Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1986-03-14
1988-05-24
Schain, Howard E.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
2041826, 424 85, 424 88, 424 92, 424 11, 514 2, 514 21, 530300, 530303, 530350, 530371, 530413, 530417, C07K 114, C07K 312, C07K 314, C12N 1500
Patent
active
047466472
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns the purification of biological material, more particularly the purification of contaminated products (peptides/proteins) which have been produced using recombinant DNA (rDNA) technique.
In the preparation of products (proteins/peptides) by rDNA technique, use is made of microbial cloning hosts, primarily bacteria or yeast.
During the working-up and purification of these rDNA-produced products, one has mainly used permeation gel chromatography utilising e.g. Sephadex.RTM., ion exchange chromatography and/or HPLC with different types of columns, including reverse phase columns. All of these purification methods have aimed at the removal of microbial contaminants originating from the cloning hosts (so-called E. coli peptides, EPC) (cf. "Journalen", Vol. 3, No. 12, 1983, pp. 331-334, published by Kabi-Vitrum Sverige AB). These methods have up to now been considered efficient enough to remove the above-mentioned contaminating host contaminants. Checking the purity of such products has most often been performed by different types of polyacrylamide gel electrophoresis techniques including the use of dissociating agents such as SDS etc. Also, different types of immuno-assays, such as ELISA and RIA, have been used. Moreover, one has frequently performed pyrogenicity tests and Limulus amoebocyte lysate (LAL) tests.
In spite of the above-mentioned purification methods, the products produced by rDNA technique have still been shown to elicit antibodies directed against these products (proteins/peptides), even though this formation of antibodies has been deemed to be small and, perhaps, medically acceptable. Nevertheless, experts within this field have put forward that caution should be exercised in the registration of these products. ("Lakartidningen", Vol. 81, No. 8, 1984, p. 636 "Proceed slowly in registering biosynthetic drugs!"). However, in those instances where the protein is to be administered over long periods of time, such as in life-long diseases (e.g. diabetes), not even a slight antibody formation is acceptable.
The basis of the present invention is the insight that also contaminants other than proteins/peptides from the microbial cloning host may give rise to immune reactions.
The invention is based upon research results which show that, due to hydrophobic interaction, minor amounts of hydrophobic contaminants from the microbial cloning host may stick to the product (peptide/protein) during the working-up thereof from the rDNA production environment employed. These hydrophobic contaminants which firmly attach to the product will then constitute epitopes and create fusion epitopes which will elicit antibody formation, not only against the contaminant in question, but also against the product produced by rDNA technique.
The purification methods previously employed have been too "mild" to free the product produced by rDNA technique from these hydrophobic contaminants, and the contaminants which most often are smaller than the product (the peptide/protein) have therefore been carried along during the working-up and purification without being detected.
In the method according to the present invention, these hydrophobic contaminants which result in the formation of epitopes and fusion epitopes, are removed by electroseparation, such as electroseparation using gel-forming substances or electrodialysis, whereby the hydrophobic contaminants are separated from the product (the peptide/protein) such that the purified product will not give rise to immune reactions upon administration to mammals, including man.
The invention relates to a method of purifying a contaminated product (protein/peptide) which has been produced by rDNA technique in that the contaminated product is purified from hydrophobic contaminants which are charged and which originate from the microbial cloning host employed, such that the product is subjected to electroseparation to remove the contaminants. In addition, the invention comprises that the hydrophobic contaminants may obtain at least part of their charge by a
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