CS255356B1 - Method of blood styptic mammalian gamma globulin preparation for intervascular application - Google Patents
Method of blood styptic mammalian gamma globulin preparation for intervascular application Download PDFInfo
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- CS255356B1 CS255356B1 CS847531A CS753184A CS255356B1 CS 255356 B1 CS255356 B1 CS 255356B1 CS 847531 A CS847531 A CS 847531A CS 753184 A CS753184 A CS 753184A CS 255356 B1 CS255356 B1 CS 255356B1
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- gamma globulin
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Description
Vyná'ez sa týká spósobu přípravy krvného zvieracieho savčieho gamaglobulínu preThe invention relates to a method for preparing a blood animal mammalian gamaglobulin for
i. v. podanie, z normálnej alebo hyperimunnej plazmy připadne z normálného alebo retroplacentárneho připadne hyperimunného séra, ktorý je určený pre terapeutické účely.i. in. administration, from normal or hyperimmune plasma, respectively from normal or retroplacental or hyperimmune serum, which is intended for therapeutic purposes.
Súčasná situácia priemyselnej produkcie masa, mlieka a kožušín změnila životné podmienky chovných zvierat od narodenia až po dosiahnutie jatočnej hmotnosti. Nesprávná výživa zvieracích samic a nie vždy dostatočná starostlivost o novonarodené mláďata ovplyvňujú životnost a imunologická odolnost voči bakteriologickým alebo virologickým infekciam.The current situation of industrial production of meat, milk and fur has changed the welfare of breeding animals from birth to carcass weight. Improper nutrition of animal females and not always sufficient care for newborn chicks affect the longevity and immunological resistance to bacteriological or virological infections.
Mechanizmus imunitnej obranyschopnosti sa mimo iného opiera o systém nespecifických obranných mechanizmov, medzi ktoré možeme zahrnúť systém komplementu, ako aj systém špecifickej imunity, kde možeme zařadit' imunoglobulínový systém.The immune defense mechanism relies, inter alia, on a system of non-specific defense mechanisms, which may include a complement system as well as a system of specific immunity, including an immunoglobulin system.
Přenos imunoglobulínov vo vztahu samica — mláďa a saturácia krvného séra mláďata imunoglobulínami po narodení je predovšetkým závislá od včasného podania kolostra v dostatočnom množstve. Podl'a Klaussa a spol. (Klauss G. G. B., Bennet A., JonesThe transfer of immunoglobulins in the female-cub relationship and saturation of the blood serum of the pups with immunoglobulins after birth is mainly dependent on the timely administration of colostrum in sufficient quantities. According to Klauss et al. (Klauss G.G.B., Bennet A., Jones
E. V.: Immunology 16, 293, 1969) dochádza aj pri optimálnej kolostrálnej výživě ku vý2 skytu imunodeficientného stavu u 20 % teliat. Zistenia našich autorov publikované v 1983 uvádzajú však rozdielné údaje. Normálny obsah imunoglobulínov málo podlá Kudlača a spol. (Kudlač E., Schulz J., Vedral V., Vollhardt V.: Veterinární medicína 28, 7, 401, 1983J iba 24 % a pódia Paulika a spol. (Paulik Š., Slanina li., Bomba A., Poláček M.: Veterinární medicína 28, 11, 669,E. V .: Immunology 16, 293, 1969), even with optimal colostral nutrition, an immunodeficiency condition occurs in 20% of calves. However, the findings of our authors published in 1983 show different data. The normal content of immunoglobulins is low according to Kudlača et al. (Kudlač E., Schulz J., Vedral V., Vollhardt V .: Veterinary Medicine 28, 7, 401, 1983J only 24% and stage Paulika et al. (Paulik Š., Bacon li., Bomba A., Poláček M .: Veterinary medicine 28, 11, 669,
1983) iba 30 % novonarodených teliat.1983) only 30% of newborn calves.
Jednou z hlavných příčin úhynu teliat bol prevážne pozitivny bakteriologický nález a nízký obsah imunoglobulínov u uhynulých kusov (I-Ioryna B., Draganov D.: Veterinární medicína 25, 9, 537, 1980; Horyna B., Lavička M., Kabelík V., Krpata V.: Veterinární medicína 25, 9, 545, 1980).One of the main causes of calf death was mainly a positive bacteriological finding and low immunoglobulin content in dead animals (I-Ioryna B., Draganov D .: Veterinary Medicine 25, 9, 537, 1980; Horyna B., Lavicka M., Kabelik V. , Krpata V .: Veterinary Medicine 25, 9, 545, 1980).
Hladina imunoglobulínov naviac kolíše v závislosti na čase od podania kolostra u teliat (Vajda V., Slanina L·.: Veterinární medicína 25, 9, 527^ 1980) a žriebat (Komárek J., Hamáčková J.: Veterinářství 34, 5, 209,In addition, immunoglobulin levels vary with time from colostrum administration in calves (Vajda V., Slanina L .: Veterinary Medicine 25, 9, 527 ^ 1980) and foals (Komarek J., Hamackova J .: Veterinarstvi 34, 5, 209 .
1984) pričom bol pozorovaný aj sezónny pokles imunoglobulínov v krvi v období február — máj a vzostup v období máj — október.1984) and a seasonal decrease in blood immunoglobulins was observed in February-May and an increase in May-October.
Využívanie doterajších poznatkov sa začalo realizovat tým sposobom, že imunologická odolnost zvieracích savčích mláďat sa zvyšuje v období výskytu znížených hla255356 din imunoglobulínov podáváním homológnych imunoglobulínov prevažne i. m. cestou.The use of the prior art has begun to be realized in such a way that the immunological resistance of animal mammalian pups increases during the period of reduced hla255356 din immunoglobulins by administering homologous immunoglobulins predominantly i. m. way.
Pri podávání homológneho gamaglobulínového preparátu i. m. cestou si musíme uvedomiť, že preparáty tohoto typu majú zpravidla vysokú antikomplementárnu aktivitu, ktorá může ovplyvňovať nešpecifickú imunitu blokováním komplementového systému, v důsledku čoho může byť ohrozený terapeutický efekt tých imunoglobulínov, ktoré ku svojej činnosti potrebujú komplement.When administering a homologous gamaglobulin preparation i. m. It should be appreciated that preparations of this type generally have high anti-complementary activity, which may affect non-specific immunity by blocking the complement system, and as a result the therapeutic effect of those immunoglobulins that need complement to function may be compromised.
