Top > Search of International Patents > Method for suppressing aggregation of polypeptide

Method for suppressing aggregation of polypeptide commons meetings

Foreign code F190009848
File No. (2017001685)
Posted date Jul 25, 2019
Country United States of America
Application number 201816184028
Gazette No. 20190135860
Date of filing Nov 8, 2018
Gazette Date May 9, 2019
Priority data
  • P2017-216409 (Nov 9, 2017) JP
Title Method for suppressing aggregation of polypeptide commons meetings
Abstract The present invention relates to a method for suppressing aggregation of polypeptide. Specifically, the present invention relates to a method for suppressing, in a solution comprising an antibody or an Fc region-containing protein, formation of an aggregate derived from an antibody or an Fc region-containing protein having a non-native conformation, the method comprising: the steps of (i) binding an AF.2A1 polypeptide or an analog thereof with an aggregate derived from the antibody or Fc region-containing protein having a non-native conformation in the solution; and (ii) collecting the aggregate bound to the polypeptide or analog thereof from the solution.
Outline of related art and contending technology BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a method for suppressing aggregation of polypeptide.
Description of the Related Art
So-called antibody drug, which makes use of monoclonal antibodies in the therapeutic application and annual sales of which exceed 30 billion dollars, is the largest segment in biopharmaceuticals and also the fastest growing segment in the whole pharmaceutical industry. 23 full-size monoclonal antibodies and three monoclonal antibody fragments have been marketed so far and some of them have already become blockbusters whose annual sales exceed 1 billion dollars. Between 1995 and 2007, the number of drug candidate monoclonal antibodies entering clinical trials increased three or more times and the number further continues increasing (“Preclinical Development of Monoclonal Antibodies and Related Biologicals: Emerging technologies and new therapeutic candidates” (2010)1)).
Antibody drugs are required to be formulations that can stably be stored over a long period of time. Antibody drugs, which are protein origin, have concerns on their immunogenicity from the viewpoint of efficacy and safety. Actually, some antibody drugs that had been confirmed on their safety caused adverse events due to the induction by their immunogenicity and use of them were stopped (Casadevall (2002)2); Gershon (2002)3)). The immunogenicity of an antibody drug means that antibodies are produced against the antibody drug. Factors that influence the immunogenicity include those derived from components from products and the manufacturing process and those derived from the immune response of the individual patient. Those derived from components from products and the manufacturing process include the primary structure, modification, and structure of the antibody, host cell proteins (HCPs), aggregates of the antibody, addition of non-human sugar chain, properties of additives and containers, and the like. Those derived from the immune response of the individual patient include the health status of the patient, the route of administration, the dose and the duration of administration, genetic factors, and the like. These factors cause not only the reduction of the therapeutic effect on the patient, but also anaphylaxis and cytokine release syndrome, and crossreaction with endogenous protein. As a result, the immunogenicity leads to severe adverse events.
In recent years, increasing findings implicate aggregates of antibodies, which is one of the risk factors of the immunogenicity, with the immunogenicity (Fradkin (2009)4); Carpenter (2009)5); Hermeling (2006)6)). Since antibody drugs are preparations of protein origin, their properties are largely dependent on the manufacturing process. Actually, the possibility of structural change of such antibodies by a variety of stress that occurs to the antibodies during culture and purification process has been suggested. Moreover, antibody drugs are desired to be stored stably for a long period of time while they are at high concentrations. Antibody drugs at high concentrations are difficult to be maintained stable in aqueous solutions and aggregate due to the change in structure from their native conformation or the association by environmental stress during the storage. Therefore, when aggregates of antibody drugs, which are known to have the advantage of less side effects, are associated with side effects, it is considered to be essential to take measures to remove them as much as possible and/or to have improvement in the technology of analysis. The technology for suppressing aggregation of protein in general will be described below.
In the current technology of aggregation suppression, aggregation is suppressed by adding a denaturing agent such as urea or a particular amino acid(s) to aqueous protein solutions. Furthermore, surfactants that weakly interact with protein, such as Tween80 or TritonX-100, are used as means for improving the solubility of protein. However, the existing technology has problems such as denature of protein structure and inhibition of activity. There are many proteins that may become therapeutic agents for many diseases, but it is not easy to formulate such a protein while maintaining its pharmacological effect in the development of protein pharmaceutical preparations. A sugar or a sugar alcohol may be added to formulations for the purpose of increasing structural stability of protein (Carpenter (2002)7)). An inhibitor for aggregation of a protein (JP2014-208669A), formulations that inhibit protein aggregation (JP2009-502972T), and methods for reducing protein aggregation (JP2009-530380T) will be introduced below.
JP2014-208669A discloses a technique for suppressing protein aggregation by lowering the viscosity of a protein solution using a peptidic surfactant AAAAAAD or AAAAAAK to suppress protein aggregation during the formulation for the purpose of medical application of proteins containing an antibody. The peptidic surfactants to be used are known to have the effect of stabilizing membrane proteins such as G protein-coupled receptor bovine rhodopsin and the property of self-assembling (Zhao (2006)8); Nagai (2007)9)), but the use as a suppressor of protein aggregation had been unknown before JP2014-208669A.
