IGG separation medium

Details IGG separation medium IGG separation medium

1997-06-09

2002-06-04

Ponnaluri, Padmashri (Department: 1627)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Separation or purification

C530S412000, C530S387100, C530S389100, C530S391100, C530S812000, C530S813000, C530S814000, C530S815000, C530S816000, C530S817000, C530S402000, C436S518000, C436S528000, C436S529000, C436S530000, C436S531000, C436S532000

Reexamination Certificate

active

06399750

ABSTRACT:

1. FIELD OF THE INVENTION
The present invention relates generally to methods of preparing and using a separation medium for purifying IgG. In particular, the present invention relates to the preparation and use of Protein A and variants of Protein A, including recombinantly produced variants, to purify IgG by coupling the Protein A or variant thereof to a base matrix with a bridging group.
2. BACKGROUND OF THE INVENTION
Adsorbents which exhibit IgG-binding proteins have been used to capture IgG in aqueous media for more than twenty years. Initially, native Protein A (GB 1,441,979 (Sjöqvist)) was used. Later recombinantly produced forms of Protein A and were developed (WO 8400773 to Lofdahl, et al.; EP 262,192 to Guss, et al.; and U.S. Pat. No. 5,082,773 to Fahnestock).
Protein A has a broad IgG-specificity with respect to animal species, but the specificity may vary with respect to subclasses (for instance, human IgG3 will not bind to Protein A). Protein G binds to all IgG subclasses of a majority of important mammalian species. The advantage of Protein A compared to Protein G is that the binding of IgG is weaker, and consequently milder conditions can be used to release IgG from Protein A. This is of importance for the purification of individual monoclonal antibodies.
Recombinant techniques enable simple mapping of IgG-binding proteins with regard to the functionality of different domains. In the case of Protein A, it was found that the native form contained five consecutively ordered IgG-binding C domains (E, D, A, B and C), followed by an X-domain which did not bind IgG. The new technique facilitated the preparation of IgG-binding fragments and variants where one or more amino acids was/were replaced, added or removed. Unless otherwise indicated, reference to Protein A indicates the native form, or IgG-binding fragments and variants of Protein A that have the same IgG specificity as native Protein A. Variants of Protein A which contain cysteine were produced relatively early on, and the inserted cysteine residue was used for binding to base matrices. It was considered important not to place cysteine as a C-terminal residue. A variant having cysteine as the penultimate amino acid in the C-terminal part was bonded to activated Thiol SEPHAROSE® (Pharmacia Biotech AB, Uppsala, Sweden) via disulfide bond formation and studied as an IgG-separation medium (T. Profy (Repligen); EP 284,368 and U.S. Pat. No. 5,084,559). Similar studies were also presented in FASEB 87, Mar. 29-Apr. 2, 1987 (Poster N44, Profy, et al). The results obtained with three other variants (1, 2 and 5 domains) of Protein A with cysteine in a C-terminal linker sequence (amino acid 10 from the C-terminal) (Ljungquist, et al, Eur. J. Biochem. 186 (1989) 557-561) were later presented. These latter variants were also coupled covalently via disulfide bound formation to thiopropyl SEPHAROSE®. Immobilization to tresyl chloride or tosyl chloride activated gels was suggested as an alternative, with the intention of avoiding reductively sensitive linking groups. An equimolar relationship was found between IgG binding capacity and the number of domains for one-domain and two-domain variants. The five-domain variant never bonded more than the double molar amount of IgG. IgG-capacities comparable to those achieved earlier with soluble forms of native Protein A were obtained it was later found that in certain applications, non-cys-containing variants can give molar binding ratios which lie between two and three).
