Separation Matrix

Patent metadata

Field	Value
Publication number	US-2019119318-A1
Application number	US-201716096952-A
Country	US
Kind code	A1
Filing date	May 10, 2017
Priority date	May 11, 2016
Publication date	Apr 25, 2019
Grant date	—

Abstract

The invention relates to a separation matrix comprising at least 11 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein: a) the ligands comprise multimers of alkali-stabilized Protein A domains, and b) the porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml.

First claim

1 . A separation matrix comprising at least 11, such as at least 15, 15-21, 17-21 or 18-20 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:
a) said ligands comprise multimers of alkali-stabilized Protein A domains, b) said porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml, and c) said separation matrix has a max pressure of at least 0.58 MPa when packed at 300+/−10 mm bed height in a FineLine™ 35 column.

2 . The separation matrix of claim 1 , wherein said cross-linked polymer particles comprise cross-linked polysaccharide particles.

3 . The separation matrix of claim 1 , wherein said cross-linked polymer particles comprise cross-linked agarose particles.

4 . The separation matrix of claim 1 , wherein said cross-linked polymer particles have a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa.

5 . The separation matrix of claim 1 , wherein said multimers comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains.

6 . The separation matrix of claim 1 , wherein said multimers comprise hexamers of alkali-stabilized Protein A domains.

7 . The separation matrix of claim 1 , having a 10% breakthrough dynamic binding capacity for IgG of at least 45 mg/ml, such as at least 50 or at least 55 mg/ml at 2.4 min residence time.

8 . The separation matrix of claim 1 , having a 10% breakthrough dynamic binding capacity for IgG of at least 60 mg/ml, such as at least 65, at least 70 or at least 75 mg/ml at 6 min residence time.

9 . The separation matrix of any preceding claim 7 , wherein the 10% breakthrough dynamic binding capacity for IgG at 2.4 min or 6 min residence time is reduced by less than 20% after incubation 31 h in 1.0 M aqueous NaOH at 22+/−2 C.

10 . The separation matrix of claim 1 , having a dissociation constant for IgG2 of below 0.2 mg/ml, such as below 0.1 mg/ml, in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.

11 .- 17 . (canceled)

18 . The separation matrix of claim 1 , wherein said alkali-stabilized Protein A domains comprise an Fc-binding polypeptide having an amino acid sequence as defined by, or having at least 80% or at least 90, 95% or 98% identity to SEQ ID NO 53

(SEQ ID NO 53)

X 1 Q X 2 AFYEILX 3 LP NLTEEQRX 4 X 5 F IX 6 X 7 LKDX 8 PSX 9

SX 10 X 11 X12LAEAKX 13 X 14 NX 15 AQ

wherein individually of each other:
X 1 =A or Q or is deleted
X 2= E,K,Y,T,F,L,W,I,M,V,A,H or R
X 3= H or K
X 4= A or N
X 5= A,G,S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K
X 6 =Q or E
X 7 =S or K
X 8 =E or D
X 9 =Q or V or is deleted
X 10 =K,R or A or is deleted
X 11 =A,E or N or is deleted
X 12 =or L
X 13 =K or R
X 14 =L or Y
X 15 =D,F,Y,W,K or R.

19 . The separation matrix of claim 18 , wherein individually of each other:
X 1 =A or is deleted, X 2 =E, X 3 =H, X 4 =N, X 6 =Q, X 7 =S, X 8 =D, X 9 =V or is deleted, X 10 =K or is deleted, X 11 =A or is deleted, X 12 =I, X 13 =K, X 14 =L.

20 . (canceled)

21 . The separation matrix of claim 1 , wherein said multimers comprise hexamers of alkali-stabilized Protein A domains.

22 . The separation matrix of claim 1 , wherein the polypeptides are linked by linkers comprising up to 25 amino acids, such as 3-25 or 3-20 amino acids.

23 . The separation matrix of claim 1 , wherein at least two polypeptides are linked by linkers comprising or consisting essentially of a sequence having at least 90% identity with an amino acid sequence selected from the group consisting of APKVDAKFDKE, APKVDNKFNKE, APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK, APKYEDGVDAKFDKE and YEDG.

24 . A method of isolating an immunoglobulin, comprising the steps of:
a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to claim 1 , b) washing said separation matrix with a washing liquid, c) eluting the immunoglobulin from the separation matrix with an elution liquid, and d) cleaning the separation matrix with a cleaning liquid.

25 . The method of claim 24 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH, such as 0.4-1.0 M NaOH or KOH.

26 . The method of claim 25 , wherein steps a)-d) are repeated at least 10 times, such as at least 50 times or 50-200 times.

27 .- 29 . (canceled)

Assignees

• Ge Healthcare Bioprocess R&D Ab

Inventors

• Rodrigo Gustav José
• Bjorkman Tomas
• Ander Mats
• Hansson Jesper Ulf

Classifications

• B01J20/286

  Phases chemically bonded to a substrate, e.g. to silica or to polymers · CPC title

• B01J20/28016

  Particle form · CPC title

• B01D15/3809

  of the antigen-antibody type, e.g. protein A, G or L chromatography · CPC title

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K17/10

  the carrier being a carbohydrate · CPC title