Táto skutočnosť by dokázala vysvětlit popisovaný kladný terapeutický účinok hovádzieho i. m. gamaglobulínu ako ho popisuje Tlučhoř a Komárek (Tlučhoř V., Komárek J.: Veterinářství 33, 5, 221, 1983) a nie celkom terapeuticky přesvědčivý účinok gamaglobulínového i. m. preparátu od toho istého výrobců ako ho popisuje Jílek a spol. (Jílek F., Dobšinský O., Novotný B.: Veterinářství 33, 5, 218, 1983), ktorí použili dvojnásobná dávku ako Tlučhoř a Komárek.This could explain the reported positive therapeutic effect of bovine i. m. gamaglobulin as described by Tlučhoř and Komárek (Tlučhoř V., Komárek J .: Veterinarstvo 33, 5, 221, 1983) and not quite therapeutically convincing effect of gamaglobulin i. m. preparation from the same manufacturers as described by Jílek et al. (Jílek F., Dobšinský O., Novotný B .: Veterinarstvi 33, 5, 218, 1983), who used a double dose such as Tlučhoř and Komárek.
Až doposia! sa realizuje příprava zvieracích savčích gamaglobulínov pre terapeutické účely prevažne podl'a kritérií pre intramuskulárne podanie t. j. kladie sa důraz na výťažnosť, elektroforetickú čistotu a na obsah látok so schopnosťou vyvolat pyretickú reakciu. Preparáty tohoto typu majú dlhú dobu vstrebatelnosti a zpravidla vykazujú antikomplementárnu a hypetenzívnu aktivitu.Until now! For example, the preparation of animal mammalian gamaglobulins for therapeutic purposes largely according to the criteria for intramuscular administration t. j. Emphasis is placed on recovery, electrophoretic purity and the content of substances with the ability to induce a pyretic reaction. Preparations of this type have a long absorption time and generally exhibit anti-complementary and hypetensive activity.
Na precipitáciu gamaglobulínu zo zmesi zvieracích plazmatických alebo sérových bielkovín může byť použitý síran amonný a hydroxid hlinitý (Schultze Η. E., Matheka H. D.: Behringwerke Mitt. 28, 9, 1954; Schultze Η. E., Schonenberger M., Matheka H. D.: Behringwerke Mitt. 26, 21, 1952), rivanol (Hořejší J., Smetana R.: Acta Med. Scand. 155, 65, 1956), síran sodný (Amiraian K., Leikhim E. J.: J. Immunol. 87, 301, 1961), chlorid hlinitý (Lewln J.: Therapie 9, 523, 1954), DEAE celulóza (Sober H. A., Peterson E. A.: Fed. Proč. 17, 1 116, 1958; Peterson E. A., Sober H. A.: J. Am. Chem. Soc. 78, 756, 1956; Fahey J. L., Herbett A. P.: J. Biol. Chem. 234, 2 645, 1959; Stanworth D. H.: Nátuře 188, 156, 1960), kyselina kaprylová (Steinbuch M., Audran R.: Rev. Franc. D‘etud. Clin. Biol. 14, 1054, 1969), ionty Zn+ + a Al+ + + (Rejnek J., Svařil F.: Coll. Czechoslov. Chem. Commun. 22, 1 489, 1957; Rejnek J., Škvařil F.: Coll. Czechoslov. Chem. Commun. 23, 773, 1958; Krauze R., Naimski K., Zakrewski K.: Acta Biochim. Pol. 8, 209, 1961), polymetaf osfát (Nitschman H. S., Rickli R., Kistler P.: Vox Sang. 5, 232, 1969), éter (Pennell R. B. v PutnámAmmonium sulfate and aluminum hydroxide can be used to precipitate gamaglobulin from a mixture of animal plasma or serum proteins (Schultze, E., Matheka HD: Behringwerke Mitt. 28, 9, 1954; Schultze, E., Schonenberger M., Matheka HD: Behringwerke Mitt. 26, 21, 1952), rivanol (Horejsi J., Smetana R .: Acta Med. Scand. 155, 65, 1956), sodium sulfate (Amiraian K., Leikhim EJ: J. Immunol. 87, 301, 1961), aluminum chloride (Lewln J .: Therapie 9, 523, 1954), DEAE cellulose (Sober HA, Peterson EA: Fed. Proc. 17, 1116, 1958; Peterson EA, Sober HA: J. Am. Chem. Soc., 78, 756, 1956; Fahey JL, Herbett AP: J. Biol. Chem. 234, 2645, 1959; Stanworth DH: Nature 188, 156, 1960), caprylic acid (Steinbuch M., Audran R .: Rev. Biol. 14, 1054 (1969), Zn + + and Al + + + ions (Rejnek J., Svařil F .: Coll. Czechoslov. Chem. Commun. 22, 1 489, 1957). Rejnek J., Skvaril F .: Coll. Czechoslov. Chem. Commun. 23, 773, 1958; Krauze R., Naimski K., Zakrewski K: Acta Biochim. Pol., 8, 209, 1961), polymetaph osphate (Nitschman HS, Rickli R., Kistler P .: Vox Sang. 5, 232 (1969), ether (Pennell RB in Putnam
F. W.) ed (The Plasma Proteine vol I Academie Press New York 1960, str. 3) a etanol (Cohn (E. J., Gurd F. N. R., Surgenor D.F. W.) ed (The Plasma Protein Vol I Academic Press New York 1960, p. 3) and ethanol (Cohn (E.J., Gurd F.N.R., Surgenor D.
M., Barnes B. A., Brown R. K., Derounaux G., Gillespie J. M., Kahnt F. W., Lever W. F., LiuM., Barnes B. A., Brown R. K., Derounaux G., Gillespie J. M., Kahnt F. W., Lever W. F., Liu
C. H., Mittelman D., Mouton R. F., SchmidC. H., Mittelman D., Mouton R. F., Schmid
K., Uroma E.: J. Am. Chem. Soc. 72, 465, 1950; Deutsch H. F., Gostling G. L., Alberty R. A., Williams J. W.: J. Biol. Chem. 164, 109, 1946; Oncley J. L., Melin M., Rickert D.K., Uroma E., J. Am. Chem. Soc. 72, 465 (1950); Deutsch H. F., Gostling G. L., Alberty R. A., Williams J. W., J. Biol. Chem. 164, 109 (1946); Oncley J.L., Melin M., Rickert D.
A., Cameron J. W., Gross P. M.: J. Am. Chem. Soc. 71, 541, 1949; Nitschman H. S., Kistler P., Lergier W.: Helv. Chim. Acta 37, 866, 1954; Kistler P., Nitschman H. S.: Vox Sang. 7, 414, 1962; Taylor H. L., Bloom F. C., McCall K. B., Hydman L. A., Anderson H. D.: J. Am. Chem. Soc. 78, 1 356, 1956).A., Cameron, J. W., Gross, P. M., J. Am. Chem. Soc. 71, 541 (1949); Nitschman, H. S., Kistler, P., Lergier, W., Helv. Chim. Acta 37, 866 (1954); Kistler P., Nitschman H. S .: Vox Sang. 7, 414 (1962); Taylor H. L., Bloom F. C., McCall K. B., Hydman L. A., Anderson H. D., J. Am. Chem. Soc. 78, 1356 (1956).
