EP0224532A1 - Process for preparing gamma-globulins supplied by intravenous administration and gamma-globulins obtained - Google Patents

Process for preparing gamma-globulins supplied by intravenous administration and gamma-globulins obtained

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Publication number
EP0224532A1
EP0224532A1 EP86903414A EP86903414A EP0224532A1 EP 0224532 A1 EP0224532 A1 EP 0224532A1 EP 86903414 A EP86903414 A EP 86903414A EP 86903414 A EP86903414 A EP 86903414A EP 0224532 A1 EP0224532 A1 EP 0224532A1
Authority
EP
European Patent Office
Prior art keywords
gamma
carried out
globulins
peg
pepsin
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
EP86903414A
Other languages
German (de)
French (fr)
Inventor
Marie-France Makula
Jacques Liautaud
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.)
Sanofi Pasteur SA
Original Assignee
Institut Merieux SA
Pasteur Merieux Serum et Vaccines SA
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 Institut Merieux SA, Pasteur Merieux Serum et Vaccines SA filed Critical Institut Merieux SA
Publication of EP0224532A1 publication Critical patent/EP0224532A1/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation

Definitions

  • the present invention relates to a process for the preparation of gamma globulin which can be administered intravenously as well as to the gamma globulin obtained by this process.
  • a first method seeks to eliminate the aggregates and consists in carrying out a pepsin digestion of the preparation of gamma globulins, so as to cause a break in heavy chains near the junction of the Fab and Fc fractions. See, for example, patent FR-A-2,433,342.
  • Another method which had been developed by the applicant, consists in treating a preparation of gamma-globulins with plasmin, so as to cause controlled digestion retaining a large percentage (from 30 to 50) of intact gamma-globulin and giving Fab and Fc fragments. This preparation is active and very well tolerated.
  • the patent FR-A-2,301,266 recommends not to carry out an enzymatic treatment, in particular with pepsin, of gamma-globulins and to carry out, instead, a fractionation with polyethylene glycol.
  • a process for the preparation of gamma globulins by fractionation with polyethylene glycol (PEG) has been developed which has the advantage of preventing the formation of aggregates or of eliminating them, while retaining intact globulins.
  • PEG polyethylene glycol
  • the different preparations described above can present shock factors by activation of the kallikrein-bradykinin system, these factors appear to be linked to a residual content of prekallikrein activator.
  • Another technique which makes it possible to obtain good quality gamma globulins, consists in carrying out a treatment at acid pH, in the presence of small amounts of pepsin. See, for example, J.J. Walsh: Purification of normal immunoglobulin for intravenous use. DEVELOP. BIOL. STAND. 1974, 27, 31-6. However, there are still aggregates and dimers at a relatively large rate.
  • the present invention proposes to provide preparations of gamma globulin which can be administered intravenously, both without aggregates and dimers, without anti-complementary power and without kallikrein and prekallikrein activator and with a profile of subclasses comparable to that of normal Human Serum.
  • Another objective of the invention is to provide such a process which, applied to gamma ⁇ globulin preparations prepared by fractionation with low levels of PEG or similar substances, improves these preparations by notably reducing the anti-complementary power, the content of kallikrein and activator of prekallikrein, as well as the residual PEG.
  • the subject of the invention is a process for the preparation of gamma-globulins which can be administered intravenously, characterized in that it comprises a fractionation step with polyethylene glycol (PEG) or substances similar and a mild enzymatic treatment step, in which the pH is dependent on the nature of the enzyme used, this being preferably added in the form of traces, the treatment being carried out to avoid sensitive proteolysis.
  • PEG polyethylene glycol
  • the enzyme used is chosen from the group formed by pepsins, fibrinolysins (plasmin), and papains.
  • the starting material is a fraction of serum origin rich in gamma-globulins such as fraction II Technique 6.9 of Cohn, known to give products free of hepatitis, or a fraction of pla ⁇ centary origin such as the technical fraction 6.9 of Cohn modified by Taylor.
