Site-Specific Antibody Conjugation via Low-Ionic Strength Buffer
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Solution Overview
Problem
Current methods for site-specific conjugation of antibodies using transglutaminase require deglycosylation, which affects the immunological and biophysical properties of antibodies, and there is a need for methods that preserve N-linked glycans and do not introduce Gln-containing peptide tags.
Innovation Solution
Conjugating glycosylated antibodies with a primary amine compound in the presence of transglutaminase under low-ionic strength conditions, allowing for site-specific conjugation without the need for deglycosylation, using buffers with low sodium phosphate, potassium phosphate, sodium acetate, or Tris at concentrations of 10 mM or less.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If deglycosylation is performed to enable site-specific conjugation with transglutaminase, then conjugation efficiency is improved, but immunological and biophysical properties of the antibody are affected
Solution Approach 1:
The patent changes the ionic strength parameter of the buffer system from conventional high ionic strength to low ionic strength (10 mM or less of sodium phosphate, potassium phosphate, sodium acetate, or Tris). This parameter change enables transglutaminase to catalyze conjugation at Gln295 of glycosylated antibodies without requiring deglycosylation, thereby maintaining both conjugation efficiency and antibody properties
Solution Approach 2:
The patent introduces a specific buffer system with low ionic strength as an intermediary medium that facilitates the interaction between transglutaminase and glycosylated antibodies. This buffer system acts as a mediator that allows the enzyme to access and conjugate at the Gln295 site despite the presence of N-linked glycans, resolving the contradiction between maintaining glycans and achieving efficient conjugation
2Reliability
If traditional buffer conditions are used for transglutaminase conjugation, then enzyme activity is maintained, but conjugation at Gln295 is blocked by N-linked glycans
Solution Approach 1:
The patent modifies the ionic strength parameter of the buffer system, reducing it to 10 mM or less of sodium phosphate, potassium phosphate, sodium acetate, or Tris. This parameter change creates low ionic strength conditions that enable transglutaminase to effectively conjugate at Gln295 of glycosylated antibodies, overcoming the blocking effect of N-linked glycans while preserving enzyme activity
3Productivity
If deglycosylation is performed to remove N-linked glycans, then site-specific conjugation is enabled, but manufacturing complexity increases
Solution Approach 1:
The patent extracts and eliminates the deglycosylation step from the manufacturing process. By using low ionic strength buffer conditions, the method enables direct conjugation of transglutaminase to glycosylated antibodies at Gln295 without requiring the removal of N-linked glycans. This extraction of the deglycosylation step simplifies the manufacturing process while maintaining conjugation specificity
Solution Approach 2:
The patent changes the buffer ionic strength parameter to low levels (10 mM or less), which fundamentally alters the reaction conditions to allow direct conjugation without deglycosylation. This parameter change eliminates the need for additional process steps, thereby reducing manufacturing complexity while achieving site-specific conjugation
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach achieves a high degree of labeling (DOL) of at least 1.8, preserves the biophysical and immunological properties of antibodies, and improves the manufacturability and stability of antibody-drug conjugates by maintaining intact glycans, comparable to traditional methods but with enhanced stability and activity.
Implementation Method 1
These enzymes catalyze covalent bond formation between the ε-amino group of lysine and the γ-carboxamide group of glutamine sidechain of proteins resulting in an isopeptide bond
Implementation Method 2
catalyze covalent bond formation between the ε-amino group of lysine and the γ-carboxamide group of glutamine sidechain of proteins resulting in an isopeptide bond
Implementation Method 3
reacting a glycosylated antibody or a glycosylated Fc fusion protein with an amine compound in the presence of transglutaminase in low-ionic strength conditions
Implementation Method 4
buffers with low sodium phosphate, potassium phosphate, sodium acetate, or Tris at concentrations of 10 mM or less
Data Source
AI summary
Provided herein are methods for site-specific conjugation of glycan intact antibodies by a transglutaminase. According to particular embodiments, the reaction conditions are maintained or reduced to a low-ionic strength condition, which allows for efficient and fast conjugation without the need for antibody deglycosylation. Also described are pharmaceutical compositions and uses related to the conjugation method.