Preparáty gamaglobulínu z homolognej savčej krvnej plazmy alebo séra připravené podlá doterajších kritérií pre i. m. podanie majú zpravidla hypotenzívny a antikomplementárny účinok čo je pre kvality intravenóznych preparátov nepřípustné. Podanie preparátu týchto kvalit intravenózne může v důsledku náhlého poklesu krvného tlaku zviera ohrozit a v důsledku antikomplementárnej aktivity oslabit jeho nešpecifickú obranu.Gamaglobulin preparations from homologous mammalian blood plasma or serum prepared according to prior criteria for i. m. administration usually have a hypotensive and anticomplementary effect which is unacceptable for the quality of intravenous preparations. Administration of a preparation of these qualities intravenously may endanger the animal as a result of a sudden drop in blood pressure and weaken its non-specific defense due to anticomplementary activity.
Napriek tomu, že hypotenzívny účinok pri intramuskulárnom podaní nemá taký priebeh ako pri intravenóznom podaní, možno ho označit spolu s vysokým antikomplementárnym účinkom za vlastnosti nežiaduce pre preparáty savčieho gamaglobulínu, ktoré sú určené pre terapeutické účely či už pre intramuskulárne alebo pre intravenózne podanie.Although the hypotensive effect of intramuscular administration is not as successful as that of intravenous administration, it can be described, together with a high anti-complementary effect, as undesirable for mammalian gamaglobulin preparations intended for therapeutic purposes, whether intramuscular or intravenous.
Na odstraňovanie antikomplementárnej aktivity gamaglobulínu, ktorý bol připravený zo zmesi savčích p’azmatických alebo sérových bielkovín pri zachovaní homológnosti může byť použitá úprava hodnoty pH na 4,0 pri 24 hodinovej inkubácii pri 37 stupňoch Celzia (Barandun S., Kistler P., Jeunet F., Isliker H.: Vox Sang. 7, 157, 1962).A pH adjustment of 4.0 for 24 hours incubation at 37 degrees Celsius (Barandun S., Kistler P., Jeunet F) can be used to remove the anti-complementary activity of gamaglobulin prepared from a mixture of mammalian plasmatic or serum proteins while maintaining homology. , Isliker H., Vox Sang., 7, 157 (1962).
Pokles antikomplementárnej aktivity gamaglobulínu bol zaznamenaný po přidaní aktívneho uhlia (Steinbuch M., Audran R., Amouch P., Blatrix C.: Vox Sang. 13, 103, 1967), po přidaní trikalciumfosfátu (Steinbuch M., Audran R., Amouch P., Blatrix C.: Rev. med Tours, suppl. No 3, 1968) a po přidaní albuminu (Auerswald W., Kiesewetter E.: Wien. med. Wschr. 115, 690, 1965).A decrease in the anti-complementary activity of gamaglobulin was noted after the addition of activated carbon (Steinbuch, M., Audran, R., Amouch, P., Blatrix, C .: Vox Sang. 13, 103, 1967), after the addition of tricalcium phosphate (Steinbuch, M., Audran, R., Amouch. P., Blatrix C .: Rev. med Tours, suppl. No 3, 1968) and after the addition of albumin (Auerswald W., Kiesewetter E .: Wien. Med. Wschr. 115, 690, 1965).
Znižovanie antikomplementárnej aktivity gamaglobulínového roztoku pomocou proteolytického štiepenia bolo realizované pepsínom (Schultze Η. E., Schwick G.: Dtsch. med. Wschr. 87, 1 643, 1962) a plazmínom (Sgouris J. T.: Vox Sang. 13, 71, 1967; Barandun S., Castel F., Makula M. F., MorellThe reduction of the anti-complementary activity of the gamaglobulin solution by proteolytic cleavage was realized by pepsin (Schultze, E., Schwick, G .: Dtsch. Med. Wschr. 87, 1643, 1962) and plasmin (Sgouris JT: Vox Sang. 13, 71, 1967; Barandun S., Castel F., Makula MF, Morell
A., Pian R., Škvařil F.: 28, 157, 1975).A., Pian R., Skvaril F .: 28, 157 (1975).
Antikomplementárnu aktivitu gamaglobulínovej molekuly je možné znížiť jej chemickou redukciou beta merkaptoetanolom (Wiedermann G., Miescher P. A., Franklin E. C.: Proč. Soc. exp. Biol. Med. 113, 609, 1963), modifikáciou beta propiolaktonom (Stephan W.: Z. kliň. Chem. 7, 262, 1969; Stephan W.: Vox Sang. 28, 422, 1975), redukciou s dithioerythritolom (Isenman D.The anti-complementary activity of the gamaglobulin molecule can be reduced by its chemical reduction by beta mercaptoethanol (Wiedermann G., Miescher PA, Franklin EC: Proc. Soc. Exp. Biol. Med. 113, 609, 1963), by modification of beta propiolactone (Stephan W .: Z. Chem., 7, 262, 1969; Stephan W .: Vox Sang. 28, 422 (1975), by reduction with dithioerythritol (Isenman D.
25535S25535S
E., Dorrington K. J., Painter R. H.: J. Immun. 114, 1 726, 1975], sulfonáciou (Masuho Y., Tomibe K., Matsuzawa K., Ohtsu A.: Vox Sang. 32, 175, 1977), amidáciou (Schidtberger R.: US Patent No 4 118 379 — 1978), redukciou s dithiothreitolom a alkyláciou s jodacetamídom (Fernandes P. M., Lundblad J. L.: Vox Sang. 39, 101, 1980) a redukciou s dithiothreitolom v kombinácii s dithioerythritolom s následnou alkyláciou pomocou jodacetamidu alebo metyljodidu, akrylonitrilu, benzylbromidu připadne etylénoxidu (Schroeder D. D,, Tankersley D. I,., Lundblad J. L.: Vox Sang. 40, 373 1931).E., Dorrington, K. J., Painter, R. H., J. Immun. 114, 1726, 1975], by sulfonation (Masuho Y., Tomibe K., Matsuzawa K., Ohtsu A .: Vox Sang. 32, 175, 1977), by amidation (Schidtberger R .: US Patent No 4,118,379 - 1978) ), reduction with dithiothreitol and alkylation with iodoacetamide (Fernandes PM, Lundblad JL: Vox Sang. 39, 101, 1980), and reduction with dithiothreitol in combination with dithioerythritol followed by alkylation with iodoacetamide or methyl iodide, acrylonitrile dibromide, acrylonitrile, ethyl acrylonitrile D., Tankersley D.I., Lundblad JL: Vox Sang. 40, 373 (1931).