  • This fraction has an IgG purity greater than or equal to 90%. It may contain varying amounts of albumin which can be up to 10%. 1st example:
  • the starting raw material consists in dissolving the precipitate and in clarifying this solution, a solution which generally contains from 1 to 4% of proteins.
  • a precipitation step is carried out by adding polyethylene glycol (PEG), of molecular weight 4,000, so as to obtain a PEG concentration of 5. .
  • PEG polyethylene glycol
  • the pH is adjusted 5.8 with acetic acid N or HCl 0.1 N and the temperature maintained between 0 and 4 * C.
  • the ionic strength is very low, of the order of 0.02.
  • a precipitate is thus obtained which contains the aggregates which are then separated from the solution by simple decantation, by filtration, or by centrifugation. The precipitate is removed.
  • the gamma globulins then precipitate and the precipitate thus formed is separated from the liquid phase by decantation or centrifugation.
  • This precipitate contains gamma globulins devoid of high molecular weight aggregates, but which may still contain a level close to 10% of dimerized proteins.
  • the electrophoretic purity of this product is greater than or equal to 90 II may contain albumin.
  • the anti-complementary activity is very weak. The level of kallikrein and of the prekallikrein activator is reduced, but these can still exist at variable rates, depending on the content of the starting material used.
  • pepsin in solution, in insolubilized form on conventional supports. Incubation is carried out for 10 to 96 hours, at this temperature, depending on the quantity of enzymes used. After treatment, the pH is neutralized.
  • An ultrafiltration is carried out so as to bring the concentration of the gamma globulin solution to 70 g / l.
  • This ultrafiltration step is followed by a diafiltration intended for the removal of PEG.
  • This diafiltration is carried out with a NaCl solution 4.5 g / 1.
  • the volume of diafiltration solution to be used depends on the level of PEG to be removed. -Generally, a volume corresponding to 8 times that of the IgG solution makes it possible to remove more than 90% of the PEG initially contained in the solution and thus go from a rate of 1.5 g / 1 to 0.10 g / 1.
  • the solution obtained is then adjusted to 50 g / l of protein, 50 g / l of sucrose, 4.5 g / l of sodium chloride, at pH 7.0.
  • the substance is then divided into bottles of 0.5 - 2.5 or 5 g of gamma globulin, then lyophilization is carried out.
  • the starting raw material consists in dissolving the precipitate and in clarifying this solution which contains from 1 to 12% of proteins and, moreover, preferably 6% to 10% of proteins.
  • Sparse enzyme treatment Weak treatment with human fibrinolysin is performed.
  • the concentration of human fibrinolysin is 0.5 to 4 caseinolytic units per gram of protein.
  • the incubation temperature is from 0 ° to 37 ° C, preferably 4 ° C.
  • the pH is 6.0 to 8.0, preferably 7.4.
  • the incubation time is dependent on the temperature and the quantity of enzyme used: from 2 days to 10 days.
  • the protein solution which generally contains 1 to 4% protein, is precipitated by adding polyethylene glycol (PEG), of molecular weight 4,000, so as to obtain a concentration 5% PEG.
  • PEG polyethylene glycol
  • the pH is adjusted to 5.8 with acetic acid N or HCl 0.1 N and the temperature maintained between 0 and 4 ° C.
  • the ionic strength is very low, of the order of 0.02.
  • An ultrafiltration is carried out so as to bring the concentration of the gamma globulin solution to 70 g / l.
  • This ultrafiltration step is followed by a diafiltration intended for the removal of PEG.
  • This diafiltration is carried out with a NaCl solution 4.5 g / 1.
  • the volume of diafiltration solution to be used depends on the level of PEG to be removed. Generally, a volume corresponding to 8 times that of the IgG solution makes it possible to eliminate more than 90% of the PEG initially contained in the solution and thus to go from a rate of 1.5 g / 1 to 0.10 g / 1.
  • the solution obtained is then adjusted to 50 g / l of protein, 50 g / l of sucrose, 4.5 g / l of sodium chloride, at pH 7.0.
  • the substance is then distributed in vials of 0.5 - 2.5 or 5 g of gamma globulin, then lyophilization is carried out.
  • Residual PEG level reduced by more than 10 times compared to the original content.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicinal Preparation (AREA)

