Antibody purification
Antibody compositions and methods for the purification of antibodies are provided. Purification methods provided involve the use of hydroxyapatite resin (HA) to separate an antibody of interest from one or more impurities.
1 . A composition comprising at least two proteins, an intact anti-BCMA/anti-CD3 bispecific antibody and a clipped version of the bispecific antibody, wherein
(a) the bispecific antibody comprises an anti-BCMA arm and an anti-CD3 arm, the anti-CD3 arm comprises a VH region comprising an amino acid sequence as shown in SEQ ID NO: 1 and a VL region comprising an amino acid sequence as shown in SEQ ID NO: 3, and the anti-BCMA arm comprises a VH region comprising an amino acid sequence as shown in SEQ ID NO: 5 and a VL region comprising an amino acid sequence as shown in SEQ ID NO: 7;
(b) the clipped version of the bispecific antibody has a molecular mass that is less than 1% different from the intact bispecific antibody;
and that the composition comprises at least 90% by mass of the intact bispecific antibody of the proteins in the composition, and 0.1 to 5% by mass of the clipped version of the bispecific antibody of the proteins in the composition.
2 . The composition of claim 1 , wherein in the clipped version of the bispecific antibody has the same amino acid sequences as the intact bispecific antibody, except that the peptide bond between amino acid residue 56 and amino acid residue 57 of the N terminal of the VH region of the CD3 arm of the clipped version of the bispecific antibody is cleaved, and the clipped off 56 amino acid portion of the VH region of the CD3 arm of the clipped version of the bispecific antibody remains bound to the rest of the clipped version of the bispecific antibody through a disulfide bond.
3 . The composition of claim 2 , wherein the clipped version of the bispecific antibody has a molecular mass that is 18 Dalton more than the intact bispecific antibody.
4 . The composition of claim 1 , wherein the composition comprises at least 95% by mass of the intact bispecific antibody of the proteins in the composition.
5 . The composition of claim 1 , wherein the composition comprises at least 95% by mass of the intact bispecific antibody of the proteins in the composition, and 0.1 to 3% by mass of the clipped version of the bispecific antibody of the proteins in the composition.
6 . A composition comprising at least two proteins, an intact anti-BCMA/anti-CD3 bispecific antibody and a clipped version of the bispecific antibody, wherein
(a) the bispecific antibody comprises an anti-BCMA arm and an anti-CD3 arm, the anti-CD3 arm comprises a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 2 and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 4, and the anti-BCMA arm comprises a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 6 and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 8;
(b) the clipped version of the bispecific antibody has a molecular mass that is less than 1% different from the intact bispecific antibody;
and that the composition comprises at least 90% by mass of the intact bispecific antibody of the proteins in the composition, and 0.1 to 5% by mass of the clipped version of the bispecific antibody of the proteins in the composition.
7 . The composition of claim 6 , wherein the clipped version of the bispecific antibody has the same amino acid sequences as the intact bispecific antibody, except that the peptide bond between amino acid residue 56 and amino acid residue 57 of the N terminal of the VH region of the CD3 arm of the clipped version of the bispecific antibody is cleaved, and the clipped off 56 amino acid portion of the VH region of the CD3 arm of the clipped version of the bispecific antibody remains bound to the rest of the clipped version of the bispecific antibody through a disulfide bond.
8 . The composition of claim 7 , wherein the clipped version of the bispecific antibody has a molecular mass that is 18 Dalton more than the intact bispecific antibody.
9 . The composition of claim 6 , wherein the composition comprises at least 95% by mass of the intact bispecific antibody of the proteins in the composition.
10 . The composition of claim 6 , wherein the composition comprises at least 95% by mass of the intact bispecific antibody of the proteins in the composition, and 0.1 to 3% by mass of the clipped version of the bispecific antibody of the proteins in the composition.
11 . A method of purifying an antibody preparation comprising at least two proteins, an intact anti-BCMA/anti-CD3 bispecific antibody and a clipped version of the bispecific antibody, wherein:
(a) the anti-CD3 arm of the intact bispecific antibody comprises (i) a VH region comprising an amino acid sequence as shown in SEQ ID NO: 1 and a VL region comprising an amino acid sequence as shown in SEQ ID NO: 3, or (ii) a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 2 and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 4;
(b) the anti-BCMA arm of the intact bispecific antibody comprises (i) a VH region comprising an amino acid sequence as shown in SEQ ID NO: 5 and a VL region comprising an amino acid sequence as shown in SEQ ID NO: 7, or (ii) a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 6 and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 8, and
(c) that the clipped version of the bispecific antibody has a molecular mass that is less than 1% different from the intact bispecific antibody;
comprising the steps of:
A) loading the antibody preparation in a load buffer onto a hydroxyapatite (HA) resin; and
B) eluting the intact antibody of interest from the HA resin with an elution buffer.
12 . The method of claim 11 , wherein in the clipped version of the bispecific antibody has the same amino acid sequences as the intact bispecific antibody, except that the peptide bond between amino acid residue 56 and amino acid residue 57 of the N terminal of the VH region of the CD3 arm of the clipped version of the bispecific antibody is cleaved, and the clipped off 56 amino acid portion of the VH region of the CD3 arm of the clipped version of the bispecific antibody chain remains bound to the rest of the clipped version of the bispecific antibody through a disulfide bond.
13 . The method of claim 12 , wherein the clipped version of the bispecific antibody has a molecular mass that is 18 Dalton more than intact bispecific antibody.
14 . The method of claim 11 , wherein the HA resin is ceramic hydroxyapatite (cHA) resin.
15 . The method of claim 11 , wherein after loading the antibody preparation onto the HA resin but prior to eluting the intact bispecific antibody the resin is washed with a wash buffer comprising phosphate ions at concentration between 10 and 50 mM.
16 . The method of claim 11 , wherein the elution buffer comprises phosphate ions at a concentration of of 36-44 mM, 45-55 mM or 54-66 mM.
17 . The method of claim 11 , wherein the elution buffer comprises phosphate ions at a concentration increased during the elution from 40 mM to 100 mM.
18 . The method of claim 11 , wherein the pH of at least one of the load buffer, wash buffer, and elution buffer is at or between pH 7.0 and 8.0.
19 . The method of claim 11 , wherein the antibody preparation contains proteins that were previously loaded onto and eluted from at least one of: i) a protein A resin and ii) an ion exchange resin.