Antikomplementárna aktivita savčlebo gainaglobuínu súvisí jednak so vznikom polymerov gamaglobulíuu, ktoré můžu vznikat v procese technologické] izolácie a jednak vzniká v procese lyofilizácie v důsledku interakcie nízkomolekulárnych látok s molekulami gamaglobulínu. Ponžitie deští lovanej apyrogennej vody pri separácii nízkomolekulárnych látok z gamaglobulínu jednak spůsobuje precipitáciu najma vyš ších polymerov gamaglobulínu a jednak odstránenie nízkomolekulárnych látok v důsledku čoho pri lyotilizácii nemůže důjs!' ku ich interakcii s molekulami gamaglobulínu, číže nedochádza ani ku vzniku antikoniplementárnej aktivity. Použitie vodného roztoku chloridu sodného, v koncsntrácii do 30 g na liter, zaručuje polymérom varna globulínu v roztoku potřebná stabilitu a dochádza len ku separácii nízkomolekulárnych látok, ktoré spůsobujú vznik antikomplementárnej aktivity v procese Izolácie.The anti-complementary activity of mammalian or gainaglobulin is related, firstly, to the formation of gamma globulin polymers, which may arise in the process of technological isolation, and secondly to the lyophilization process due to the interaction of low molecular weight substances with gamma globulin molecules. The use of distilled pyrogen-free water in the separation of low-molecular-weight substances from gamaglobulin causes precipitation of the smallest gamma-globulin polymers and, on the other hand, removal of low-molecular substances as a result of which lyophilization cannot occur. their interaction with gamma globulin molecules, and thus there is no anticoniplementary activity. The use of an aqueous solution of sodium chloride, at a concentration of up to 30 g per liter, guarantees the necessary stability in the solution of the brewing globulin polymer and only separates the low molecular weight substances which give rise to anticomplementary activity in the isolation process.
Antikomplementárna aktivita bola testovaná na základe úbytku aktivity komplementu. Úbytok známej hodnoty aktivity komplementu po přidaní známého množstva roztoku lyofilizovaného gamaglobulínového preparátu bol stanovený metodou podlá Kabata a Mayera (Kabat E. A., Mayer Μ. M.: Experimentální imunochemie ČSAV, Praha 1965 str. 180).Anti-complement activity was tested for complement activity loss. The decrease in the known value of complement activity after addition of a known amount of lyophilized gamaglobulin preparation solution was determined by the method of Kabat and Mayer (Kabat E.A., Mayer, M.: Experimental Immunochemistry of the Czechoslovak Academy of Sciences, Prague, 1965, p. 180).
Efekt dešti1 ovanej apyrogénnej vody pri odstráňovaní antikomplementárnej aktivity je možné dokumentovat u 5 experimentálnych šarží gamaglobulínu, ktorý bol izolovaný z frakcie III podlá Cohna a Oncleya. Pokial' bola použitá samotná destilovaná voda, bolo dosiahnuté zníženie antikomplementárnej aktivity gamaglobulínu po lyofilizácii o 80 až 95 °/o. Použitie vodného roztoku chloridu sodného, za použitia toho istého gamaglobulínového materiálu, v koncentrácii do 30 g na liter spůsobio zníženie antikomplementárnej aktivity gamaglobulínu po lyofilizácii iba o 30% až 45%. Po kial niesu v roztoku gamaglobulínu přítomné jeho vyššie polyméry, sú výsledky testován ia antikomplementárnej aktivity v lyofilizovanom gamaglobulínovom preparáte, z ktorého bolí odstráňované nízkomolekulárne látky za použitia destilovanej apyrogénnej vody alebo za použitia vodného roztoku chloridu sodného do 30 gramov na liter prakticky rovnaké.The effect of the rain 1 ortoformiate pyrogen-free water in removing the anticomplementary activity can be documented in 5 experimental batches of gamma globulin, which was isolated from fraction III according to Cohn and Oncley. When distilled water alone was used, a reduction in the anti-complementary activity of gamaglobulin after lyophilization of 80-95% was achieved. The use of aqueous sodium chloride solution, using the same gamaglobulin material, at a concentration of up to 30 g per liter causes a reduction in the anti-complementary activity of gamaglobulin after lyophilization of only 30% to 45%. When its higher polymers are present in the gamaglobulin solution, the results are tested for the anticomplementary activity in the lyophilized gamma globulin preparation from which low molecular weight substances are removed using distilled pyrogen-free water or using aqueous sodium chloride solution up to 30 grams per liter practically the same.
Hypotenzívny účinok bol testovaný na základe poklesu krvného tlaku králika priamou krvnou metodou podlá ČSL 3 (ČSL 3, svazok I, vydanie III, Avicenum Praha 1970 str. 149) s tým, že králíkovi o hmotnosti 2 až 3 kg sa aplikujú 2 ml gama globulínového roztoku na kg hmotnosti. Obsah gama globulínu v aplikovanom roztoku je 50 gramov na 1 000 ml roztoku. U preparátov připravených podlá navrhovaného postupu sa hypotenzívna aktivita nevyskytovala v tak výraznej intenzitě, čo je možné dokumentovat údajom, že z 5 připravených experimentálnych šarží podlá předloženého vynálezu bol stanovený pokles systolického tlaku maximálně o 4 až 8 % v porovnaní s tým istým materiálom v tej istej dávke bez separácie nízkomolekulárnych zlúčenín, kde bol stanovený pokles systolického prvného tlaku od 20 do 30 %.The hypotensive effect was tested by decreasing rabbit blood pressure by a direct blood method according to CSL 3 (CSL 3, Volume I, Edition III, Avicenum Prague 1970 p. 149) with 2 ml to 3 kg of rabbit globulin administered 2 ml of gamma globulin. solution per kg of weight. The gamma globulin content of the applied solution is 50 grams per 1000 ml solution. The formulations prepared according to the proposed procedure did not exhibit hypotensive activity to such an appreciable intensity, as evidenced by the data that from a total of 5 experimental batches of the present invention a systolic pressure drop of at most 4-8% was determined compared to the same material in the same material. dose without separation of low molecular weight compounds, where a decrease in systolic first pressure of 20 to 30% was determined.
Podstatou vynálezu je odstranenie balastných negamaglobulínových příměsí, ktoré majú vzhladom ku gamaglobulíuu nižšiu molekulárnu hmotnost. Za hraničně hodnohy možno považovat údaje okolo 50 000 molekulárnej hmotnosti.It is an object of the present invention to remove ballast negamaglobulin admixtures having a lower molecular weight relative to gamma globulin. Data about 50,000 molecular weight can be considered as marginal values.