Abstract

Une solution de gamma-globulines, provenant par exemple du fractionnement de Cohn, est soumise à une étape de fractionnement au polyéthylène-glycol, à une étape de traitement ménagé enzymatique à la pepsine, fibrinolysine ou papaïne, et à une étape d'élimination du PEG.A solution of gamma-globulins, resulting for example from Cohn fractionation, is subjected to a fractionation step with polyethylene glycol, to a step of enzymatic treatment with pepsin, fibrinolysin or papain, and to a step of elimination of the PEG.

Description

Procédé de préparation de gamma-globulines administrées par voie intraveineuse et gamma-globulines obtenues. Process for the preparation of gamma globulin administered intravenously and gamma globulin obtained.
La présente invention a trait à un procédé de préparation de gamma-globulines administrables par voie intraveineuse ainsi qu'aux gamma-globulines obtenues par ce procédé.The present invention relates to a process for the preparation of gamma globulin which can be administered intravenously as well as to the gamma globulin obtained by this process.
Il existe actuellement sur le marché un cer¬ tain nombre de préparations de gamma-globulines obtenues par des procédés différents et qui présentent à la fois des qualités et des inconvénients liés à ces procédés. Parmi les inconvénients que ces procédés s'efforcent d'atténuer le plus possible, se trouve en bonne place le pouvoir d'activation du complément qui peut entraîner des réactions allant jusqu'au choc anaphylactique. D'une façon générale, ce pouvoir' anti-complémentaire est attribué à l'existence d'agrégats globuliniques qui seraient inhérents aux différents procédés de préparation et notamment aux étapes de fractionnement alcoolique.There are currently on the market a certain number of gamma globulin preparations obtained by different processes and which have both the qualities and the drawbacks associated with these processes. Among the drawbacks that these methods strive to mitigate as much as possible, there is in good place the power of activation of the complement which can cause reactions going as far as anaphylactic shock. In general, this anti-complementary power is attributed to the existence of globulin aggregates which would be inherent in the various methods of preparation and especially in alcoholic fractionation stages.
C'est pourquoi, il a été déjà développé différents procédés de préparation cherchant à supprimer, ou du moins à limiter cet inconvénient. Un premier procédé cherche à éliminer les agrégats et consiste à effectuer une digestion à la pepsine de la préparation de gamma-globulines, de façon à provoquer une rupture de chaînes lourdes à proximité de la jonction des fractions Fab et Fc. Voir, par exemple, le brevet FR-A- 2.433.342.This is why various preparation methods have already been developed seeking to eliminate, or at least limit this drawback. A first method seeks to eliminate the aggregates and consists in carrying out a pepsin digestion of the preparation of gamma globulins, so as to cause a break in heavy chains near the junction of the Fab and Fc fractions. See, for example, patent FR-A-2,433,342.
Un autre procédé, qui avait été mis au point par la déposante, consiste à traiter une préparation de gamma-globulines à la plasmine, de façon à provoquer une digestion ménagée conservant un pourcentage important (de 30 à 50 ) de gamma-globuline intacte et donnant des fragments Fab et Fc. Cette préparation est active et très bien tolérée.Another method, which had been developed by the applicant, consists in treating a preparation of gamma-globulins with plasmin, so as to cause controlled digestion retaining a large percentage (from 30 to 50) of intact gamma-globulin and giving Fab and Fc fragments. This preparation is active and very well tolerated.
Ces différents procédés qui cherchent à réduire le pouvoir anti-complémentaire présentent cependant l'inconvénient d'une digestion plus ou moins poussée des gamma-globulines et consistent finalement à rechercher un compromis entre l'innocuité et l'activité de la préparation.These various methods which seek to reduce the anti-complementary power, however, have the drawback of more or less extensive digestion of gamma globulins and ultimately consist in seeking a compromise between the safety and the activity of the preparation.
On peut également citer divers procédés de préparation formant des gamma-globulines modifiées chimique- ment : alkylation, réduction, sulfonation.Mention may also be made of various preparation processes forming chemically modified gamma globulins: alkylation, reduction, sulfonation.
Le brevet FR-A-2.301.266 préconise de ne pas effectuer de traitement enzymatique, notamment à la pepsine, des gamma-globulines et d'effectuer, à la place, un frac¬ tionnement au polyéthylène-glycol. Ainsi, il a été mis au point un procédé de préparation de gamma-globulines par fractionnement au polyéthylène-glycol (P.E.G.) qui présente l'avantage d'empêcher la formation des agrrégats ou de les éliminer, tout en conservant des globulines intactes. En outre, bien qu'à des degrés divers, les différentes préparations décrites ci-dessus peuvent présenter des facteurs de choc par activation du système kallicréine-bradykinine, ces facteurs paraissent liés à une teneur résiduelle en activateur de prékallicréine.The patent FR-A-2,301,266 recommends not to carry out an enzymatic treatment, in particular with pepsin, of gamma-globulins and to carry out, instead, a fractionation with polyethylene glycol. Thus, a process for the preparation of gamma globulins by fractionation with polyethylene glycol (PEG) has been developed which has the advantage of preventing the formation of aggregates or of eliminating them, while retaining intact globulins. In addition, although to varying degrees, the different preparations described above can present shock factors by activation of the kallikrein-bradykinin system, these factors appear to be linked to a residual content of prekallikrein activator.
