Compositions comprising polypeptides

Patent metadata

Field	Value
Publication number	US-10000574-B2
Application number	US-58066004-A
Country	US
Kind code	B2
Filing date	Nov 26, 2004
Priority date	Nov 28, 2003
Publication date	Jun 19, 2018
Grant date	Jun 19, 2018

Abstract

The present invention relates to compositions comprising polypeptides, especially polypeptides capable of specifically binding predetermined antigens. The polypeptide in the composition comprises at least two antigen binding sites. These at least two antigen binding sites are located on a single polypeptide chain. One of the at least two antigen binding sites specifically binds the human CD3 antigen. The polypeptide may exist in both monomeric form and multimeric form. The multimeric form of the polypeptide constitutes no more than 5% of the total weight of the combined monomeric and multimeric forms of said polypeptide.

First claim

The invention claimed is:

1. A pharmaceutical composition that simultaneously binds CD3 and CD19, the composition comprising a single polypeptide chain comprising the amino acid sequence set forth in any one of SEQ ID NOS: 1-6 and a citrate/lysine buffer pH 6.0-7.5, wherein the polypeptide is present in the composition
in both monomeric form and multimeric form, said monomeric form being a single polypeptide chain and said multimeric form comprising at least two single polypeptide chains non-covalently associated with one another, wherein said multimeric form of said polypeptide constitutes no more than 3% of the total weight of the combined monomeric and multimeric forms of said polypeptide, wherein said pharmaceutical composition is obtained by
a) providing a composition comprising said polypeptide in both multimeric and monomeric form obtained from expression of said polypeptide in CHO cells;
b) isolating said polypeptide in both multimeric and monomeric form from said composition, said isolating is accomplished by
i) applying said composition to a first chromatographic material comprising a metal ion, which is a Zn 2+ or Ni 2+ ion;
ii) removing any components of said composition which have not bound to said first chromatographic material by washing said first chromatographic material with a first buffer;
iii) eluting said polypeptide in both multimeric and monomeric forms from said first chromatographic material by applying imidazole to said first chromatographic material in a concentration of at least 60 mM; and
iv) collecting a first eluate comprising said polypeptide in multimeric form and said polypeptide in monomeric form;

c) performing a precursor step that is preparatory for the separation of said polypeptide in multimeric form from said polypeptide in monomeric form to occur in step (d), said precursor step accomplished by
i) applying said first eluate to a second chromatographic material, which is an ion exchange material;
ii) removing any components of the first eluate which have not bound to said second chromatographic material by washing said second chromatographic material with a second buffer;
iii) eluting said polypeptide in multimeric and monomeric form from said second chromatographic material by applying sodium chloride to said second chromatographic material in a concentration ranging from 200 mM to 500 mM; and
iv) collecting a second eluate;

d) performing a separation of said polypeptide in multimeric form from said polypeptide in monomeric form, said separation accomplished by
i) applying said second eluate to a third chromatographic material allowing separation on the basis of molecular weight;
ii) translocating components of the applied second eluate along said third chromatographic material by applying a citrate/lysine buffer pH 6.0-7.5 to said third chromatographic material; and
iii) collecting a third eluate in fractions;

e) analyzing said fractions of said third eluate individually to obtain a measure of the amount of said polypeptide in monomeric form relative to the amount of polypeptide in multimeric form in each fraction; and
f) combining fractions of said third eluate which contain the polypeptide in monomeric form to obtain a composition such that the multimeric form of said polypeptide constitutes no more than 3% of the total weight of the combined monomeric and multimeric forms of said polypeptide.

2. The composition of claim 1 , wherein steps (b)(ii) and/or (c)(ii) is/are performed by means of chromatography on a column or by means of a batch process.

3. The composition of claim 1 , wherein said first chromatographic material comprises the Zn 2+ or the Ni 2+ ion.

4. The composition of claim 1 , wherein said second chromatographic material allows separation on the basis of anion exchange.

5. The composition of claim 1 , wherein said washing of steps (b)(ii) and (c)(ii) are performed using a volume of first and/or second buffer which is 6 to 10 times greater than the volume of the first and/or second chromatographic material, respectively.

6. The composition of claim 1 , wherein said translocating of step (d)(ii) is accomplished by applying a volume of said running buffer equivalent to 3 to 7 times the volume of the third chromatographic material.

7. The composition of claim 1 , wherein said first and second buffer are each phosphate buffer pH 8.

8. The composition of claim 1 , wherein said running buffer in step (d)(ii) comprises phosphate buffer pH 7.0-7.5 or citrate/lysine buffer pH 6.0-7.5.

9. The composition of claim 1 , wherein said analyzing is performed using a chromatographic method which separates substances on the basis of their molecular weight.

10. The composition of claim 9 , wherein said chromatographic method is size exclusion chromatography.

11. The composition of claim 1 , wherein:
said imidazole is applied either as a concentration gradient or as a single concentration and/or
said sodium chloride is applied either as a concentration gradient or as a single concentration.

12. The composition of claim 11 , wherein:
said imidazole is applied in a single concentration selected from the group consisting of: 70 mIVI, 80 mM, 90 mIVI, 100 mIVI, 110 mIVI and 120 mM; and
said sodium chloride is applied in a single concentration selected from the group consisting of: 370 mM, 380 mM, 390 mM, 400 mIVI, 410 mM and 420 mM.

13. The composition of claim 12 , wherein said imidazole is applied in a concentration of 80 mM and/or said sodium chloride is applied in a concentration of 400 mM.

14. The pharmaceutical composition of claim 1 , wherein said multimeric form of said polypeptide constitutes no more than 2% of the total weight of the combined monomeric and multimeric forms of said polypeptide.

15. The pharmaceutical composition of claim 1 , wherein said multimeric form of said polypeptide constitutes no more than 1% of the total weight of the combined monomeric and multimeric forms of said polypeptide.

Assignees

• Hofmeister Robert
• Prang Nadja
• Wolf Andreas
• Hanakam Frank
• Urbig Thomas
• Itin Christian
• Baeuerle Patrick
• Amgen Res Munich Gmbh

Inventors

• Hofmeister Robert
• Prang Nadja
• Wolf Andreas
• Hanakam Frank
• Urbig Thomas
• Itin Christian
• Baeuerle Patrick

Classifications

• C07K2317/34

  Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues · CPC title

• C07K16/30 Primary

  from tumour cells · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title

• C07K2317/31

  multispecific · CPC title

• C07K16/2809

  against the T-cell receptor (TcR)-CD3 complex · CPC title