 혼합물의 제조 방법
CN113584006A (zh) 一种冻干人凝血酶原复合物及其制备方法
JP4886358B2 (ja) エンドウホエー由来の可溶性ポリペプチドを有効成分とする起泡剤およびこれを含む炭酸飲料
CN106474461B (zh) 制备冻干人凝血酶的方法
RU2828847C1 (ru) Способ получения гепарина
CN120060220A (zh) 一种凝乳蛋白酶及其制备方法
SU1671691A1 (ru) Способ получени @ - N - ацетилглюкозаминидазы BacILLUS SUвтILIS ВКПМ В-2471

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21782608

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21782608

Country of ref document: EP

Kind code of ref document: A2