Une autre technique, qui permet d'obtenir des gamma-globulines de bonne qualité, consiste à effectuer un traitement à pH acide, en présence de faibles quantités de pepsine. Voir, par exemple, J.J. Walsh : Purification of normal immunoglobulin for intravenous use. DEVELOP. BIOL. STAND. 1974, 27, 31-6. Cependant, il reste encore des agrégats et des dimères à un taux relativement important.Another technique, which makes it possible to obtain good quality gamma globulins, consists in carrying out a treatment at acid pH, in the presence of small amounts of pepsin. See, for example, J.J. Walsh: Purification of normal immunoglobulin for intravenous use. DEVELOP. BIOL. STAND. 1974, 27, 31-6. However, there are still aggregates and dimers at a relatively large rate.
On a également proposé, dans la demande de brevet EP-A-0 120 385, d'éviter d'utiliser des traitements à la pepsine ou à la plasmine, que ces enzymes soient insolubilisées ou non, pour proposer une digestion aux enzymes pancréatiques accompagnée d'un traitement au polyéthylène glycol à concentration relativement élevée. Cependant, cette gamma-globuline n'est pas équilibrée en sous-classes .It has also been proposed, in patent application EP-A-0 120 385, to avoid using pepsin or plasmin treatments, whether or not these enzymes are insolubilized, to propose digestion with pancreatic enzymes accompanied treatment with relatively high concentration polyethylene glycol. However, this gamma globulin is not balanced in subclasses.
La présente invention se propose de fournir des préparations de gamma-globulines administrables par voie intraveineuse, à la fois dépourvues d'agrégats et de dimères, dépourvues de pouvoir anti-complémentaire et dépourvues de kallicréine et d ' activateur de prékallicréine et avec un profil des sous-classes comparable à celui du Sérum Humain normal.The present invention proposes to provide preparations of gamma globulin which can be administered intravenously, both without aggregates and dimers, without anti-complementary power and without kallikrein and prekallikrein activator and with a profile of subclasses comparable to that of normal Human Serum.
Un autre objectif de l'invention est de four¬ nir un tel procédé qui, appliqué aux préparations de gamma¬ globulines préparées par un fractionnement avec de faibles teneurs en PEG ou substances analogues, améliore ces préparations en diminuant notamment le pouvoir anti¬ complémentaire, la teneur en kallicréine et en activateur de la prékallicréine, ainsi que le PEG résiduel.Another objective of the invention is to provide such a process which, applied to gamma¬ globulin preparations prepared by fractionation with low levels of PEG or similar substances, improves these preparations by notably reducing the anti-complementary power, the content of kallikrein and activator of prekallikrein, as well as the residual PEG.
L'invention a pour objet un procédé de préparation de gamma-globulines administrables par voie intraveineuse, caractérisé en ce qu'il comporte une étape de fractionnement au polyéthylène-glycol (PEG) ou substances similaires et une étape de traitement enzymatique ménagé, dans lequel le pH est dépendant de la nature de 1'enzyme utilisée, celle-ci étant ajoutée de préférence sous forme de traces, le traitement étant conduit pour éviter une protéolyse sensible.The subject of the invention is a process for the preparation of gamma-globulins which can be administered intravenously, characterized in that it comprises a fractionation step with polyethylene glycol (PEG) or substances similar and a mild enzymatic treatment step, in which the pH is dependent on the nature of the enzyme used, this being preferably added in the form of traces, the treatment being carried out to avoid sensitive proteolysis.
L'enzyme utilisée est choisie dans le groupe formé par les pepsines, fibrinolysines (plasmine) , et papaines .The enzyme used is chosen from the group formed by pepsins, fibrinolysins (plasmin), and papains.
Le matériau de départ est une fraction d'origine sérique riche en gamma-globulines telle que la fraction II Technique 6.9 de Cohn, connue pour donner des produits exempts d'hépatite, ou une fraction d'origine pla¬ centaire telle que la fraction technique 6.9 de Cohn modifiée par Taylor. Cette fraction a une pureté en IgG supérieure ou égale à 90 % . Elle peut contenir des quantités variables en albumine qui peuvent aller jusqu'à 10 % . 1er exemple :The starting material is a fraction of serum origin rich in gamma-globulins such as fraction II Technique 6.9 of Cohn, known to give products free of hepatitis, or a fraction of pla¬ centary origin such as the technical fraction 6.9 of Cohn modified by Taylor. This fraction has an IgG purity greater than or equal to 90%. It may contain varying amounts of albumin which can be up to 10%. 1st example:
1 - Précipitation au PEG :1 - Precipitation at PEG:
La matière première de départ consiste en une mise en solution du précipité et en une clarification de cette solution, solution qui contient en général de 1 à 4 % de protéines . On procède à une étape de précipitation par adjonction de polyéthylène-glycol (PEG), de poids moléculaire 4 000, de façon à obtenir une concentration en PEG de 5 . . Le pH est ajusté 5,8 par de l'acide acétique N ou HC1 0,1 N et la température maintenue entre 0 et 4*C. La force ionique est très basse, de l'ordre de 0,02.The starting raw material consists in dissolving the precipitate and in clarifying this solution, a solution which generally contains from 1 to 4% of proteins. A precipitation step is carried out by adding polyethylene glycol (PEG), of molecular weight 4,000, so as to obtain a PEG concentration of 5. . The pH is adjusted 5.8 with acetic acid N or HCl 0.1 N and the temperature maintained between 0 and 4 * C. The ionic strength is very low, of the order of 0.02.