Odstranenie balastných negaraaglobu'lnových příměsí je možné previest:Removal of ballast negaraaglobulin additives can be carried out by:
A. Gélovou filtráciouA. Gel filtration
B. DialýzouB. Dialysis
C. UltrafiltráciouC. Ultrafiltration
D. DiafiltráciouD. Diafiltration
A. V súčasnosti existuje celá rada komerčných materiálov pre gélovou filtráciu na báze dextranových gélov, pod označením Sephadex, polyakry amidových gélov, pod označením Biogel, hydroxyalkylmetakrylátových gélov, pod označením Spheron, polystyrénových gélov, pod označením Styragel, polyvinylacetátových gélov, pod označením Merskogel,. poréznych silikátov, pod označením Porasi', a poréznych sídel pod označením Bioglas, ktoré majú odstupňované velkosti pórov v gélových časticiach a umožňujú gélovou filtráciu v poměrně širokých rozmedziacli molekulárnych hmotností.A. There are currently a variety of commercial dextran gel filtration materials under the designation Sephadex, polyacrylamide gels, under the designation Biogel, hydroxyalkylmethacrylate gels, under the designation Spheron, polystyrene gels, under the designation Styragel, polyvinyl acetate gels, under the designation . porous silicates, under the name Porasi, and porous sites under the name Bioglas, which have graduated pore sizes in the gel particles and allow gel filtration in relatively wide molecular weight ranges.
B. Dialýza sa v poslednom desaťročí znovu začína uplatňovat najma pri výrobě liečiv ako technologická metoda separácie tých zmesí prírodných alebo syntetických látok, ktorých molekuly majú podstatné rozdielnú hmotnost.B in the final dialysis tent Rocio recurs apply in particular to the manufacture of medicaments such as those technological methods of separation of mixtures of natural and synthetic compounds, which have substantially different molecular weight.
V súčasnosti existuje celá rada komerčných membrán pre dialýzu či už v podobě rovinnéj membrány a'ebo v podobě potrubia o menlivom priemere a menlivej velkosti pórov na báze regenerovanej celulózy alebo jej derivátov, připadne na báze kolódia a taktiež na báze vinylových polymérov.There are now a number of commercial dialysis membranes, either in the form of a planar membrane or in the form of pipes of varying diameter and pore size based on regenerated cellulose or its derivatives, possibly on the basis of collodions and also based on vinyl polymers.
C. Separácia nízkomolekulárnych zlúčenín do molekulové) hmotnosti 50 000 sa mo255356 že uskutočniť aj pomocou ultrafiltrácie, ktorú možno realizovat v podobě dutých nití pričom gamaglobulínový roztok přetéká pod tlakom do 800 kP vo vnútri niťovej membrány a cez póry v stene niťovej membrány prestupujú nízkomolekulárne látky v vod nom roztoku do zbernej nádoby.C. Separation of low molecular weight compounds to a molecular weight of 50,000 can also be accomplished by ultrafiltration, which can be carried out in the form of hollow yarns, with the gamma globulin solution flowing under a pressure of up to 800 kP inside the yarn membrane and passing through the pores in the yarn membrane wall of aqueous solution into a collection vessel.
Ultrafiltráciu je možno realizovat aj v podobě kazetového systému kde v rovinnom usporiadaní je alebo vyměnitelná membrána alebo doska z umelej hmoty s definovanými vetkosťami pórov. Gamaglobulínový roztok pod tlakom do 800 kP preteká například nad membránou a cez póry v stene rovinnej membrány alebo došky z ume lej hmoty prestupujú relativné nízkomolekulárne látky vo vodnom roztoku do zbernej nádoby.The ultrafiltration can also be carried out in the form of a cassette system where in a planar configuration there is or a replaceable membrane or plastic plate with defined pore sizes. The gamma globulin solution flows under a pressure of up to 800 kP, for example, above the membrane, and through the pores in the wall of the planar membrane or plastic thatch, the relatively low molecular weight substances in the aqueous solution enter the collecting vessel.
D. Odstráňovanie relativné nízkomolekulárnych zlúčenín do molekulárnej hmotnosti 50 000 je možno uskutočniť aj pomocou diafiltrácie, čo jě kombinácia dialýzy a ultrafiltrácie. Diafiltráciu je možné realizovat jednak v podobě dutých nití pričom gamaglobulínový roztok preteká pod tlakom do 200 kP vo vnútri niťovej membrány a cez póry v stene niťovej membrány prestupujú reálne nízkomolekulárne látky do z vonkajšiej strany pretekajúceho roztoku, ktorý relativné nízkomolekulárne látky odplavuje od niťovej membrány do odpadu.D. Removal of relatively low molecular weight compounds to a molecular weight of 50,000 can also be accomplished by diafiltration, a combination of dialysis and ultrafiltration. Diafiltration can be carried out in the form of hollow yarns whereby the gamma globulin solution flows under pressure up to 200 kP inside the yarn membrane and through the pores in the yarn membrane wall real low-molecular substances pass into the outer side of the flowing solution, which releases the relatively low molecular substances into the yarn waste. .
Diafiltráciu je možné realizovat aj v podobě kazetového systému kde v rovinnom usporiadaní je alebo vyměnitelná membrána připadne doska z umelej hmoty s definovanou vefkosťou pórov. Gamaglobulínový roztok pod mierným tlakom do 200 kP preteká například nad membránou a cez póry v stene rovinnej membrány alebo poréznej došky prestupujú reálne velmi pomalým prietokom relativné nízkomolekulárne látky do pod membránou pretekajúceho roztoku, ktorý odplavuje relativné nízkomolekulárne látky do odpadu.The diafiltration can also be realized in the form of a cassette system where in a planar configuration there is or a replaceable membrane or plastic plate with a defined pore size. For example, the gamma globulin solution under a moderate pressure of up to 200 kP flows above the membrane and passes through the pores in the wall of the planar membrane or porous thatch at a relatively slow rate of relatively low molecular weight material flowing into the membrane flowing solution that washes the relatively low molecular weight waste.
Východzlou surovinou može byť pasta frakcie II izolovaná metodou podlá Cohna a Oncleya z normálnej alebo hyperimunnej savčej plazmy připadne z normálneho alebo retroplacentárneho připadne hyperimunného séra savcov. Taktiež však možu byt použité bielkovinné gamaglobulínové materiály izolované pomocou iných metod.The starting material may be a Fraction II paste isolated by the method of Cohn and Oncley from normal or hyperimmune mammalian plasma, respectively from normal or retroplacental or hyperimmune serum of mammals. However, protein gamma globulin materials isolated using other methods may also be used.
