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)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Enzymes And Modification Thereof (AREA)
• Medicinal Preparation (AREA)

Applications Claiming Priority (2)

Publications (1)

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ID=9319669

Family Applications (1)

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Family Cites Families (4)

• 1985
  • 1985-05-30 FR FR8508094A patent/FR2582515B1/fr not_active Expired
• 1986
  • 1986-05-28 IL IL78959A patent/IL78959A/xx not_active IP Right Cessation
  • 1986-05-28 GR GR861380A patent/GR861380B/el unknown
  • 1986-05-29 CA CA000510329A patent/CA1269629A/fr not_active Expired - Lifetime
  • 1986-05-29 ES ES555452A patent/ES8705230A1/es not_active Expired
  • 1986-05-30 JP JP61503111A patent/JPS62503036A/ja active Pending
  • 1986-05-30 EP EP86903414A patent/EP0224532A1/fr not_active Ceased
  • 1986-05-30 WO PCT/FR1986/000184 patent/WO1986006963A1/fr not_active Application Discontinuation
  • 1986-05-30 US US07/012,662 patent/US4874708A/en not_active Expired - Fee Related
  • 1986-05-30 AU AU59542/86A patent/AU586869B2/en not_active Ceased
  • 1986-05-30 ZA ZA864074A patent/ZA864074B/xx unknown
• 1987
  • 1987-01-29 DK DK046587A patent/DK46587A/da not_active Application Discontinuation

Non-Patent Citations (1)

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