JP2009-502972T discloses an invention of formulations that inhibit protein aggregate formation induced by one or more freeze/thaw cycles and stirring, comprising an inhibitor of insoluble aggregate formation in predetermined buffer conditions. Specifically, the amount of aggregates is reduced by contacting a solution comprising a protein or protein fragment with an amount of an inhibitor of insoluble aggregate formation effective to inhibit insoluble aggregate formulation. The buffer conditions are those of a phosphate buffer having a pH range of about 4.0 to about 8.0. The inhibitor includes MgCl2, propylene glycol, Pluronic-F68, Poloxamer 188, ethanol, or a combination thereof.
JP2009-530380T discloses methods for reducing protein aggregation involving adding methionine to a formulation (comprising a surfactant and any of citric acid, succinic acid, histidine, and tris buffer) to a concentration of about 0.5 mM to about 145 mM. It is shown that protein aggregation is reduced in such a formulation in comparison with that in a formulation lacking methionine.
For antibody drugs, efforts to reduce the formation of aggregates, other than the approaches described above, including the optimization of antibody-producing cell lines and media, studies on buffer solutions and additives in antibody solutions and purification conditions, and elucidation of the mechanism of aggregate formation are in progress (Bowers (2014)10); Handlogten (2017)11); Yueh (2007)12); Imamura (2016)13); Imamura (2017)14)). However, there is still the risk of adverse events due to aggregation. Some precedent studies indicate that aggregation reaction evolves over time. Therefore, if there are aggregates in an antibody solution, then it is considered that there is a risk that immunogenicity is induced by the formation of large aggregates during the storage. Therefore, it is ideal to remove aggregates from antibody solutions just before administration of the solutions.
The aggregation of an antibody is considered to occur during the production process, the purification process, and the storage of the antibody (Cromwell (2006)15)). In particular, the purification of antibodies involves a pH change to very low pH in conditions for the elution from Protein A and this is associated with the risk of conformational change and aggregation of antibodies (Welfle (1999)16); Thies (2001)17)). Therefore, various approaches to removing aggregates during the production and purification processes have been tried. Examples of such approaches include methods involving change the conditions for the antibody extraction to relatively mild ones (U.S. Pat. No. 9,382,297B, JP2009-297018A, WO 2012/165544, JP2010-081866A; Varady (1988)18); Watanabe (2009)19); Watanabe (2013)20); Tsukamoto (2014)21), use of Low affinity matrix (Gulick (2000)22); Li (1998)23)), and techniques involving addition of arginine for suppressing aggregation (Arakawa (2004)24); Tsumoto (2005)25); Arakawa (2007) 26); Ejima (2005)22)).
There are various steps in a production process for removing antibody aggregates. Generally, monoclonal antibodies are produced by cell culturing of cell lines derived from animals. In usual operations for purifying an antibody from a liquid cell culture, the liquid cell culture is centrifuged and suspended components are first precipitated and removed. Then, cell debris having a size of 1 μm or less, which cannot be eliminated by centrifugation, is removed by size filtration with a microfiltration membrane. Furthermore, sterile filtration with a filtration membrane having a maximum pore size of 0.22 μm or less is conducted for sterilization to obtain a sterile solution containing the protein of interest (harvest step). Subsequently, also contaminates such as host cell protein (HCP), deoxyribonucleic acid (DNA), endotoxin, virus, and protein A released from the column, but not only the aggregates of the antibody, are removed from this sterile solution using a purification process with a combination of plural chromatographic techniques, represented by the affinity chromatography using protein A to separate and purify the protein of interest (downstream step). The purity of a protein is increased through plural purification processes but it is difficult to separate and remove non-native conformational forms of the protein and assemblies of the protein. Moreover, aggregates are usually removed by filtration with a filtration membrane having a pore size of 0.22 μm or less or a method of removal employing plural types of chromatography in current production processes and there are size ranges of aggregates that cannot be removed.
There are a wide variety of aggregates from oligomers to visible particles. The reasons for such a variety include sizes and forms of stress-induced aggregates that vary depending on the environmental stress. These aggregates are classified into <100 nm, 100 to 1000 nm (submicrometer), 1 to 100 μm, >100 μm by the classification by Narhi (Narhi (2012)28)). Such aggregates that are difficult to be removed by existing methods are aggregates in the ranges of <100 nm and 100 to 220 nm, which is a part of the submicrometer range.
Based on plural precedent studies, small aggregates are considered to be likely to form larger aggregates (Weiss (2007)29); Joubert (2011)30)). Therefore, even if a protein preparation of a high purity is obtained through a production process, the presence of small sizes of aggregates is considered to result in the induction of larger aggregates during the storage for a long period of time. Thus, it is important to evaluate the rate of aggregate formation in antibody solutions after the removal of sizes of aggregates that cannot be removed by any existing technique by employing one of the artificial proteins and probes that specifically interacts with aggregates having a size of 220 nm or less, which are difficult to be removed or prevented from the formation by existing techniques.
The present inventors made an artificial protein AF.2A1 having 25 residues and exhibiting an affinity to the Fc region of human IgG using the 10-residue microprotein chignolin and an artificial protein library (WO 2014/103203) containing randomized amino acid sequences (Watanabe (2014)31)). AF.2A1 exhibits specific high affinity to the Fc region in a non-native structure generated by acid treatment, heat-treatment, reduction, or the like and a technique that can strictly distinguish a non-native conformational from and a native conformational form of the Fc region was developed (WO 2014/115229).
Scope of claims [claim1]
1. A method for suppressing, in a solution comprising an antibody or an Fc region-containing protein, formation of an aggregate derived from an antibody or an Fc region-containing protein having a non-native conformation, the method comprising: the steps of
(i) binding an AF.2A1 polypeptide or an analog thereof with a monomer and an aggregate derived from the antibody or Fc region-containing protein having a non-native conformation in the solution; and
(ii) collecting the monomer and aggregate bound to the polypeptide or analog thereof from the solution.

