Amine pegylation methods for the preparation of site-specific protein conjugates

A polyethylene glycol aldehyde and protein technology, applied in the fields of peptide/protein components, chemical instruments and methods, animal/human proteins, etc., can solve the problem of reducing patient compliance rate, increasing patient inconvenience, and unsatisfactory patient treatment outcomes And other issues

Inactive Publication Date: 2018-02-16
REZOLUTE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A more frequently administered dosing regimen increases patient inconvenience, can reduce patient compliance, and can lead to suboptimal patient outcomes
If the pharmaceutically active agent is administered by injection, one more injection increases the frequency of pain, risk of infection, and probability of immune response in the patient

Method used

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  • Amine pegylation methods for the preparation of site-specific protein conjugates
  • Amine pegylation methods for the preparation of site-specific protein conjugates
  • Amine pegylation methods for the preparation of site-specific protein conjugates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] At a range of sodium cyanoborohydride concentrations ([NaBH 3 CN]) to determine operating conditions and sodium cyanoborohydride limits for achieving consistent yields of monoPEGylated insulin-PEG conjugates ("mPEGIns"). The following parameters (values ​​in parentheses) were kept constant over a series of experiments: [rhI] 0(0.86mM), [mPEGpropald] 0 / [rhI] 0 (1.04), [EDTA] / [rhI] 0 (0.17-0.18), temperature (28°C), buffer strength (30 mM), and pH (4.0). [rhI] 0 is the initial concentration of recombinant human insulin; [mPEGpropald] 0 is the initial concentration of methoxypropanediol aldehyde; [EDTA] is the concentration of ethylenediaminetetraacetic acid. The starting materials were also the same for each reaction. exist Figure 4 Yields of mono-PEGylated insulin-PEG conjugates are graphed as a function of sodium cyanoborohydride. in [rhI] 0 At this value, mPEGIns yields are shown in [NaBH 3 CN] = 1.0 mM and [NaBH 3 CN] = optimal concentration between 1.5 ...

Embodiment 2

[0050] Chelating Zn in rhI Raw Materials by EDTA 2+ ion, thus dissolving rhI. In order to comply with the United States Pharmacopoeia (USP), the Zn contained in the rhI raw material 2+ Must be less than or equal to 1.00% (w / w). Mimic Zn by substituting an appropriate amount of zinc acetate for a small portion of sodium acetate in the reaction buffer 2+ concentration.

[0051] For a narrow range of [EDTA] / [rhI] 0 experimenting. The following parameters (values ​​in parentheses) were held constant for this series of reactions: [rhI] 0 (0.86-0.88mM), [mPEGpropald] 0 / [rhI] 0 (1.04), [NaBH 3 CN] (1.2 mM), temperature (28°C), buffer strength (30 mM), and pH (4.0). The yield of mPEGIns in each reaction was monitored by RPHPLC analysis, and in Figure 5A This result is shown graphically in [EDTA] / [rhI] 0 The function. Figure 5A The data shown in the [EDTA] / [rhI] of 0.25 0 There is a maximum yield of PEGIns at . [EDTA] / [rhI] greater than 0.175 0 In terms of the value o...

Embodiment 3

[0053] For a larger range of [EDTA] / [rhI] 0 Example 2 was repeated. For repeatability experiments the following parameters were held constant (values ​​in square brackets): [rhI] 0 (0.86mM), [mPEGpropald] 0 / [rhI] 0 (1.07), [NaBH 3 CN] (2.0 mM), temperature (28°C), buffer strength (40 mM), and pH (4.0). exist Figure 5B This set of experiments is given in [EDTA] / [rhI] 0 mPEGIns yield as a function. exist Figure 5B In the range shown in, an increase in [EDTA] / [rhI] was observed 0 Reduced mPEGIns yield.

[0054] With 0.36% (w / w) Zn 2+ rhI to complete the experiments of Examples 2 and 3. Figure 5A Indicates [EDTA] / [rhI] in the range of 0.175 to 0.50 0 Should result in approximately the same yield of mPEGIns. Said Zn 2+ content, [EDTA] / [Zn 2+ ] The corresponding range is 0.55 to 1.56. For this case, [EDTA] / [Zn 2+ ] = 0.55 corresponds to [EDTA] / [rhI] 0 = 0.48. based on Figure 5B Data in [EDTA] / [Zn 2+ ] can have a range upper limit of 2.0. However, when cons...

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Abstract

Examples include a method of making a protein-PEG conjugate. The method may include providing an aqueous protein solution. The aqueous protein solution may include a protein, a pH buffer, and a chelating agent. The chelating agent may be chosen from the group consisting of an aminopolycarboxylic acid, a hydroxyaminocarboxylic acid, an N-substituted glycine, 2-(2-amino-2-oxocthyl) aminoethane sulfonic acid (BES), and deferoxamine (DEF). The method may also include introducing sodium cyanoborohydride and a methoxy polyethylene glycol aldehyde to the aqueous protein solution. The sodium cyanoborohydride in the methoxy polyethylene glycol aldehyde may have a molar ratio ranging from about 5:1 to about 1.5:1. The method may further include reacting the methoxy polyethylene glycol aldehyde withthe protein to form the protein-PEG conjugate. The pH buffer may maintain a pH of the aqueous protein solution ranging from 4.0 to 4.4 during the reaction.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Patent Application Serial No. 62 / 170,933 to Mary S. Rosendahl et al., entitled "Amine Pegylation Process for the Preparation of Site-Specific Protein Conjugates," filed June 4, 2015 Priority to Mary S. Rosendahl et al., entitled "Proteins and Protein Conjugates with Increased Hydrophobicity," U.S. Provisional Patent Application Serial No. 62 / 086,294, filed December 2, 2014 , the entire disclosure of this patent application is incorporated herein by reference for all purposes as if set forth herein. Background technique [0003] Delivering drugs, hormones, proteins, or other pharmaceutically active agents into patients presents several challenges. A pharmaceutically active agent has been delivered into a patient. Two such methods are ingestion and injection. Following ingestion, the drug passes through the patient's digestive system before reaching the bloodstream or targeted a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/00C07K14/62C07K14/00
CPCA61K38/26A61K38/27A61K38/28A61K38/29A61K47/60A61K9/5031A61K31/00C07K14/62
Inventor 玛丽·S·罗森代尔桑卡兰·B·曼崔普拉咖达
Owner REZOLUTE INC
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