On obtient ainsi un précipité qui contient les agrégats que l'on sépare alors de la solution par simple décantation, par filtration, ou par centrifugation. Le précipité est éliminé.A precipitate is thus obtained which contains the aggregates which are then separated from the solution by simple decantation, by filtration, or by centrifugation. The precipitate is removed.
Sur le surnageant qui en résulte, on effectue ensuite une nouvelle précipitation, sans nouvelle addition de PEG, à la même température, mais en augmentant la force ionique à 0,05 par addition de NaCl de 0,05 % à 0,2 % et de préférence 0,1 % et en portant le pH de 7 à 8,5 et de préférence 8,0.On the resulting supernatant, a further precipitation is then carried out, without further addition of PEG, at the same temperature, but by increasing the ionic strength to 0.05 by adding NaCl from 0.05% to 0.2% and preferably 0.1% and bringing the pH from 7 to 8.5 and preferably 8.0.
Les gamma-globulines précipitent alors et l'on sépare de la phase liquide par décantation ou centrifu- gation le précipité ainsi formé. Ce précipité contient les gamma-globulines dépurvues d'agrégats de haut poids moléculaire, mais pouvant encore contenir un taux voisin de 10 % de protéines dimérisées. La pureté électrophorétique de ce produit est supérieure ou égale à 90 II peut contenir de l'albumine. L'activité anti-complémentaire est très faible. Le taux de kallicréine et de 1 'activateur de prékallicréine est réduit, mais celles-ci peuvent encore e ister à des taux variables, suivant la teneur de la matière de départ utilisée.The gamma globulins then precipitate and the precipitate thus formed is separated from the liquid phase by decantation or centrifugation. This precipitate contains gamma globulins devoid of high molecular weight aggregates, but which may still contain a level close to 10% of dimerized proteins. The electrophoretic purity of this product is greater than or equal to 90 II may contain albumin. The anti-complementary activity is very weak. The level of kallikrein and of the prekallikrein activator is reduced, but these can still exist at variable rates, depending on the content of the starting material used.
2 - Traitement enzymatique ménagé : Le précipité de gamma-globuline est repris dans de l'eau apyrogene de façon à obtenir une concentration de 20 g/1 en gamma-globulines. On ajoute 50 g/1 de sac¬ charose et 0,002 g/1 de pepsine, la température étant amenée et maintenue à 37'C, le pH étant réglé à 4,1 et on incube pendant 24 heures.2 - Sparse enzymatic treatment: The precipitate of gamma-globulin is taken up in pyrogen-free water so as to obtain a concentration of 20 g / 1 in gamma-globulins. 50 g / l of sacose and 0.002 g / l of pepsin are added, the temperature being brought to and maintained at 37 ° C., the pH is adjusted to 4.1 and incubation is carried out for 24 hours.
On préfère utiliser la pepsine en solution, sous forme insolubilisée sur des supports classiques. L'incubation s'effectue pendant 10 à 96 heures, à cette température, suivant la quantité d'enzymes utilisée. Après traitement, le pH est neutralisé.It is preferred to use pepsin in solution, in insolubilized form on conventional supports. Incubation is carried out for 10 to 96 hours, at this temperature, depending on the quantity of enzymes used. After treatment, the pH is neutralized.
3 - Traitement ultérieur :3 - Subsequent processing:
On effectue une ultrafiltration, de façon à porter la concentration de la solution de gamma-globulines à 70 g/1. Cette étape d'ultrafiltration est suivie d'une diafiltration destinée à l'élimination du PEG. Cette diafiltration s'effectue par une solution de NaCl 4,5 g/1. Le volume de solution de diafiltration à utiliser est fonc¬ tion du taux de PEG à éliminer. -Généralement, un volume correspondant à 8 fois celui de- la solution d'IgG permet d'éliminer plus de 90 % du PEG contenu initialement dans la solution et de passer ainsi d'un taux de 1,5 g/1 à 0,10 g/1.An ultrafiltration is carried out so as to bring the concentration of the gamma globulin solution to 70 g / l. This ultrafiltration step is followed by a diafiltration intended for the removal of PEG. This diafiltration is carried out with a NaCl solution 4.5 g / 1. The volume of diafiltration solution to be used depends on the level of PEG to be removed. -Generally, a volume corresponding to 8 times that of the IgG solution makes it possible to remove more than 90% of the PEG initially contained in the solution and thus go from a rate of 1.5 g / 1 to 0.10 g / 1.
On effectue alors l'ajustement de la solution obtenue à 50 g/1 de protéine, 50 g/1 de saccharose, 4,5 g/1 de chlorure de sodium, à pH 7,0. On répartit ensuite la substance en flacons de 0,5 - 2,5 ou 5 g de gamma-globulines, puis on effectue une lyophilisation. 2ème exemple :The solution obtained is then adjusted to 50 g / l of protein, 50 g / l of sucrose, 4.5 g / l of sodium chloride, at pH 7.0. The substance is then divided into bottles of 0.5 - 2.5 or 5 g of gamma globulin, then lyophilization is carried out. 2nd example:
La matière première de départ consiste en une mise en solution du précipité et en une clarification de cette solution qui contient de 1 à 12 % de protéines et, de plus, préférentiellement 6 % à 10 % de protéines.The starting raw material consists in dissolving the precipitate and in clarifying this solution which contains from 1 to 12% of proteins and, moreover, preferably 6% to 10% of proteins.
Traitement enzymatique ménagé : On procède à un faible traitement par de la fibrinolysine humaine. La concentration en fibrinolysine humaine est de 0,5 à 4 unités caséinolytiques par gramme de protéines .Sparse enzyme treatment: Weak treatment with human fibrinolysin is performed. The concentration of human fibrinolysin is 0.5 to 4 caseinolytic units per gram of protein.