[claim2]
2. The method according to claim 1, wherein
the aggregate comprises an aggregate precursor having a particle size less than 0.22 μm.

[claim3]
3. The method according to claim 1, wherein
the aggregate comprises an aggregate having a size equal to or larger than that of a dimer.

[claim4]
4. The method according to claim 1, wherein
the AF.2A1 polypeptide or analog thereof consists of an amino acid sequence set forth in (A) or (B) below:
(A) the amino acid sequence set forth in SEQ ID NO: 1; or
(B) an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 1 by substitution, addition, or deletion of one or several amino acids, wherein the polypeptide consisting of the amino acid sequence exhibits binding activity to an aggregate comprising an antibody or an Fc region-containing protein having a non-native conformation.

[claim5]
5. The method according to claim 4, wherein
the AF.2A1 polypeptide or analog thereof consists of an amino acid sequence set forth in any of SEQ ID NOs: 1 and 3 to 25.

[claim6]
6. The method according to claim 1, wherein
the antibody or Fc region-containing protein having a non-native conformation has a non-native conformation caused by stress selected from the group consisting of acid treatment, heating, reduction, oxidization, freeze-thawing, and a physical stimulation.

[claim7]
7. The method according to claim 1, wherein
the antibody is any of human immunoglobulin G1 to 4.

[claim8]
8. The method according to claim 1, wherein
the antibody is a human antibody, a chimeric antibody, a humanized antibody, or a murine antibody.

[claim9]
9. The method according to claim 1, wherein
the AF.2A1 polypeptide or analog thereof is immobilized onto a solid-phase carrier.

[claim10]
10. The method according to claim 9, wherein the solid-phase carrier is a particle having a particle size of 1 to 10 μm.

[claim11]
11. The method according to claim 9, wherein the solid-phase carrier is a magnetic particle or a porous particle made of a polymer resin.

[claim12]
12. The method according to claim 9, wherein the AF.2A1 polypeptide or analog is immobilized onto a solid-phase carrier via any binding selected from the group consisting of biotin-avidin, biotin-streptavidin, and biotin-neutravidin.

[claim13]
13. A method of producing an antibody, comprising the method for suppressing formation of an aggregate derived from an antibody or an Fc region-containing protein having a non-native conformation according to claim 1.

[claim14]
14. A method of producing an antibody drug, comprising the method for suppressing formation of an aggregate derived from an antibody or an Fc region-containing protein having a non-native conformation according to claim 1.

[claim15]
15. An aggregate formation suppressor for suppressing formation of an aggregate in a solution comprising an antibody or an Fc region-containing protein,
comprising a solid-phase carrier onto which an AF.2A1 polypeptide or an analog thereof consisting of an amino acid sequence set forth in (A) or (B) below is immobilized, wherein the solid-phase carrier is a particle having a particle size of 1 to 10 μm:
(A) an amino acid sequence set forth in SEQ ID NO: 1; or
(B) an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 1 by substitution, addition, or deletion of one or several amino acids, wherein the polypeptide consisting of the amino acid sequence has a function of binding specifically to an aggregate comprising an antibody or an Fc region-containing protein having a non-native conformation.

[claim16]
16. The aggregate formation suppressor according to claim 15, wherein
the AF.2A1 polypeptide or analog thereof consists of an amino acid sequence set forth in any of SEQ ID NOs: 1 and 3 to 25.

[claim17]
17. The aggregate formation suppressor according to claim 15, wherein
the solid-phase carrier is a magnetic particle or a porous particle made of a polymer resin.

[claim18]
18. The aggregate formation suppressor according to claim 15, wherein
the AF.2A1 polypeptide or analog thereof is immobilized onto a solid-phase carrier via any binding selected from the group consisting of biotin-avidin, biotin-streptavidin, and biotin-neutravidin.
  • Inventor, and Inventor/Applicant
  • Senga Yukako
  • Watanabe Hideki
  • Honda Shinya
  • NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY
IPC(International Patent Classification)
Reference ( R and D project ) Biomedical Research Institue,Molecular and Cellular Breeding Research Group

PAGE TOP

close
close
close
close
close
close