Bielkovinný materiál sa rozpúšťa v takom množstve destilovanej apyrogénnej vody o teplote 0 °C až +6 °C, aby sa hodnota koncentrácie bielkovín nachádzala v rozmedzí 10 až 180 gramov na liter, s optimom 60 až 100 gramov na liter. Rozpúšťanie sa urýchluje miešaním. Po rozpuštění sa gamaglobulínový roztok vyčíri například filtráciou.The proteinaceous material is dissolved in an amount of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C such that the protein concentration value is in the range of 10-180 grams per liter, with an optimum of 60-100 grams per liter. Dissolution is accelerated by stirring. After dissolution, the gamaglobulin solution is clarified, for example, by filtration.
V technologických pomeroch sú vyššie u vedené optimálně podmienky zachované vtedy ak rozpúšťame 1 kg gamaglobulínovej pasty v 2 000 ml destilovanej apyrogénnej vody o teplote 0 °C až +6 °C. Hodnota pH takto připraveného gamaglobulínového roztoku bývá zpravidla v rozmedzí pH 4,0 až 8,0 a koncentrácia bielkovín v rozmedzí 60 až 100 gramov na liter. Takto připravený roztok gamaglobulínu sa vyčíri například filtráciou a nízkomolekulárne zlúčeniny do 50 000 molekulárnej hmotnosti sa odstránia například gélovou filtráciou, dialýzou, ultrafiltráciou alebo diafiltráciou pomocou elúcie alebo narieďovania s destilovanou apyrogénnou vodou alebo s vodným roztokom chloridu sodného o koncentrácii maximálně do 30 gramov na liter, s optimom 3,0 až 9,0 gramov na liter, o teplote 0 °C až +6 °C s hodnotou pH v rozmedzí 4,0 až 8,0. Gamaglobulínový roztok sa po oddělení nízkomolekulárnych látok sterilizuje, například filtráciou alebo pomocou iných metod, a potom sa zakoncentrováva například lyofilizáciou a’ebo pomocou membrán, vakuovou destiláciou, vymrazovaním alebo inými metodami a v případe potřeby sa podrobí ďalšiemu spracovaniu na konečný produkt.In the technological conditions, the above-mentioned optimum conditions are maintained if we dissolve 1 kg of gamaglobulin paste in 2000 ml of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C. The pH of the thus prepared gamma globulin solution is generally in the range of pH 4.0 to 8.0 and the protein concentration in the range of 60 to 100 grams per liter. The gamma globulin solution thus prepared is clarified, for example, by filtration, and low molecular weight compounds up to 50,000 molecular weight are removed, for example, by gel filtration, dialysis, ultrafiltration or diafiltration by elution or dilution with distilled pyrogen-free water or aqueous sodium chloride at concentrations up to 30 grams per liter. with an optimum of 3.0 to 9.0 grams per liter, at a temperature of 0 ° C to + 6 ° C with a pH in the range of 4.0 to 8.0. The gamma globulin solution, after separation of the low molecular weight substances, is sterilized, for example by filtration or other methods, and then concentrated, for example, by lyophilization or by means of membranes, vacuum distillation, freeze drying or other methods, and subjected, if necessary, to further processing to the final product.
Vynález je dalej objasněný na príkladoch prevedenia, ktorými jeho rozsah nieje ani obmedzený ani vyčerpaný.The invention is further elucidated by means of exemplary embodiments in which its scope is neither limited nor exhaustive.
Příklady prevedeniaExamples of design
Příklad 1Example 1
Východziou surovinou móže byť pista frakcie II izolovaná metodou podlá Cohna a Oncleya z normálnej alebo hyperimunnej savčej plazmy připadne z normálneho alebo retroplacentárneho připadne hyperimunného séra savcov s antibakteriálnou, antitoxickou alebo antivirovou protilátkovou aktivitou. Bielkovinný gamaglobulínový materiál sa rozpúšťa v takom množstve destilovannej apyrogénnej vody o teplote 0 °C až +6 °C aby hodnota koncentrácie bielkovín sa nachádzala v rozmedzí 10 až 180 gramov na liter, s optimom 60 až 100 gramov na liter. Rozpúšťanie sa urýchluje miešaním. Po rozpuštění sa gamaglobulínový roztok vyčíri například filtráciou.The starting material may be a fraction II pista isolated by the Cohn and Oncley method from normal or hyperimmune mammalian plasma, respectively from normal or retroplacental or hyperimmune serum of mammals with antibacterial, antitoxic or antiviral antibody activity. The proteinaceous gamma globulin material is dissolved in an amount of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C such that the protein concentration value is in the range of 10-180 grams per liter, with an optimum of 60-100 grams per liter. Dissolution is accelerated by stirring. After dissolution, the gamaglobulin solution is clarified, for example, by filtration.
V technologických pomeroch sú vyššie uvedené podmienky optima zpravidla zachované vtedy ak rozpúšťame 1 kg gamaglobulínovej pasty v 2 000 ml destilovanej apyrogénnej vody o teplote 0 CC až +6 °C. Hodnota pH gamaglobulínového roztoku bývá zpravidla v rozmedzí 4,0 až 8,0 a koncentrácia bielkovín v rozmedzí 60 až 100 gramov na liter. Takto připravený roztok purifikovaného savčieho gamaglobulínu sa vyčíri například filtráciou a nízkomolekulárne zlúčeniny sa zo savčieho gamaglobulínového roztoku odstránia:In technological conditions, the above optimum conditions are generally maintained when dissolving 1 kg of gamma globulin paste in 2000 ml of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C. The pH of the gamma globulin solution is generally in the range of 4.0 to 8.0 and the protein concentration in the range of 60 to 100 grams per liter. The purified mammalian gamaglobulin solution thus prepared is clarified, for example, by filtration, and the low molecular weight compounds are removed from the mammalian gamaglobulin solution:
A. Gélovou filtráciouA. Gel filtration
B. DialýzouB. Dialysis
C. UltrafiltráciouC. Ultrafiltration
D. DiafiltráciouD. Diafiltration
A. Odstránenie nízkomolekulárnych zlúčenín sa prevádza elučnou gélovou chromato grafiou s destilovanou apyrogénnou vodou alebo s roztokom chloridu sodného v destilované] apyrogénnej vodě o koncentráeii do 30 gramov na liter pri teplote 0 °C až +6°C pri pH 4,0 až 8,0 s vylučovacou medzou gélu vyjádřenou moleku’árnou hmotnosťou do 50 000 pričom je potřebné uvedené materiály používat v optlmálnom zrnění zaručujúcom prietok v technologických podmienkách.A. Removal of low molecular weight compounds is performed by elution gel chromatography with distilled pyrogen-free water or brine in distilled pyrogen-free water at a concentration of up to 30 grams per liter at 0 ° C to + 6 ° C at pH 4.0 to 8, 0 with a gel exclusion limit expressed as a molecular weight of up to 50,000, and it is necessary to use said materials in an optimum grain to guarantee a flow rate in the process conditions.
