US 12,116,414 B2
Method of modifying isoelectric point of antibody via amino acid substitution in CDR
Tomoyuki Igawa, Shizuoka (JP); Hiroyuki Tsunoda, Shizuoka (JP); Tatsuhiko Tachibana, Shizuoka (JP); and Taichi Kuramochi, Shizuoka (JP)
Assigned to Chugai Seiyaku Kabushiki Kaisha, Tokyo (JP)
Filed by Chugai Seiyaku Kabushiki Kaisha, Tokyo (JP)
Filed on Feb. 5, 2024, as Appl. No. 18/432,567.
Application 18/432,567 is a division of application No. 18/425,859, filed on Jan. 29, 2024.
Application 18/432,567 is a division of application No. 17/578,524, filed on Jan. 19, 2022.
Application 18/425,859 is a division of application No. 17/578,524, filed on Jan. 19, 2022.
Application 17/578,524 is a division of application No. 15/725,692, filed on Oct. 5, 2017, granted, now 11,248,053, issued on Feb. 15, 2022.
Application 15/725,692 is a continuation of application No. 14/741,786, filed on Jun. 17, 2015, granted, now 9,828,429, issued on Nov. 28, 2017.
Application 14/741,786 is a continuation of application No. 12/679,922, granted, now 9,096,651, issued on Aug. 4, 2015, previously published as PCT/JP2008/067534, filed on Sep. 26, 2008.
Claims priority of application No. 2007-250165 (JP), filed on Sep. 26, 2007; and application No. 2007-256063 (JP), filed on Sep. 28, 2007.
Prior Publication US 2024/0239906 A1, Jul. 18, 2024
Int. Cl. C07K 16/28 (2006.01); A61K 39/395 (2006.01); C07K 16/00 (2006.01); G01N 33/68 (2006.01)
CPC C07K 16/2866 (2013.01) [A61K 39/39591 (2013.01); C07K 16/00 (2013.01); C07K 16/28 (2013.01); G01N 33/6854 (2013.01); C07K 2317/24 (2013.01); C07K 2317/565 (2013.01); C07K 2317/732 (2013.01)] 21 Claims
 
1. A method for producing a multispecific antibody comprising a first arm comprising a first heavy chain variable region (VH) and a first light chain variable region (VL) and a second arm comprising a second VH and a second VL, the method comprising using a standard chromatographic technique to purify the multispecific antibody from a mixture of the multispecific antibody, a homomultimer of the first arm, and a homomultimer of the second arm, wherein:
the amino acid residue at Kabat numbering position 55 in the first VL is glutamic acid or aspartic acid;
the amino acid residue at Kabat numbering position 55 in the second VL has no charge or has a positive charge;
the isoelectric point of the first arm is different from the isoelectric point of the second arm;
the standard chromatographic technique separates the multispecific antibody from the two homomultimers based at least in part on the different isoelectric points of the first and second arms; and
the standard chromatographic technique comprises cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, hydrophobic charge interaction chromatography, or chromatofocusing.