Hybridoma Cell Selection via Intracellular BirA Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The existing methods for selecting hybridoma cells for monoclonal antibody production are inefficient and time-consuming due to the heterogeneity of cell pools and the need for external biotin-protein ligase, which requires optimization for each batch, leading to unstable and uncontrollable processes.
Innovation Solution
Integration of biotin protein ligase (BirA) into hybridoma cells via expression vectors for intracellular biotinylation of surface proteins, allowing for in vivo biotinylation under natural conditions, eliminating the need for external enzymes and optimizing cell viability and selection processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If external biotin-protein ligase is used for biotinylation, then biotinylation can be achieved, but the process becomes unstable and requires optimization for each batch
Solution Approach 1:
The patent merges the biotin-protein ligase function with the hybridoma cell by introducing a polynucleotide encoding BirA that integrates into the cell genome. This combination eliminates the need for external enzyme addition and ensures consistent intracellular biotinylation across all batches, directly resolving the instability issue.
Solution Approach 2:
The hybridoma cell becomes self-sufficient by producing its own biotin-protein ligase through the integrated polynucleotide. The cell autonomously performs biotinylation of surface proteins without requiring external enzymes, eliminating batch-to-batch optimization needs and improving process reliability.
2Ease of manufacture
If external biotin-protein ligase is added, then biotinylation occurs, but additional reagents and optimization steps are required
Solution Approach 1:
The hybridoma cell produces its own biotin-protein ligase internally, eliminating the need to add external biotin-protein ligase reagents. This self-service approach simplifies the manufacturing process by removing multiple addition steps and optimization requirements while reducing the quantity of external substances needed.
3Productivity
If hybridoma cells are selected from heterogeneous pools, then antigen-specific cells can be identified, but the process is time-consuming and inefficient
Solution Approach 1:
The patent uses fluorescence-labeled detection antibodies that bind to biotinylated surface proteins, creating a fluorescent signal that allows rapid identification of antigen-specific hybridoma cells. This fluorescent labeling enables quick visual differentiation and selection from heterogeneous pools, dramatically improving selection efficiency and reducing time loss.
Solution Approach 2:
The patent replaces manual or mechanical cell selection methods with fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). These automated sorting systems use fluorescent or magnetic labels to rapidly separate antigen-specific cells from heterogeneous pools, significantly increasing productivity and reducing selection time compared to traditional manual methods.
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 standardizes biotinylation, reduces the need for external reagents, and enhances the stability and efficiency of hybridoma cell selection, enabling more reliable and efficient identification and isolation of antigen-specific hybridoma cells.
Implementation Method 1
the binding of an adapter ligand can take place, namely preferably biotin, that is enzymatically conjugated with the ligation peptide sequence (or biotinylation peptide) under addition from full medium under gentle conditions by means of a BirA (biotin protein ligase) ligase
Implementation Method 2
The antibody released from the hybridoma cell binds to biotin via an antigen-(strept)avidin complex containing an epitope, whereby the antibody binds to the epitope of the antigen and, via the streptavidin-biotin bond, can in turn be assigned to the hybridoma cell
Data Source
AI summary
The invention relates to an improved method for the selection of hybridoma cells from a plurality of hybridoma cells for the generation of monoclonal antibodies as well as hybridoma cells together with suitable expression vectors by means of intracellular biotinylation and the use thereof.