US 12,135,265 B2
Chromatographic isolation of cells and other complex biological materials
Herbert Stadler, Niemetal (DE)
Assigned to Juno Therapeutics GmbH, (DE)
Filed by Juno Therapeutics GmbH, Munich (DE)
Filed on Dec. 21, 2018, as Appl. No. 16/231,193.
Application 16/231,193 is a division of application No. 14/380,699, granted, now 10,228,312, previously published as PCT/EP2013/053650, filed on Feb. 25, 2013.
Claims priority of provisional application 61/602,150, filed on Feb. 23, 2012.
Prior Publication US 2019/0234844 A1, Aug. 1, 2019
Int. Cl. G01N 33/543 (2006.01); B01D 15/38 (2006.01); G01N 1/40 (2006.01); G01N 33/569 (2006.01)
CPC G01N 1/405 (2013.01) [B01D 15/3809 (2013.01); B01D 15/3823 (2013.01); G01N 33/54306 (2013.01); G01N 33/56966 (2013.01); G01N 33/56972 (2013.01); G01N 2333/7051 (2013.01); G01N 2333/70517 (2013.01)] 20 Claims
 
1. A chromatography column comprising an affinity chromatography stationary matrix suitable for a CD4+ lymphocyte cell separation,
wherein the affinity chromatography stationary matrix comprises immobilized thereon both (1) an affinity reagent that comprises a streptavidin mutein and (2) a receptor binding reagent,
wherein the streptavidin mutein is immobilized on the chromatography stationary matrix,
wherein the receptor binding reagent comprises: (1) a Fab monomer fragment that binds CD4 on the surface of lymphocytes with (i) a dissociation constant (KD) of 10−3 to 10−7 M and/or (b) a dissociation rate constant (koff) of 3×10−5 sec−1 or greater, and (2) a binding partner C comprising a streptavidin-binding peptide fused to the C-terminus of the Fab monomer fragment,
wherein the binding partner C of the receptor binding reagent is bound to a binding site Z of the immobilized streptavidin mutein of the affinity reagent, thereby immobilizing the receptor binding reagent on the chromatography matrix;
wherein the Fab fragment comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 1 and a light chain comprising the sequence set forth in SEQ ID NO: 2,
wherein the streptavidin-binding peptide comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 3, and
wherein the binding site Z has a lower dissociation constant (Kd) for biotin or a biotin analog than for the binding partner C.