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US 10,118,962 B2Granted
Methods for purification of single domain antigen binding molecules
AssigneeAblynx N.V.
InventorsPaul R. Brown, Scott Andreas Tobler, Andrew M. Wood, Austin Wayne Boesch
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Abstract
Processes and methods of purifying or separating Single Domain Antigen Binding (SDAB) molecules that include one or more single binding domains (e.g., one or more nanobody molecules), substantially devoid of a complementary antibody domain and an immunoglobulin constant region, using Protein A-based affinity chromatography, are disclosed.
Claims40
A method of separating or purifying an antigen-binding fusion polypeptide from cell culture medium comprising:
- obtaining cell culture medium containing the antigen-binding fusion polypeptide that is produced as a product of a eukaryotic cell culture and secreted into the cell culture medium,
- adjusting the cell culture medium containing the antigen-binding fusion polypeptide to a conductivity between about 12 and 9 mS/cm and a pH equal to or less than about 4.5,
- contacting the medium containing the antigen-binding fusion polypeptide with a cation exchange-based support, under conditions that allow the antigen-binding fusion polypeptide to bind or absorb to the support, wherein the cation exchange-based support is a cation exchange (CEX) resin, wherein the CEX resin has a capacity of at least about 40 g/L;
- and
- selectively eluting the antigen-binding fusion polypeptide from the support by washing the support with at least one cation exchange washing buffer, thereby removing one or more contaminants;
- and eluting the antigen-binding fusion polypeptide with an elution buffer;
- wherein said antigen-binding fusion polypeptide is devoid of a complementary antibody domain and an immunoglobulin Fc region, and wherein the antigen-binding fusion polypeptide is not pegylated, wherein said antigen-binding fusion polypeptide comprises one or more VHHs.
4 more elements
A method of separating or purifying an antigen-binding fusion polypeptide that comprises one or more VHHs from cell culture medium, comprising:
- obtaining cell culture medium containing an antigen-binding fusion polypeptide that is produced as a product of a eukaryotic cell culture and secreted into the cell culture medium,
- adjusting the cell culture medium containing the antigen-binding fusion polypeptide to a conductivity between about 12 and 9 mS/cm and a pH equal to or less than about 4.5;
- contacting the medium containing the antigen-binding fusion polypeptide and one or more contaminants with a cation exchange support,
- allowing the antigen-binding fusion polypeptide to flow through the support,
- washing the support with at least one cation exchange washing buffer;
- removing one or more contaminants;
- selectively eluting the antigen-binding fusion polypeptide from the support, thereby obtaining an eluted antigen-binding fusion polypeptide preparation;
- contacting the eluted antigen-binding fusion polypeptide preparation with a hydroxyapatite resin;
- and
- selectively eluting the antigen-binding fusion polypeptide from the hydroxyapatite resin, wherein said antigen-binding fusion polypeptide does not have a complementary antibody variable domain or an immunoglobulin Fc region, and wherein the antigen-binding fusion polypeptide is not pegylated.
7 more elements
A method or process of providing a recombinant antigen-binding fusion polypeptide that comprises one or more VHHs, comprising:
- providing a mammalian host cell comprising a nucleic acid that encodes the recombinant antigen-binding fusion polypeptide;
- maintaining the host cell under conditions in which the recombinant antigen-binding fusion polypeptide is expressed;
- obtaining a medium of the recombinant antigen-binding fusion polypeptide and one or more contaminants;
- purifying or separating the recombinant antigen-binding fusion polypeptide from said medium using cation exchange based chromatography support, wherein said purifying or separating step comprises
- adjusting the medium containing the antigen-binding fusion polypeptide to a conductivity between about 12 and 9 mS/cm and a pH equal to or less than about 4.5;
- contacting the medium with the support, under conditions that allow the antigen-binding fusion polypeptide to bind or absorb to the support;
- removing one or more contaminants;
- and
- selectively eluting the antigen-binding fusion polypeptide from the support, thereby obtaining an eluted antigen-binding fusion polypeptide preparation, wherein said antigen-binding fusion polypeptide is devoid of a complementary antibody domain and an immunoglobulin Fc region, and wherein the antigen-binding fusion polypeptide is not pegylated.
6 more elements
Dependencies are inferred from the claim text.