Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-06-10
2002-08-06
Cochrane Carlson, Karen (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S008100, C424S178100, C424S134100, C530S350000, C530S402000, C530S391700, C435S069100, C435S235100, C435S173300, C435S091500, C435S320100
Reexamination Certificate
active
06429192
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a novel purification process for production of mannan-binding lectin (MBL) (formerly designated mannan binding protein, MBP) preferably from donor plasma, to be used as an MBL medicinal product. The product is to be used for substitution or replacement therapy in patients with inherited or acquired MBL-deficiency associated with functional and/or clinical symptoms, i.e. where it is contemplated that said patients would benefit from the administration of MBL, e.g. for the treatment or prevention of infections.
INTRODUCTION
Innate (also named natural or non-anticipatory) immune functions have recently received an increasing interest as important elements in defence mechanisms against potentially pathogenic microorganisms. Thus, attention has especially been given to a group of lectins, the collecting, which are believed to play an important role in the immediate defence against a wide range of microorganisms. The serum protein MBL is a collectin, i.e. it is built up as oligomeric structures, characterized by calcium-dependent, C-type carbohydrate-recognition domains (CRDs) attached to collagenous rods. The precise oligomerization of circulating MBL remains unclear; however, higher order oligomeric, bouquet-like structures such as hexameric MBL with multiple binding sites appear to be essential for the functional activity of MBL (for recent reviews, see references 1, 2) (a list of references is given at the end of this specification).
The cumulative knowledge about MBL indicates a future role for this protein in interventional therapy against serious infections. MBL is structurally similar to the complement component C1q, an essential component in the activation of the classical pathway of complement. MBL appears to activate the complement system by a mechanism analogous to that of C1q, i.e. via associated serine proteases, termed MASPs (MBL-associated serine proteases). This antibody-independent complement activation has been named the “MB-lectin pathway of complement activation” (3, 4).
MBL binds to carbohydrate structures on surfaces of bacteria, yeast, parasitic protozoa and viruses, and has been found to exhibit antibacterial activity through killing of the bacteria via the terminal, cytolytic pathway of the complement system, or through promotion of phagocytosis by opsonization. The level of MBL in plasma is genetically determined. Each individual has a constitutional MBL level reflected by the genomic structure in the controlling region as well as in the coding region. The concentration of MBL in plasma thus varies from about 10 &mgr;g/ml to less than 10 ng/ml. Infants or adults with deficiency or very low levels of MBL are especially susceptible to infections. Recent information points to a role of MBL deficiency as a susceptibility factor in HIV infection, and also to MBL deficiency being associated with more rapid death following development of AIDS (1). MBL deficiency may also predispose to recurrent spontaneous abortions (5).
Mannan-binding lectin was first isolated from human serum in 1983 (6) by affinity chromatography on mannan-Sepharose (mannan coupled to a Sepharose matrix) in the presence of Ca-ions. Elution of MBL from the affinity column was performed by means of EDTA.
It appears from later publications that MBL has been purified essentially by the same procedure from serum and plasma. The purified MBL preparations recovered from this one-step procedure were heavily contaminated by antibodies with specificity for carbohydrates and serum amyloid p-component (SAP). To obtain MBL of higher purity, further chromatographic steps were included in the purification procedures, such as a Sepharose precolumn to the affinity column, and additional affinity steps using different carbohydrates either coupled to the matrix or added to the elution buffer; other chromatographic principles as ion exchange and gel filtration chromatography were employed as well (7, 8, 9, 10). In general, at least two affinity chromatographic steps have been employed in the procedures for obtaining highly purified MBL. Recently a procedure has been described, where a plasma protein fraction obtained by precipitation of human plasma with 7% PEG was used as the starting material for MBL purification (11). This procedure differed from those previously described in that the affinity chromatography was performed on non-conjugated Sepharose (Sepharose without immobilized carbohydrate-ligands): first the solubilized PEG-precipitate was subjected to batch adsorption on Sepharose, and after elution of MBL by EDTA, a subsequent affinity chromatography step on a Sepharose column was performed, with eluting of MBL by mannose. By this procedure employing two consecutive affinity steps, MBL was obtained at high purity. DNA encoding human mannose binding protein is disclosed in WO98/01519.
DETAILED DISCLOSURE OF THE INVENTION
The present invention relates to a process for purifying mannan-binding lectin (MBL), preferably from a crude plasma protein fraction. The process of the invention will, among other elements, include at least two key elements: performing one affinity chromatography step on a non-conjugated polysaccharide matrix, and performing at least one validated virus-reduction step.
MBL can be purified from a wide range of starting materials containing MBL. In one embodiment, the starting material for the process of the invention is an MBL containing supernatant or a lysed cell suspension from a yeast or mammalian cell culture expressing MBL, said cell culture comprising cells coding for mammalian (e.g. human) MBL and optionally coding for the MBL Associated Serine Proteases (MASPs). The MBL expressing cell culture is grown in a medium providing the cell culture the nutrients needed with or without serum added to the culture medium. In another embodiment, MBL is purified from milk and/or colostrum from a mammal expressing a mammal (e.g. human) MBL gene. In one embodiment, the mammal is a transgenic non-human animal. In a preferred embodiment of the invention, the starting material for the process of the invention is a crude plasma protein fraction obtainable from industrial scale ethanol fractionation procedures, such as Cohn fraction I, II and III; Cohn fraction II and III; or Cohn fraction III. In a preferred embodiment, the plasma protein fraction is Cohn fraction II and III, where filter aid may or may not be present depending on the method employed for isolation of the Cohn fraction, i.e. by filtration or centrifugation. The use of Cohn fraction II and III as starting material has several advantages. These comprise, but are not limited to: no need of further ethanol fractionation, immunoglobulins can be recovered for an immunoglobulin product, and MBL is recovered from a fraction usually discarded.
Each of the starting materials will require a few pre-processing steps to obtain an MBL containing solution. The pre-processing steps will be discussed below.
The first key element of the present process, the affinity chromatography on a non-conjugated polysaccharide matrix, has several advantages. These comprise, but are not limited to: no need for prior protein precipitation, selectivity for functionally active MBL, a high degree of purification, removal of viruses, concentration by volume reduction.
The MBL containing solution is a complex protein mixture, where MBL may constitute less than 0.05% of the total proteins from the starting materials. Purification by means of chromatographic methods alternative to affinity chromatography would require further protein fractionation of the MBL containing solution e.g. by protein precipitation. The advantage of employing an affinity step is that no prior protein fractionation steps, such as precipitation and resuspension steps are needed, thus allowing the MBL containing solution to be applied directly to the column.
As a consequence of the pre-processing steps, e.g. the ethanol fractionation or the nature of the MBL expression system used, it is expected that the MBL containing solution contains MBL as
Birch Stewart Kolasch & Birch, LLP.
Carlson Karen Cochrane
Robinson Hope A.
Statens Serum Institut
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