Ko kolóny naplnenej s nepučeným gélo vým materiálom necháme nasávat gamaglobulínový roztok takou rýchlosťou, aby prietok spodnou plochou kolóny sa pohyboval medzi 0,01 ml až 3,0 ml za minutu na plochu 1 cm2. Množstvo savčielio gamaglobulínového roztoku naseděného na kolonu nemá překročit 25 až 30 % objemu gélovej Časti kolóny. Účinnost delenia je možné sledovat podta stupňa oddelenia etylalkoholu, chloroformu a ninhydrin pozitívnych látok z gamaglobulínového roztoku. Pokial' by nedošlo ku dokonalému oddeleniu je po trebné zmenšit objem vkládaného gamaglobulínového roztoku na kolonu a spoma iť prietok. Po separácii nízkomolekulárnych zlúčenín zo savčieho zvieracieho gamaglobulínového roztoku sa tento ďalej spracováva.Allow the gamma-globulin solution to be sucked into the co-filled columns of the non-swollen gel material at a rate such that the flow through the bottom of the column is between 0.01 ml and 3.0 ml per minute per area of 1 cm 2 . The amount of mammalian gamma globulin solution mounted on the column should not exceed 25 to 30% of the volume of the gel portion of the column. Separation efficiency can be monitored by the degree of separation of ethanol, chloroform and ninhydrin positive substances from the gamaglobulin solution. If complete separation is not necessary, it is necessary to reduce the volume of the loaded gamaglobulin solution per column and slow down the flow rate. After separation of the low molecular weight compounds from the mammalian animal gamaglobulin solution, this is further processed.
B. Sterilný roztok puntíkovaného gamaglobulínu sa opatrné přesaje do sterilného dialyzačného potrubia, ktoré sa před zaha jením presavania v spodnej časti uzatvorí uzlom a po přesátí sa vrchná část uzatvorí uzlom so smyčkou za ktorá sa naplněné po trubie zavěsí na háčik, ktorý je tak umiestnený, aby roztok gamaglobulínu v dia yzacnom potrubí bol v stálom styku s pretekajácou destilovanou apyrogénnou vodou alebo s roztokom chloridu sodného v destilované] apyrogénnej vodě o koncentráeii do 30 gramov na liter pri teplote 0 C až +6 °C pri pH 4,0 až 8,0 pričom vefkosf pó rov v dialyzačnej membráně by mala byt tak vetká, aby umožnila separáciu nízkomo lekulárnych látok vyjádřená moleku árnou hmotnosťou do 50 000.B. The sterile solution of purified gamma globulin is carefully transferred to a sterile dialysis tubing, which is closed with a knot before starting to draw in the lower part and, after sieving, the upper part is closed with a knot with a loop behind which is hung up on the hook. that the gamma globulin solution in the diaphragm is in continuous contact with overflowing distilled pyrogen-free water or sodium chloride solution in distilled pyrogen-free water at a concentration of up to 30 grams per liter at 0 ° C to + 6 ° C at pH 4.0 to 8; Wherein the phosphor pores in the dialysis membrane should be large enough to allow the separation of low-molecular weight substances, expressed in molecular weights up to 50,000.
Koniec dialýzy je indikovaný negativnou reakciou na etanol, chloroform a na ninhydrin pozitivně látky v pretekajácej dialyzačnej tekutině ako aj negativnou reakciou na etanol a chloroform v gamaglobulínovom roztoku. Po skončení dialýzy sa dialyzačne potrubie zvesí z háčika, opatrné sa vyberie a po rozstrihnutí sa gamaglobulínový roztok preleje do zbernej nádoby a ďalej sa spracováva.The end of dialysis is indicated by a negative reaction to ethanol, chloroform and ninhydrin positive substances in the overflow dialysis fluid as well as a negative reaction to ethanol and chloroform in the gamma globulin solution. When dialysis is complete, the dialysis tubing is suspended from the hook, carefully removed and, after shearing, the gamaglobulin solution is poured into a collection vessel and further processed.
C. Odstraňovanie re!atívne nízkomolektilárnych zláčenín do molekulárnej hmotnosti 50 000 je možné uskutočniť pomocou ultrafiltrácie, pričom relativné nízkomolekulárne látky v podobě roztoku odtekajá do zbernej nádoby. Ultrafiltráciu je možné realizovat jednak v podobě dutých nití alebo v podobě kazetového systému, kde sa používajá došky alebo membrány s definovanými vefkosťami pórov.C. Removing Re ! The active low molecular weight compounds up to a molecular weight of 50,000 can be performed by ultrafiltration, with the relative low molecular weight solution flowing into the collection vessel. The ultrafiltration can be carried out either in the form of hollow threads or in the form of a cassette system where it is used thatch or membrane with defined pore sizes.
Vzhfadom na to, že roztok gamaglobulínu sa pri ultrafiltrácii zakoncentrováva prevedieme opatovné nariedenie s destilovanou apyrogénnou vodou alebo s roztokom chloridu sodného v destilovanej apyrogénnej vodě v koncentráeii do 30 gramov na liter a toto zakoncentrovávanie například na polovičný objem a narieďovanie na póvodný objem prevádzame dovtedy pokiaf je reakcia gamaglobulínového roztoku na přítomnost chloroformu a etanolu pozitivna.Because the gamaglobulin solution is concentrated by ultrafiltration, dilution is carried out with distilled pyrogen-free water or sodium chloride solution in distilled pyrogen-free water at a concentration of up to 30 grams per liter, and this concentration is, for example, half the volume and transferred to the original volume. reaction of the gamaglobulin solution to the presence of chloroform and ethanol positively.
Sterilizácia ultrafiitračného zariadenia sa prevádza alebo parou alebo chemickou cestou například prepláchnutím aparatáry vodným roztokom formalínu o koncentrácii do 50 g na liter a následným vymytím formalínu destilovanou vodou.Sterilization of the ultrafiltration device is carried out or by steam or chemical means, for example, by rinsing the apparatus with an aqueous solution of formalin at a concentration of up to 50 g per liter and subsequently washing the formalin with distilled water.