Parallel herewith, Genex (U.S. Pat. No. 4,977,247 (Fahnestock, et al) has produced a recombinant variant of rProtein G-cys in which cysteine is located at the C-terminal end of an IgG-binding domain. In preparing separation media based on this Protein G variant, the choice was to bind rprotein G-cys covalently to aminohexyl-agarose activated with the bi-functional reagent N-sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate (U.S. Pat. No. 4,977 247, Claim 1 and column 18, lines 22-37). GAMMABINDG® Plus (Pharmacia Biotech AB) is a commercially available solid phase rProtein G-cys product with cysteine as the C-terminal residue. The product is synthesized by coupling the cysteinyl residue to aminohexyl agarose activated with N-sulfosuccinimidyl 4-(N-maleimidomethyl) cyklohexane-1-carboxylate.
As far as we are aware, the variant of the matrix-bound rProtein A-cys produced by Repligen has not found favor commercially. The reason may be that the coupling to the matrix is through unstable structures (—S—S—), although the reason may also be due to factors unknown to us. Whatever the reason, however, the adsorbent that totally dominates commercially makes use of native Protein A or different forms of recombinant Protein A that lack cysteine. The market for products based on Protein G has been substantially smaller, probably because Protein A has more advantageous binding properties.
3. SUMMARY OF THE INVENTION
The object of the invention can be summarized in the desire to provide adsorption media which have a) the IgG-binding specificity of Protein A; b) at least the same stability as other adsorbents based on native Protein A; c) the same or improved capacity to bind IgG. compared to earlier known variants of matrix bound rProtein A-cys (primarily calculated as the ratio mol IgG per mol cys-variant of Protein A with one, two or more IgG binding domains). For variants with two or more domains this means molar ratios ≧2. Fulfillment of these objects will enable more effective processes to be used for purifying IgG from different starting materials.
4. DESCRIPTION OF THE PREFERRED EMBODIMENTS
The main aspect of the invention is a separation medium which comprises a base matrix substituted with groups of the formula I:
—
B—X
—rProtein
A
-cys,  I
where
a. rProtein A-cys is recombinantly produced Protein A which contains cysteine in its amino acid sequence;
b. B is a bridge which binds to the base matrix; and
c. X contains a heteroatom N or S originating from rProtein A-cys.
The characteristic feature is that X is a thioether sulphur (—S—) and/or a secondary amine (—NH—); i.e. in one and the same separation medium X may be either or both a thioether sulphur and/or a secondary amine, with preference for greater than 50%, such as essentially 100%, of all X being thioether sulphur.
The optimal molar ratio between the total IgG binding capacity and the amount of Protein A on the matrix may vary depending on the number of IgG-binding domains that are present in the Protein A of the adsorbent. For single-domain variants the ratio is 1 and for 2-domain variants the ratio is ≈2. For three-, four- and five-domain variants the ratio is ≈2 or preferably >2. The maximum value is determined by the number of IgG-binding domains and is therefore contingent on the particular Protein A construction used.
In principle, B can be anything that has satisfactory stability under the conditions applied in the adsorption/desorption of IgG (time, temperature, pH, etc.). Examples of relevant structures in the bridge —B— are amide, ester, ether, thioether, hydrocarbon chains, azo, carbamate, etc. Hydrocarbon chains present in —B— may be straight, branched or cyclic, and normally have only saturated carbon atoms (2-10 carbon atoms, preferably 2, 3 or 4 carbon atoms to retain a pronounced hydrophilic nature). It is preferred that bridge —B— binds to the base matrix via an ether structure or an amide/ester structure. It is also preferred that B comprises a straight, branched or cyclic saturated hydrocarbon chain which may optionally compromise at one or more positions in the hydrocarbon chain an inserted oxygen or nitrogen, or at one or more positions in the hydrocarbon chain a hydrogen substituted with an amino or a hydroxy group. For stability reasons one and the same carbon atom should bind at most one oxygen or nitrogen atom. The structures that are preferred in —B— are those which occur when rProtein A-cys is coupled to the matrix via an epoxy group or epihalo; i.e. —B— includes at the end nearest X, the structure
—CH
2
—CHOH—CH
2
—

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