US 12,066,365 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. 16/231,188.
Application 16/231,188 is a continuation 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 2019/0226951 A1, Jul. 25, 2019
Int. Cl. G01N 33/53 (2006.01); B01D 15/38 (2006.01); G01N 1/40 (2006.01); G01N 33/543 (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)] 13 Claims
 
1. A method of isolating a target cell, wherein the target cell is a CD4+ lymphocyte, the method comprising:
(a) contacting a stationary phase chromatographic matrix suitable for cell separation with a receptor binding reagent, wherein:
the stationary phase chromatographic matrix is a nonmagnetic or non-magnetizable material and comprising a streptavidin mutein affinity reagent immobilized thereon,
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 (ii) a koff rate of 3×10−5 sec−1 or greater, 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, and the receptor binding reagent further comprises (2) a binding partner C that comprises a streptavidin-binding peptide fused to the C-terminus of the Fab monomer fragment, wherein the binding partner C is capable of reversibly binding to a binding site Z of the streptavidin mutein affinity reagent that is immobilized on the stationary phase, the binding site Z having a lower dissociation constant (KD) for biotin or a biotin analog than for the binding partner C, thereby immobilizing the receptor binding reagent on the stationary phase chromatographic matrix;
(b) loading a sample comprising one or more CD4+ lymphocyte target cells onto a selection affinity chromatography column to form a complex in the column, wherein the selection affinity chromatography column has been formed by loading the chromatographic matrix comprising the receptor binding reagent immobilized thereof of (a) into a chromatography column, and wherein the complex comprises multimers of the monomeric Fab fragment fusion bound to the target cells, thereby reversibly immobilizing the one or more CD4+ lymphocyte target cells on the chromatographic matrix;
(c) washing the selection affinity chromatography column to remove unbound cells; and
(d) eluting the one or more CD4+ lymphocyte target cells bound on the stationary phase, comprising contacting the selection affinity chromatography column with a competition reagent selected from the group consisting of a biotin or biotin analog capable to disrupt the binding between the streptavidin-binding peptide of the receptor binding reagent and the streptavidin mutein, thereby displacing the receptor binding reagent and releasing the one or more CD4+ lymphocyte from the monomeric Fab fragment fusion of (b) in an eluate, wherein the eluate comprises the one or more CD4+ lymphocytes target cells, the Fab monomers and biotin or biotin analog,
wherein the streptavidin-binding peptide comprises the amino acid sequence of SEQ ID NO. 13 or SEQ ID NO. 3.