Hybridoma Re-cloning Service

Monoclonal antibodies (mAbs) production from established hybridoma cell lines can diminish over time due to the decrease of the number of mAb-secreting cells. In many cases, mAb production can be rescued by re-cloning the hybridoma cell line and selecting out new highly antibody-positive clones. The clone that exhibits the best growth characteristics and produces the highest antibody titer will be expanded and frozen. As a well-recognized leader in antibody preparation, Creative Biolabs is dedicated to providing hybridoma developing services and offers high-quality re-cloning services for our clients all worldwide.

Hybridoma Rescue by Re-Cloning

Identifying hybridoma cell lines that have good manufacturing potential is crucial for successful hybridoma production. To accomplish this, fused cell pools must be separated and cloned into monoclonal cultures. In general, thorough fusion screening and clone selection are the most important steps for an efficient and successful hybridoma program. The best clones should be stable and good producers that demonstrate specific attributes based on the product specifications.

Limited Dilution

Over the years, limited dilution is the most commonly used method of hybridoma cell cloning in the preparation of mAbs. Cloning by limited dilution is a method based on the Poisson distribution. Dilution of cells to an appropriate number per well can maximize the proportion of wells that contain one single clone. Two or more cloning procedures are carried out until >90% of the wells contain single clones that are positive for antibody production.

Major Advantages of Limited Dilution

Enabling visual identification of some cell characteristics. Larger colonies will mean faster-growing clones and smaller colonies mean slower-growing clones.
Supporting culture survival during expansion, as thousands of cells per colony are seeded into each well.

Semi-solid Cloning

With advancements in stable cell line development and successful alternatives available, semi-solid media has emerged as an alternative to limited dilution in liquid media. Using semi-solid re-cloning allows a great number of cells to be plated at the same time, while the high viscosity of the medium ensures individual cells grow into discrete colonies. The colonies are then isolated and moved to liquid media in 96 well plates or other vessels for expansion and further screening.

Fig.1 Clone selecting with semi-automation.¹

Automation System

Beside limited dilution and semi-solid medium approach, automation systems for monoclonal selection have become more and more popular. Automated systems couple software with imaging systems, which allows scientists to program the criteria for the cells they are interested in. The machine then identifies which cells meet the set criteria and, through the use of robotics, isolates and plates these cells of interest without the need for manual pipetting. The automated screening system developed by Creative Biolabs employs different methods for identifying clones of interest. Automating system enables high throughput, so more clones can be screened overall, increasing the probability of finding a high-producing clone.

MAb production in hybridoma cells is an important step in biopharmaceutical manufacturing, as well as being a crucial tool for large-scale antibody production for research, diagnostic and industrial applications. Obtaining a high-producing cell line by re-cloning can save your time and help you avoid the loss of valuable clones. Limited dilution, semi-solid medium, and automation system are all available at Creative Biolabs. If you are interested in our re-cloning service right now, please contact us for more information and a detailed quote.

Reference

Soitu, Cristian, et al. "Raising fluid walls around living cells." Science Advances 5.6 (2019): eaav8002.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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