La température d'incubation est de 0* à 37*C, de préférence 4*C. Le pH est de 6,0 à 8,0, de préférence 7,4.The incubation temperature is from 0 ° to 37 ° C, preferably 4 ° C. The pH is 6.0 to 8.0, preferably 7.4.
Le temps d'incubation est dépendant de la température et de la quantité d'enzyme mise : de 2 jours à 10 jours.The incubation time is dependent on the temperature and the quantity of enzyme used: from 2 days to 10 days.
2 - Précipitation au PEG : On procède à une étape de précipitation de la solution protéinique qui contient en général de 1 à 4 % de protéines par adjonction de polyéthylène glycol (PEG), de poids moléculaire 4 000, de façon à obtenir une concentra¬ tion en PEG de 5 % . Le pH est ajusté à 5,8 par de l'acide acétique N ou HCl 0,1 N et la température maintenue entre 0 et 4*C. La force ionique est très basse, de l'ordre de 0,02.2 - Precipitation with PEG: The protein solution, which generally contains 1 to 4% protein, is precipitated by adding polyethylene glycol (PEG), of molecular weight 4,000, so as to obtain a concentration 5% PEG. The pH is adjusted to 5.8 with acetic acid N or HCl 0.1 N and the temperature maintained between 0 and 4 ° C. The ionic strength is very low, of the order of 0.02.
On obtient ainsi un précipité qui contient des agrégats que l'on sépare alors de la solution par simple décantation, par filtration, ou• par centrifugation. Le précipité est éliminé.This produces a precipitate which then contains aggregate which is separated from the solution by simple decantation, filtration, or • by centrifugation. The precipitate is removed.
Sur le surnageant qui en résulte, on effectue ensuite une nouvelle précipitation, sans nouvelle addition de PEG, à la même température, mais en augmentant la force ionique à 0,05 par addition de NaCl de 0,05 % à 0,2 % et en portant le pH de 7 à 8,5 de préférence à 8,0. - Les gamma-globulines précipitent alors et l'on sépare de la phase liquide le précipité ainsi formé, par décantation ou centrifugation.On the resulting supernatant, then a new precipitation, without further addition of PEG, at the same temperature, but increasing the ionic strength to 0.05 by adding NaCl from 0.05% to 0.2% and bringing the pH from 7 to 8, 5 preferably 8.0. - The gamma globulins then precipitate and the precipitate thus formed is separated from the liquid phase, by decantation or centrifugation.
3 - Traitement ultérieur :3 - Subsequent processing:
On effectue une ultrafiltration de façon à porter la concentration de la solution de gamma-globulines à 70 g/1.An ultrafiltration is carried out so as to bring the concentration of the gamma globulin solution to 70 g / l.
Cette étape d' ultrafiltration est suivie d'une diafiltration destinée à l'élimination du PEG. Cette diafiltration s'effectue par une solution de NaCl 4,5 g/1. Le volume de solution de diafiltration à utiliser est fonc¬ tion du taux de PEG à éliminer. Généralement, un volume correspondant à 8 fois celui de la solution d'IgG permet d'éliminer plus de 90 % du PEG contenu initialement dans la solution et de passer ainsi d'un taux de 1,5 g/1 à 0,10 g/1. On effectue alors l'ajustement de la solution obtenue à 50 g/1 de protéines, 50 g/1 de saccharose, 4,5 g/1 de chlorure de sodium, à pH 7,0.This ultrafiltration step is followed by a diafiltration intended for the removal of PEG. This diafiltration is carried out with a NaCl solution 4.5 g / 1. The volume of diafiltration solution to be used depends on the level of PEG to be removed. Generally, a volume corresponding to 8 times that of the IgG solution makes it possible to eliminate more than 90% of the PEG initially contained in the solution and thus to go from a rate of 1.5 g / 1 to 0.10 g / 1. The solution obtained is then adjusted to 50 g / l of protein, 50 g / l of sucrose, 4.5 g / l of sodium chloride, at pH 7.0.
On répartit alors ensuite la substance en flacons de 0,5 - 2,5 ou 5 g de gamma-globulines, puis on effectue une lyophilisation.The substance is then distributed in vials of 0.5 - 2.5 or 5 g of gamma globulin, then lyophilization is carried out.
L'analyse des gamma-globulines obtenues selon 1 ' invention montre les avantages suivants :Analysis of the gamma globulins obtained according to the invention shows the following advantages:
. Diminution du pouvoir anti-complémentaire dans les différentes techniques de détermination utilisées. . Les sous-classes sont équilibrées.. Reduction of the anti-complementary power in the different determination techniques used. . The subclasses are balanced.
. Absence de facteurs hypotenseurs démontrée par test tensionnel sur chien et sur rat.. Absence of hypotensive factors demonstrated by blood pressure test on dogs and rats.
. Diminution du taux de protéines dimérisées de plus de 50 % par rapport à la' teneur initiale et obten- tion d'un taux de monomères très élevé (supérieur à 90 % ) .. Reduction of the level of dimerized proteins by more than 50% compared to the initial content and obtaining a very high level of monomers (greater than 90%).
. Taux de fragments inférieurs à 7 S nul ou 8 très faible.. Fragment rate less than 7 S zero or 8 very low.
. Taux de kallicréine et d'activateur de la prékallicréine nuls (inférieurs à la limite de sensibilité du test) .. Kallikrein and prekallikrein activator levels zero (below the sensitivity limit of the test).
. Taux de PEG résiduel diminué de plus de 10 fois par rapport à la teneur d'origine.. Residual PEG level reduced by more than 10 times compared to the original content.
Bien que l'invention ait été décrite à propos de formes de réalisation particulières, il est bien entendu qu'elle n'y est nullement limitée et qu'on peut lui apporter diverses modifications sans pour cela sortir ni de son cadre ni de son esprit. Although the invention has been described with regard to particular embodiments, it is understood that it is in no way limited thereto and that it can be made various modifications without departing from its scope or its spirit. .

