Proteolysis resistant antibody preparations

A protease and elastase technology, applied in the preparation of non-sialylated glycoforms of antibodies, in the field of preparation and use of substantially uniform glycoforms, and can solve the problems such as the existence and composition of glycans that are not proved

Inactive Publication Date: 2009-10-14
CENTOCOR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The relationship between glycan presence and composition and IgG susceptibility to proteolytic cleavage in humans or other species has not been ascertained so far

Method used

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  • Proteolysis resistant antibody preparations
  • Proteolysis resistant antibody preparations
  • Proteolysis resistant antibody preparations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Isolation of FC domain from IgG

[0084] Papain was obtained from Sigma and PNGase F (peptide N-glycosidase F) was obtained from NewEngland Biolabs. Sinapinic acid was obtained from Fluka. MALDI-TOF-MS analysis was performed with a VoyagerDE Biospectrometry workstation (Applied BioSystems, Foster City, CA).

[0085] Antibody samples were deglycosylated by treatment with PNGase F in 20 mM Tris-HCl buffer (pH 7.0). Deglycosylated antibody samples were purified on a protein A column (HiTrapA protein cartridges were obtained from Amersham Biosciences) and analyzed for purity by MALDI-TOF-MS.

[0086] Each antibody sample (~1 mg / ml, samples before and after deglycosylation) was treated with papain (1:50, w / w) in 20 mM Tris-HCl buffer (pH 7.0) containing 2 mM L-cysteine For treatment, aliquots were taken at regular time intervals (0, 15, 30, 60, 90 minutes, then 2, 3, 4, 5, 6, 8 and 24 hours). Each aliquot (about 2 μl) was immediately mixed with 2 μl of matrix ...

Embodiment 2

[0090] Example 2: Papain Digestion of Homogeneous Glycoforms

[0091] To evaluate the parameters of papain digestion of antibody preparations that are substantially homogeneous in their glycosylation pattern, antibody samples were enzymatically modified as described below to generate such preparations for testing.

[0092] To galactosylate purified antibody samples by enzymatic means, bovine β-1,4-galactosyltransferase (β1,4GT) obtained from Sigma Chemical Co. (St. Louis, MO) and UDP-Gal Added to antibody sample. Recombinant rat liver α-2,3-sialyltransferase (α2,3ST), recombinant α-1,3-galactosyltransferase (α1,3GT) and CMP-Sia were obtained from Calbiochem (San Diego, CA) . PNGase F was obtained from New England Biolabs (Beverly, MA) or from Prozyme (San Leandro, CA) or from SelectinBioSciences (Pleasant Hill, CA). Diplococcus pneumoniae β-galactosidase and β-glucosaminidase were obtained from ProZyme or from SelectinBioSciences. Bovine kidney β-galactosidase and all othe...

Embodiment 3

[0100] Example 3: Production of Antibody Fragments Using Matrix Metalloproteinase-3

[0101] Metalloproteases (MMPs) were purified from supernatants of cell clones expressing recombinant human MMPs. The enzyme was activated by treatment with 1 mM 4-aminophenylmercuric acetate (APMA; Sigma) at 37° C. for 1 hour or by treatment with chymotrypsin. Activated enzymes were stored at -70°C. Mix immunoglobulin preparation (0.5-1.0mg / ml) with digestion buffer (250mM Tris-HCl, pH 7.4, containing 1.5M NaCl, 50mM CaCl 2, containing 15-60 μg / ml activated MMP) were incubated at 37°C for 0-24 hours. Aliquots were extracted at 0, 15, 30, 45, 60 and 120 minutes and then at 3, 4, 5, 6, 8, 12 and 24 hours. An aliquot (about 2 μl) was mixed with matrix solution (about 2 μl), and 2 μl of the resulting mixture was applied to the MALDI-TOF-MS target plate, and then MALDI-TOF-MS was performed with a Voyager DE spectrometer. MS analysis.

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Abstract

Antibody preparations with substantially homogeneous and unsialylated glycoforms, such as G0 and G2, are prepared by enzymatic treatment, expression under certain conditions, use of particular host cells, and contact with serum. These antibody preparations resist cleavage by proteases, such as papain, ficin, bromolein, pepsin, a matrix metalloproteinase, such as MMP-7, neutrophil elastase (HNE), stromelysin (MMP-3) and macrophage elastase (MMP-12), and glycosylation modification enzymes. The antibody preparations with substantially homogeneous and unsialylated glycoforms and methods of testing for glycosylation in an antibody are useful in connection with characterization of antibody properties and / or in diseases or conditions characterized by an increase in protease activity.

Description

field of invention [0001] The present invention relates to assessing the glycoform content of antibodies, and more particularly to methods of making and using antibody preparations having substantially homogeneous glycoforms, such as non-sialylated glycoforms. technical background [0002] Antibodies are soluble serum glycoproteins that play an important role in innate immunity. The carbohydrate structures at conserved positions in the heavy chain constant region of all naturally occurring antibodies vary with isotype ( figure 1 ). Each isoform has a unique set of N-linked oligosaccharide structures that have differential effects on protein assembly, secretion or functional activity (Wright, A. and Morrison, S.L., Trends Biotech. 15:26-32( 1997)). The structure of the attached N-linked oligosaccharide ( figure 2 ) vary widely depending on the degree of processing and can include high mannose as well as complex biantennary oligosaccharides with or without bisecting GlcNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395C12Q1/37
Inventor T·S·拉于B·斯卡伦
Owner CENTOCOR
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