Purification of immunoglobulins

Details Purification of immunoglobulins Purification of immunoglobulins

1999-02-24

2000-07-25

Therkorn, Ernest G.

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

210656, 2101982, 5303871, 530413, 530416, B01D 1508

Patent

active

060933240

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to the purification of immunoglobulins from immunoglobulin-containing material, and particularly but not exclusively to the purification of immunoglobulins from plasma and plasma fractions. A particular objective of the present invention is the purification of immunoglobulins to provide a product which is suitable for intravenous administration to humans.


BACKGROUND OF THE INVENTION

Immunoglobulins derived from human plasma are used in the treatment of a number of clinical conditions such as primary agammaglobulinaemia, ITP and Kawasaki Syndrome. There is a growing demand for the product as an increasing number of studies are finding that it has application in many other autoimmune conditions. At present, immunoglobulins are purified from plasma by the Cohn fractionation procedure which separates plasma proteins by differential precipitation with ethanol. (Cohn et al., 1946).
The Cohn fractionation process has a number of disadvantages: plant design; function of antibodies; and
Therefore, there is a need for an efficient process for the recovery of immunoglobulins (IgG) at large scale.
A number of chromatographic processes for the production of pure IgG for intravenous administration have been described. Initially, ion-exchange resins such as DEAE-Sephadex were employed largely for "clean-up" purposes such as the removal of aggregates from Cohn fractionation derived product (Bjorling, 1972; Hoppe et al. 1973). However, integrated chromatographic processes for the recovery of IgG from plasma were subsequently described. Condie described a chromatographic process yielding IgG which was safe for injection (Condie, 1979; Condie 1980). One of the major issues that needed to be addressed was the potential fouling of resins by lipoproteins present in plasma. In this process, fumed colloidal silica (Aerosil) was used to remove lipoprotein from plasma prior to the chromatographic fractionation. However this treatment resulted in a marked decrease of IgG3 subclass under the chosen conditions. This altered subclass distribution is clinically undesirable in an immunoglobulin preparation. Further, removal of the silica was effected by centrifugation which is not amenable to the processing of large batches of immunoglobulin source material. The subsequent ion-exchange chromatography step was carried out using a QAE-Sephadex column at pH 7.0 with imidazole-acetate buffer at an ionic strength of 6.15 mS. Under these conditions, it would be anticipated that there would be increased interaction between immunoglobulin and the resin leading to reduced recovery. In particular, there would be losses of IgG3 and IgG4 which have a relatively anodal charge relative to IgG1 and IgG2. In addition, the use of imidazole-containing buffers or other buffers required to perform the process at pH 7 would significantly add to costs.
Bjorling has presented a procedure for isolation of gamma globulin from defibrinated serum by ion-exchange chromatography on CM-Sepharose CL6B followed by DEAE-Sepharose CL6B (Bjorling, 1985). However, in both these cases the anion-exchange step is carried out at or near pH 7.0, which would be undesirable, as described above. The use of a cation-exchange step under conditions where IgG is bound would require the use of more cycles than if anion-exchange resin was used under conditions where only contaminating proteins are bound. The binding of IgG with the use of cation-exchange chromatography would also result in greater loss of material than with the use of anion-exchange chromatography under conditions where contaminating proteins are retained and immunglobulins are unbound.
Anion-exchange chromatography has also been performed on DEAE-Trisacryl at pH 8.4 (Tousch et al 1989). Under these conditions it would not be applicable to use plasma or plasma fractions such as Cohn Supernatant 1 (SNI) as all proteins would bind thus limiting the effective capacity and through-put. Therefore partial purification by Cohn fractionation was initially performed to remove alb

REFERENCES:
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patent: 5552041 (1996-09-01), Afeyan
patent: 5679260 (1997-10-01), Boos
Journal of Chromatographya vol. 743 No. 1 Dated 1996 pp. 171-180 Yan Yan-Bo et al. "Influence of column type and chromatographic conditions on the ion-exchange chromatography of immunoglubulins".
Biotechnology of Blood Proteins vol. 227 Dated: 1993 pp. 207-212 Nourichafi N et al.; "Comparison of various chromatographic supports for purifying human plasmatic immunoglobulins from Cohn II & III fraction".
Journal of Chromatography vol. 590 Dated 1992 pp. 255-261 Neidhardt, Edith et al., "Rapid, two-step purification process for the preparation of pyrogen-free murine immunglobulin G1 monoclonal antibodies".

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