Claims

REVENDICATIONS. 1 - Procédé de préparation de gamma¬ globulines administrables par voie intraveineuse, caractérisé en ce qu'il comporte une étape de fractionnement au polyéthylène-glycol (PEG) ou substance similaire et une étape de traitement enzymatique ménagé évitant une protéolyse sensible à l'aide d'une enzyme choisie dans le groupe formé par les pepsines, les fibrinolysines et les papaïnes . 2 - Procédé selon la revendication 1, caractérisé en ce que 1 ' étape de traitement enzymatique est effectuée en présence de traces de pepsine et à pH acide.CLAIMS. 1 - Process for the preparation of gamma¬ globulins which can be administered intravenously, characterized in that it comprises a step of fractionation with polyethylene glycol (PEG) or similar substance and a step of mild enzymatic treatment avoiding sensitive proteolysis using an enzyme chosen from the group formed by pepsins, fibrinolysins and papains. 2 - Process according to claim 1, characterized in that the enzymatic treatment step is carried out in the presence of traces of pepsin and at acidic pH.
3 - Procédé selon la revendication 1 , caractérisé en ce que l'étape de traitement enzymatique est effectuée en présence de fibrinolysine humaine à pH neutre.3 - Process according to claim 1, characterized in that the enzymatic treatment step is carried out in the presence of human fibrinolysin at neutral pH.
4 - Procédé selon l'une quelconque des reven¬ dications 1 à 3, caractérisé en ce que l'étape de frac¬ tionnement s'effectue en présence d'environ 5% de PEG à pH 5 à 6, notamment 5,8, et température basse, notamment +2* à +4*C à force ionique très basse, de l'ordre de 0,02, suivie d'une seconde précipitation à force ionique plus élevée de l'ordre de 0,05 et à pH de l'ordre de 7 à 8, 5 et notamment pH 8,0.4 - Process according to any one of claims 1 to 3, characterized in that the fractionation step is carried out in the presence of approximately 5% of PEG at pH 5 to 6, in particular 5.8, and low temperature, in particular +2 * to +4 * C at very low ionic strength, of the order of 0.02, followed by a second precipitation with higher ionic strength of the order of 0.05 and at pH on the order of 7 to 8.5, and in particular pH 8.0.
5 - Procédé selon l'une quelconque des reven- dications 1 à 4, caractérisé en ce que l'étape de frac¬ tionnement s'effectue en présence d'albumine.5 - Method according to any one of claims 1 to 4, characterized in that the fractionation step is carried out in the presence of albumin.
6 - Procédé selon l'une quelconque des reven¬ dications 2, 4 et 5, caractérisé en ce que le traitement s'effectue avec la pepsine à raison de l'ordre de 0,002 g par litre de pepsine pour une solution de 20 g par litre de gamma-globuline en présence de saccharose et à pH acide supérieur ou égal à 4.6 - Method according to any one of reven¬ dications 2, 4 and 5, characterized in that the treatment is carried out with pepsin at a rate of the order of 0.002 g per liter of pepsin for a solution of 20 g by liter of gamma globulin in the presence of sucrose and at an acid pH greater than or equal to 4.
7 - Procédé selon l'une quelconque des reven¬ dications 2 et 4 à 6, caractérisé en ce que l'on utilise une pepsine sous forme insolubilisée.7 - Method according to any one of reven¬ dications 2 and 4 to 6, characterized in that one uses a pepsin in insolubilized form.
8 - Procédé selon l'une quelconque des revendications 3 à 7, caractérisé en ce que le traitement s'effectue avec la fibrinolysine humaine à raison de 0,5 à 4 CU/g de protéines et à un pH neutre.8 - Method according to any one of Claims 3 to 7, characterized in that the treatment is carried out with human fibrinolysin in an amount of 0.5 to 4 CU / g of protein and at a neutral pH.
9 - Procédé selon l'une quelconque des reven- dications 6 à 8, caractérisé en ce que l'incubation dure, suivant le taux d'enzyme, de 10 heures à 10 jours.9 - Process according to any one of claims 6 to 8, characterized in that the incubation lasts, depending on the level of enzyme, from 10 hours to 10 days.
10 - Procédé selon l'une quelconque des revendications 1 à 9, caractérisé en ce que l'on procède ensuite à une ultrafiltration pour concentrer les protéines, puis à une diafiltration pour l'élimination du PEG.10 - Process according to any one of claims 1 to 9, characterized in that one then proceeds to an ultrafiltration to concentrate the proteins, then to a diafiltration for the elimination of PEG.
11 - Préparation de gamma-globuline administrable par voie intraveineuse obtenue par le procédé selon l'une quelconque des revendications 1 à 10. 11 - Preparation of gamma globulin administered intravenously obtained by the method according to any one of claims 1 to 10.
EP86903414A 1985-05-30 1986-05-30 Process for preparing gamma-globulins supplied by intravenous administration and gamma-globulins obtained Ceased EP0224532A1 (en)

