Formulations with reduced oxidation

a technology of oxidation and formulation, applied in the field of protein formulations, can solve the problems of affecting the biological activity of the protein, unexpected and undesired consequences, and oxidation of amino acid residues, so as to prevent oxidation of the protein, reduce the oxidation potential, and reduce the effect of photosensitivity

Inactive Publication Date: 2016-04-21
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0024]In some embodiments, the compound in the formulation is from about 0.3 mM to about 1 mM. In some embodiments, the compound prevents oxidation of tryptophan, cysteine, histidine, tyrosine, and / or methionine in the protein. In some embodiments, the compound prevents oxidation of the protein by a reactive oxygen species. In some embodiments, the reactive oxygen species is selected from the group consisting of singlet oxygen, hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.
[0050]In some embodiments, the compound prevents oxidation of tryptophan, cysteine, histidine, tyrosine, and / or methionine in the protein. In some embodiments, the compound prevents oxidation of the protein by a reactive oxygen species. In some embodiments, the reactive oxygen species is selected from the group consisting of singlet oxygen, hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.
[0053]In another aspect, provided herein is a method of screening a compound that prevents oxidation of a protein in a protein composition, comprising selecting a compound that has lower oxidation potential and less photosensitivity as compared to L-tryptophan, and testing the effect of the selected compound on preventing oxidation of the protein.

Problems solved by technology

Oxidation of amino acid residues has the potential to impact the biological activity of the protein.
It is apparent from recent studies that the addition of standard excipients, such as L-Trp and polysorbates, to protein compositions that are meant to stabilize the protein can result in unexpected and undesired consequences such as ROS-induced oxidation of the protein.

Method used

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Examples

Experimental program
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Effect test

example 1

The Antioxidant L-Trp Produces ROS that Oxidize Monoclonal Antibodies in Protein Formulations

[0264]Monoclonal antibodies have been shown to produce ROS through the antibody catalyzed water oxidation pathway (ACWOP) wherein antibodies potentially catalyze a reaction between water and singlet oxygen generating hydrogen peroxide (Wentworth et al., Science 293(5536):1806-11 (2001); Wentworth et al., Proc Natl Acad Sci USA 97(20):10930-5 (2000)). In the ACWOP, a variety of ROS, including superoxide anion, dihydrogen trioxide, ozone, and even hydrotrioxy radical are generated in the pathway toward production of hydrogen peroxide (Zhu et al., Proc Natl Acad Sci USA 101(8):2247-52 (2004)). It has been shown that surface exposed tryptophans in a monoclonal anti-DR5 antibody, drozitumab (CAS number 912628-39-8), also referred to herein as mAb1, act as substrate (1O2 and O2−) generators that facilitate ACWOP even under mild light conditions in a time and concentration dependent manner (Sreedha...

example 2

Screening of Candidate Antioxidant Compounds

[0270]Tryptophan (Trp) is an electron rich amino acid that undergoes oxidative and electrophilic addition reactions in the presence of ROS such as hydroxyl radicals and singlet oxygen. Any potential antioxidant to protect Trp oxidation in proteins should have similar if not superior reactivity towards these ROS. A series of compounds that were either based on the L-Trp structure or have been reported to have antioxidant properties were evaluated. Compounds screened for antioxidant ability in this study included derivatives of tryptophan, indole, aromatic acids such as salicylic acid and anthranilic acid, and some vitamins. The chemical structures of the various compounds used were based on (A) Tryptophan derivatives (B) Kynurenine (C) Indole derivatives and (D) Aromatic acid derivatives:

(A) Tryptophan Derivatives

[0271]

NameRXAL-TryptophanCOOHHH5-Hydroxy-TryptophanCOOHOHH5-Methoxy-TryptophanCOOHOCH3H5-Amino-TryptophanCOOHNH2H5-Fluoro-Tryptop...

example 3

Candidate Antioxidant Compounds Reduced Oxidation of Monoclonal Antibody Formulations

[0284]Compounds that, compared to L-Trp, produced less H2O2 upon light treatment as well as those with lower oxidation potentials than L-Trp were chosen for evaluation of for their possible antioxidant properties using AAPH, light, and Fenton reaction as oxidative stress models (Table 3). mAb1 was used as a model protein to evaluate the effectiveness of select candidate antioxidants to protect against Trp oxidation by the different oxidation stress models. Each stress model produced oxidation through a different mechanism and therefore each was valuable in the assessment of biopharmaceutical stability. AAPH, or 2,2′-Azobis(2-Amidinopropane)Dihydrochloride, is used as a stress model to mimic alkyl peroxides potentially generated from formulation excipients such as degraded polysorbate. Decomposition of AAPH generates alkyl, alkoxyl, and alkyl peroxyl radicals that have been shown to oxidize amino aci...

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Abstract

The invention provides formulations comprising a protein in combination with a compound that prevents oxidation of the protein. The invention also provides methods for making such formulations and methods of using such formulations. The invention further provides methods of screening for compounds that prevent oxidation of a protein in a protein composition and methods of preventing oxidation of a protein in a formulation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation application of U.S. patent application Ser. No. 14 / 207,911, filed Mar. 13, 2014, which claims the benefit of U.S. Provisional Application No. 61 / 780,852, filed Mar. 13, 2013, the contents of both of which are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]This invention relates to formulations comprising a protein and further comprising a compound that prevents oxidation of said protein, methods for producing and using the formulations as well as methods of screening for compounds that prevent protein oxidation in protein compositions.BACKGROUND OF THE INVENTION[0003]Oxidative degradation of amino acid residues is a commonly observed phenomenon in protein pharmaceuticals. A number of amino acid residues are susceptible to oxidation, particularly methionine (Met), cysteine (Cys), histidine (His), tryptophan (Trp), and tyrosine (Tyr) (Li et al., Biotechnology and Bioengineer...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28G01N33/68C07K16/24
CPCC07K16/2878C07K16/241C07K2317/52C07K2317/55G01N33/6854A61K39/39591A61K31/405A61K39/39533A61K2039/505C12Q1/28
Inventor ALAVATTAM, SREEDHARAMALLANEY, MARYGREWAL, PARBIR
Owner GENENTECH INC
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