Ge Healthcare Bio-sciences Ab

Ge Healthcare Bioprocess R&d Ab

Cytiva Bioprocess R&d Ab

Hans J Johansson

Jean-Luc Maloisel

Bo-Lennart Johansson

Gunnar Glad

Annika Forss

Carina Engstrand

Sophia Hober

Tomas Bjorkman

Ronnie Palmgren

Karol Lacki

Nicolas Thevenin

Per-Mikael Aberg

Hans Berg

Gunnar Malmquist

Anna Grönberg

Anders Ljunglöf

Asa Ek

Maria Holm

Jafar Saidzadeh

Anna Groenberg

Anders Ljungloef

Per-Mikael ÅBerg

The present invention relates to a chromatography ligand defined by the following formula R&#x2014;R&#x2014;N(R)&#x2014;R&#x2014;Rwherein Ris a substituted or non-substituted phenyl group; Ris a hydrocarbon chain comprising 0-4 carbon atoms; Ris a hydrocarbon chain comprising 1-3 carbon atoms; Ris a hydrocarbon chain comprising 1-5 carbon atoms; and Ris OH or H. The invention also comprises a separation matrix, comprising the described ligands coupled to a porous support, such as particles or a membrane. The ligand and matrix according to the invention is useful for purification of biomolecules or organic compounds, such as proteins, polypeptides, DNA etc. An advantageous use according to the invention is the purification of antibodies.

Chromatography ligand

The invention discloses a polypeptide capable of binding immunoglobulins or immunoglobulin-containing proteins, which polypeptide comprises six or more domains of protein Z or the C domain of protein A or a functional variant thereof. It also discloses separation matrices comprising the polypeptide and methods of using the separation matrices for separation of immunoglobulins or immunoglobulin-containing proteins.

Affinity chromatography matrix

The present invention relates to an immunoglobulin-binding protein, wherein at least one asparagine residue has been mutated to an amino acid other than glutamine or aspartic acid, which mutation confers an increased chemical stability at pH-values of up to about 13-14 compared to the parental molecule. The protein can for example be derived from a protein capable of binding to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), such as protein A, and preferably the B-domain of Staphylococcal protein A. The invention also relates to a matrix for affinity separation, which comprises an immunoglobulin-binding protein as ligand coupled to a solid support, in which protein ligand at least one asparagine residue has been mutated to an amino acid other than glutamine.

Mutant protein

The present invention relates to a separation matrix comprised of porous particles to which antibody-binding protein ligands have been immobilized, wherein the ligand density is in the range of 5.0-10 mg/ml; the gel phase distribution coefficient of the particles expressed as Kfor a dextran of size 110 kDa is above 0.65 and the median particle diameter is between 65-84 &#x3bc;m. The carbohydrate material is preferably highly cross-linked agarose.

Purification of immunoglobulins

The present invention relates to a chromatography ligand defined by the following formula (I) R&#x2014;R&#x2014;N(R)&#x2014;R&#x2014;Rwherein Ris a substituted or non-substituted phenyl group; Ris a hydrocarbon chain comprising 0-4 carbon atoms; Ris a hydrocarbon chain comprising 1-3 carbon atoms; Ris a hydrocarbon chain comprising 1-5 carbon atoms; and Ris OH or H. The invention also comprises a separation matrix, comprising the described ligands coupled to a porous support, such as particles or a membrane. The ligand and matrix according to the invention is useful for purification of biomolecules or organic compounds, such as proteins, polypeptides, DNA etc. An advantageous use according to the invention is the purification of antibodies.

The present invention relates to a process for the purification of antibodies from one or more impurities in a liquid, which process comprises contacting said liquid with a first chromatography resin comprised of a support to which multi-modal ligands have been immobilised to adsorb the antibodies to the resin, wherein each multi-modal ligand comprises at least one cation-exchanging group and at least one aromatic or heteroaromatic ring system; adding an eluent to release the antibodies from the resin; and contacting the eluate so obtained with a second chromatography resin. In one embodiment, the ring-forming atoms of the aromatic or heteroaromatic entity are selected from the group consisting of C, S and O, and the cation exchanging group is a weak cation exchanger.

Process for the purification of antibodies

The present invention relates to a method of separating antibodies from other compound(s) in a liquid sample, wherein a mobile phase comprising said sample is contacted with a multi-modal separation matrix to adsorb undesired compounds while the antibodies remain free in the liquid, wherein the multi-modal separation matrix comprises first groups, which are capable of interacting with negatively charged sites of the target compounds, and second groups, which are capable of at least one interaction other than charge-charge interaction with said target compounds. The invention also relates to a chromatography column packed with the above-described multi-modal separation matrix and a filter having such multi-modal groups adsorbed to its surface.

Method of antibody purification

The present invention relates to the use of an alkali-stable protein, wherein at least one asparagine residue has been mutated to an amino acid other than glutamine or aspartic acid, which mutation confers an increased chemical stability at pH-values of up to about 13-14 compared to the parental molecule. The protein can for example be derived from a protein capable of binding to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), such as protein A, and preferably the B-domain of Staphylococcal protein A. The invention also relates to a matrix for affinity separation, which comprises an immunoglobulin-binding protein as ligand coupled to a solid support, in which protein ligand at least one asparagine residue has been mutated to an amino acid other than glutamine.

Protein ligands

The present invention relates to a method of separating antibodies from contaminants in a solution, which method comprises contacting the solution with a chromatography resin comprised of a support to which multi-modal ligands have been immobilised, wherein a multi-modal ligand comprises at least one cation-exchanging group and at least one aromatic or heteroaromatic ring system. In one embodiment, the ring-forming atoms of the aromatic or hereoaromatic entity are selected among C, S or O, and the cation exchanging group is a weak cation exchanger. The present method may be used as a single step procedure or as a polishing step following a capture on a Protein A column.

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Uppsala, Sweden

Hans J Johansson