D. Odstráňovanie relativné nízkomolekulárnych látok do molekulárnej hmotnosti 50 000 je možné uskutočniť pomocou diafiltrácie pričom reatívne nízkomolekulárne látky prestupujá do roztoku apyrogénnej vody alebo do roztoku chloridu sodného v destilovanej apyrogénnej vodě o koncentrácii do 30 gramov na liter pri teplote 0 °C až +6 °C pri pH 4,0 až 8,0, ktoré relativné nízkomolekulárne látky odplavujá do odpadu. Diafiltráciu je možné realizovat jednak v podobě dutých nití alebo v podobě kazetového systému kde sa používajá došky alebo membrány s definovanými vefkosťami pórov.D. Removal of relatively low molecular weight species to a molecular weight of 50,000 can be accomplished by diafiltration wherein the reactive low molecular weight substances are transferred to a solution of pyrogen-free water or sodium chloride in distilled pyrogen-free water at a concentration of up to 30 grams per liter at 0 ° C to + 6 ° C at a pH of 4.0 to 8.0, which releases the relatively low molecular weight substances into the waste. The diafiltration can be carried out either in the form of hollow threads or in the form of a cassette system where it is used thatch or membrane with defined pore sizes.
Vzhfadom na to, že roztok gamaglobulínu sa pri diafiltrácii zakoncentrováva, prevedieme opatovné nariedenie s destilovanou apyrogénnou vodou alebo s roztokom chloridu sodného v destilovanej apyrogénnej vodě o koncentráeii do 30 gramov na liter a toto zakoncentrovávanie například na polovičný objem a narieďovanie na póvodný objem prevádzame dovtedy, pokiat je reakcia gamaglobulínového roztoku na přítomnost chloroformu a etanolu pozitivna. Savčí gamaglobulínový roztok sa preleje do zbernej nádoby a ďalej sa spracováva.Since the gamaglobulin solution is concentrated during diafiltration, a dilution with distilled pyrogen-free water or sodium chloride solution in distilled pyrogen-free water with a concentration of up to 30 grams per liter is made and this concentration is, for example, half the volume and diluted to the original volume. if the reaction of the gamma globulin solution to the presence of chloroform and ethanol is positive. The mammalian gamaglobulin solution is poured into a collection vessel and further processed.
Sterilizácia diafiltračného zariadenia sa prevádza alebo parou alebo chemickou cestou naprík ad prepláchnutím aparatury vodným roztokom formalínu o koncentráeii do 50 gramov na liter a následným vymytím formalínu sterilnou destilovanou vodou.Sterilization of the diafiltration device is carried out or by steam or chemical means, for example, by rinsing the apparatus with an aqueous solution of formalin at a concentration of up to 50 grams per liter and subsequently washing the formalin with sterile distilled water.
Dokaž chloroformu sa prevádza Fujiwarovou reakciou (Fujiwara: Sitzber. Naturw. Ges. Rostock 6, 33 1916] či už v póvodnom usporiadaní, alebo v novších aplikáciach podlá Seta a Schultzeho (Seto T., Schultze M. O.: Anal. Chem. 28, 1 625, 1956), Hildebrechta (Hildebrecht C. D.: Anal. Chem. 29, 1037, 1957), Friedmana a Coopera (Friedman P. J., Cooper J. R.: Anal. Chem. 30, 1 674, 1958).Chloroform detection is carried out by the Fujiwar reaction (Fujiwara: Sitzber. Naturw. Ges. Rostock 6, 33 1916), either in the original configuration or in newer applications according to Set and Schultze (Seto T., Schultze MO: Anal. Chem. 28, 1). 625, 1956), Hildebrecht (Hildebrecht CD: Anal. Chem. 29, 1037, 1957), Friedman and Cooper (Friedman PJ, Cooper JR: Anal. Chem. 30, 1674, 1958).
Ninhydrin pozitivně látky stanovujeme spósobom podl'a Ruhemana (Ruhemann S.: J. Chem. Soc. 97, 1438, 1910). Stanovenie etanolu sa prevádza Agulhonovou reakcion (Agulhon H.: Bull. soc. chim. France[4], 9, 881,’ 1911) či už v původnom usporiadauí, alebo v novějších aplikáciach podfa Kellctta (Kellett E. G.: Analyst 62, 728 1937), alebo Webba (Webb D. A.: Sci. Proč. Roy Dublin Soc. 21, 281, 1936).Ninhydrin positives are determined by the method of Ruheman (Ruhemann S .: J. Chem. Soc. 97, 1438, 1910). The determination of ethanol is carried out by the Agulhon reaction (Agulhon H .: Bull. Soc. Chim. France [4], 9, 881, 1911), either in the original configuration or in more recent Kellctt applications (Kellett EG: Analyst 62, 728 1937). ) or Webba (Webb DA: Sci. Proc. Roy Dublin Soc. 21, 281, 1936).
Gamaglobulínový roztok po oddělení nízkomolekulárnych látok sa sterilizuje například fftráciou alebo inými metodami, a potom sa zakoncentrováva například lyofilizáciou alebo pomocou membrán, vákuovou destiláciou, vymrazovaním alebo inými metodami, a v případe potřeby sa podrobí ďalšiemu spracovaniu.After separation of the low molecular weight substances, the gamaglobulin solution is sterilized, for example, by filtration or other methods, and then concentrated, for example, by lyophilization or by means of membranes, vacuum distillation, freeze-drying or other methods, and subjected to further processing if necessary.
Příklad 2Example 2
Východziou surovinou móže byť biolkovinná pasta alebo bielkovinný roztok savčieho gamaglobulínu izolovaný aj pomocou iných metod. Bielkovinná pasta alebo bie kovinný roztok savčieho zvieracieho gamaglobulínu sa spracováva v ďalšom postupe podobné ako v příklade 1 s tým rozdielom, že kritérium odstránenia nízkomolekulárnych zlúčenín z roztoku gamaglobulínu ne súvisí s prítomnosťou etanolu alebo chloroformu, ale s prítomnosťou precipitačnej látky, ktorá bola na precipitáciu gamaglobulínu alebo jeho imobilizovanie použitá. Kritérium ninhydrin pozitívnych látok je zachované pri akejkofvek metóde, pričom proces oddelovania sa opakuje dovtedy pokial' nieje reakcia v negamaglobulínovom podiele negativna.The starting material may be a bio-protein paste or a protein solution of mammalian gamaglobulin also isolated by other methods. The protein paste or white metal solution of mammalian animal gamaglobulin is processed in a further procedure similar to Example 1, except that the criterion for removing low molecular weight compounds from the gamaglobulin solution is not related to the presence of ethanol or chloroform, but to the presence of a precipitating agent for gamaglobulin precipitation. or its immobilization used. The criterion of ninhydrin-positive substances is maintained in any method, with the separation process repeated until the reaction in the negamaglobulin fraction is negative.
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