Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1996-03-25
2000-05-30
Hutzell, Paula K.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530417, 5303871, 435 4, C07K 116
Patent
active
060692367
DESCRIPTION:
BRIEF SUMMARY
The invention relates to an immunoglobulin G concentrate suitable for therapeutic use and to the process for producing said concentrate.
Several polyvalent immunoglobulin preparations have been frequently used. In general, they are prepared from a pool of serum from 2000 to 5000 donors which ensures the presence of all the antibodies normally present in the global population of a chosen region.
Those immunoglobulin preparations are produced according to the conventional Cohn method more or less modified, thus by ethanol precipitation. This process has a major disadvantage due to the ethanol treatment which causes some protein denaturation and the formation of immunoglobulin aggregates. These aggregates induce therapeutic adverse reactions due to the activation of the complement system and to anaphylactic reactions. Those preparations are thus unsuitable for intraveinous injections and are only acceptable for intramuscular injection which restricts the amount that can be injected and thus its efficiency.
Several treatments are already performed to solve this problem: pepsin or plasmin cleavage of the immunoglobulins, treatment with .beta.-propiolactone or reducing and alkylating agents, treatment at pH 4, treatment with PEG to cause aggregate precipitation.
The Applicant has preferably avoided those enzymatic or chemical treatments and has deleted the ethanol precipitation step. He has thus set up a process which included serial chromatographic separations and does not include any precipitation step.
An immunoglobulin concentrate has already been prepared by Friesen et al. (Friesen--Joint IABS/CSL Symp. on Standardization in Blood Fractionation--Australia 1986--Develop. biol. Standard. 67, 1987, 3-13; Immunoglobulins--Pub. Central Lab. Netherlands Red Cross 1988--Ed. Krynen, Strengers, Van Aken) using one or two ion exchange chromatographic steps: of specific .gamma.-globulins from hyperimmune serums but it is only applicable to small amounts. chromatography allows processing of larger amounts and is suitable for polyvalent immunoglobulins preparation but it has not be found profitable at an industrial scale.
Immunoglobulin purification involves peculiar problems, in addition to the aforementioned, because their biological activity is linked to their structural integrity, but said activity is not easily measured, as would be the case with an enzymatic activity. As an example, for a given specific antibody, various concentrates showing identical ELISA antibody titres show various degrees of viral infectivity neutralizing capacity, according to their process of preparation.
The Applicant has thus tried to develop a non-denaturing process for immunoglobulin purification, which can be performed on an industrial scale (for example with more than 5000 liters batches of serum) and, moreover, which is compatible with the collection of other proteins of therapeutic value.
The process for producing an immunoglobulin concentrate according to the present invention does not include any ethanol precipitation step and includes a series of chromatographic steps during which the immunoglobulins always remain in liquid phase and at a pH between 5.5 and 7.8. The process also includes a viral inactivation treatment, like solvent-detergent treatment for example.
The process according to the present invention is suited for total plasma or, preferably, for cryosupernatant.
It can be used for large volumes of plasma pools for polyvalent immunoglobulin preparations as well as for smaller volumes of hyperimmune plasmas for specific immunoglobulin preparations.
The plasma or plasma fraction starting material is preferably subjected to a prepurification step, before implementing the claimed process. This prepurification is performed by filtration on a series of cartridges with a porosity of 0.5 to 0.2.mu., made of cellulose and perlites, negatively charged, and of a small amount of positively charged resin (ZETA PLUS.RTM. filters, Cuno, USA). These filters, due to their negative charges, allow Factor IX adsorption which is thus elimin
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patent: 4877866 (1989-10-01), Rudnick et al.
patent: 4880913 (1989-11-01), Doleschel et al.
Hooper et al., Central Lab. of the Netherlands Red Cross, Immunoglobulin Manufacturing Procedures, pp. 361-380 (1988).
Friesen et al., The Winnipeg Rh Institute Inc., Univ. of Manitoba, Column Ion Exchange Chromatographic Production of Human Immune Globulins and Albumin, pp. 118-126 (1982).
Friesen, Joint IABS/CSL Symp. on Standardizatino in Blood Fractionation, Australia 1986, Develop. biol. Standard. 67, 1987, 3-13.
Burnouf T. Bioseparation, 1:383-396, 1991.
Good, RA. et al. Cancer, 68(6 Suppl.) :1415-21, 1991.
Bonneel Patrick
Burnouf Thierry
Burnouf-Radosevich Miryana
Dernis Dominique
Association pour l'Essor de la Transfusion Sanguine dans la Regi
Davis Minh-Tam
Hutzell Paula K.
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