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FR8508094 1985-05-30
FR8508094A FR2582515B1 (en) 1985-05-30 1985-05-30 PROCESS FOR THE PREPARATION OF GAMMA-GOBULINS ADMINISTRABLE BY THE INTRAVENOUS ROUTE AND GAMMA-GLOBULINS OBTAINED

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ES (1) ES8705230A1 (en)
FR (1) FR2582515B1 (en)
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DE3644805A1 (en) * 1986-12-31 1988-07-14 Elkapharm Ag Bovine blood oligopeptides
US5328834A (en) * 1989-09-08 1994-07-12 Unisyn Technologies, Inc. Method for preparing immunoglobulin fragments
US5169258A (en) * 1991-01-31 1992-12-08 Raynak Larry J Pipe connectors for structure fabrication
US5219578A (en) * 1991-02-25 1993-06-15 Innovet, Inc. Composition and method for immunostimulation in mammals
US5525519A (en) * 1992-01-07 1996-06-11 Middlesex Sciences, Inc. Method for isolating biomolecules from a biological sample with linear polymers
FI952196A0 (en) * 1995-05-08 1995-05-08 Suomen Punainen Risti Veripalv Immunoglobulin production
US6124437A (en) * 1997-03-19 2000-09-26 Welfide Corporation Immunoglobulin preparation and preparation process thereof
CN1149100C (en) 1997-10-23 2004-05-12 三菱制药株式会社 Room temperature storable immunoglobulin preparation for intravenous injection
UA64742C2 (en) * 1997-12-24 2004-03-15 Альфа Терапевтик Корпорейшн Process for producing intravenously-administrable gamma globulin solution and product manufactured by this process
US6441144B1 (en) 1999-05-20 2002-08-27 Alpha Therapeutic Corporation Method for repairing dual virally inactivated immune globulin for intravenous administration
KR20020010921A (en) * 1999-06-15 2002-02-06 콜튼 에드워드 에이. Manufacturing method for intravenous immune globulin and resultant product
EP2121057A4 (en) * 2007-02-06 2012-10-10 Incept Llc Polymerization with precipitation of proteins for elution in physiological solution

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CA1064396A (en) * 1975-02-18 1979-10-16 Myer L. Coval Fractional precipitation of gamma globulin with polyethylene glycol
DE2835843A1 (en) * 1978-08-16 1980-02-28 Blutspendedienst Dt Rote Kreuz METHOD FOR PRODUCING A GAMMAGLOBULIN SOLUTION SUITABLE FOR INTRAVENOUS APPLICATION
JPS567721A (en) * 1979-07-02 1981-01-27 Mochida Pharmaceut Co Ltd Preparation of immunoglobulin for intravenous injection
AT383739B (en) * 1983-03-16 1987-08-10 Immuno Ag METHOD FOR INACTIVATING SUBSTANCES CAUSING INCOMPATIBILITY REACTIONS IN IMMUNALLOBULIN BLOOD FRACTIONS

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ES555452A0 (en) 1987-05-01
AU5954286A (en) 1986-12-24
IL78959A (en) 1991-06-30
US4874708A (en) 1989-10-17
DK46587D0 (en) 1987-01-29
FR2582515B1 (en) 1988-11-04
DK46587A (en) 1987-01-29
WO1986006963A1 (en) 1986-12-04
IL78959A0 (en) 1986-09-30
GR861380B (en) 1986-08-28
ES8705230A1 (en) 1987-05-01
AU586869B2 (en) 1989-07-27
FR2582515A1 (en) 1986-12-05
ZA864074B (en) 1987-01-28
JPS62503036A (en) 1987-12-03

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