mFISH/FISH analysis
-As an evaluation technique for cells used in biopharmaceutical production-

Chromosome Analysis and mFISH/FISH analysis will be handled by Sumitomo Chemical Co., Ltd. from January 10, 2023.

contents

1. Applications of mFISH/FISH analysis 1 ( Evaluation of monoclonality study in cell banks )
2. Applications of mFISH/FISH analysis 2 ( Confirmation of transgenes in various stages of the cell bank construction )
3. An example data of mFISH/FISH analysis
4. To whom interested in monoclonality study
5. Frequently asked questions
6. Order Flow
7. Partner Company

CHO cells and HEK293 cells, which are typically used in biopharmaceutical production, have structurally abnormal chromosomes. Such abnormal chromosomes hamper the detection of gene integration sites upon the chromosomes. The combinatorial use of multi-color FISH (mFISH) and FISH enables the accurate evaluation of gene integration sites even in the cells with these abnormal chromosomes.

Applications of mFISH/FISH analysis
1. Evaluation of monoclonality study in cell banks

According to ICH^＊ Q5D guideline, monoclonality is defined as production cells start from a single cell. Recently, FDA describes that information should be included in the regulatory submission to support the clonality of the production cell bank. mFISH/FISH analysis was introduced in the FDA presentation published in 2016, as one of the methods to obtain monoclonality data based on gene integration sites. Therefore, it is recommended to apply mFISH/FISH analysis for obtaining these data for the cell banks.

＊ICH stands for International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use. The representatives from regulatory sections and pharmaceutical industrial sections co-operates to create regulatory guidelines related to pharmaceutical products.

Applications of mFISH/FISH analysis
2. Confirmation of transgenes in various stages of the cell bank construction

Following are some examples for mFISH/FISH analysis

• Investigation of new culture media and cloning methods
• Confirmation of transfection such as target-specific gene transfection
• Pre-screening of cell bank development
• Comparison of cell banks during pre- and post-production (MCB, WCB and EPCB^＊）
＊Post-production cells are generally defined as EPCB, PPCB and CAL.

An example data of mFISH/FISH analysis

mFISH: to visualize structural changes of chromosomes
FISH: to detect a target gene on the chromosomes
The combination of both techniques maps transgenes in the cells with structurally abnormal chromosomes.
The example case of mFISH/FISH analysis in CHO cells is shown below.

A: An image of post-mFISH metaphase. The structurally abnormal chromosome is represented with multiple colors in one chromosome.
B: An mFISH karyogram. Each chromosome is defined as chromosome A, B, C, etc. and arrayed to create a karyogram.
C: An image of post-FISH metaphase. mFISH and FISH results could be compared chromosome-to-chromosome on the same metaphase image.
D: A FISH karyogram. Each chromosome is arrayed in the same manner as in mFISH.
E: The position of FISH signal is mapped. In this cell, the FISH signal was mapped at the chromosome 8-derived region on the short arm of chromosome M.

* A white arrow indicates the chromosome in which the FISH signal was detected.

To whom interested in monoclonality study

We provide consultation with clients for a study plan in comply with your schedule, criteria, objectives and budget. After the consultation, we present the experimental design which fulfills your priorities. We also explain the degisn in details in case such as,

・Planning investigation before starting the experiments
・Considering long-term projects
・Requesting an express report of the analysis result
・Considering studies in accordance with guidelines or approval documents
・Requiring documents or asking questions related to karyotypic analysis

Frequently asked questions

Q: How many metaphase cells are analyzed for a study?
A: The number varies from one study to another according to their purposes. The study generally starts from 20-cell analysis. Many clients request the analysis for more than 100 cells to evaluate monoclonality study. Some clients require the 100-cell analysis after confirming the results from the 20-cell analysis.

Q: Which type of cell banks is suitable for monoclonality study?
A: Please refer to the following information in your request.
(1) Master Cell Bank (MCB) only
(2) A set of MCB and Working Cell Bank (WCB)
(3) A set of MCB, WCB and post-production cell bank (EPCB, PPCB, CAL)

Q: What do I need to provide for the analysis?
Please deliver test cells, culture media with supplements (if necessary) and DNA for FISH probes.
Refer the description below for more details.

[Test Cells]
We receive test cells in cryopreserved stocks. Please provide culture medium and supplements.

[DNA for FISH probes]
Please prepare DNA for FISH probe production.
We receive DNA as follows.
(1) plasmid DNA
(2) DNA fragments which are processed from PCR or restriction enzyme digestion and isolated by an agarose-gel extraction.

We produce FISH probes from these DNA, so please dissolve DNA with a sterile water. DO NOT use elution buffer from a purification kit.

・If you want us to perform PCR, please send a DNA template and primers. Outsourcing of primers is also available. We produce FISH probes after performing PCR and isolating the PCR fragment from an agarose-gel.
・If you can provide a PCR product, we will isolate the PCR fragments from an agarose-gel and produce FISH probes.
・If you can provide a labeled FISH probe i.e. ready-to-use. Please send control cells along with test cells.
Price and fees vary with experiments, please consult us for the request.

Q: Which type of cell culture is performed during the analysis?
A: Standing culture and shaking culture are available.Please inform us of culture conditions and cell seeding density.

Q: Is your test facility available for a tour?
A: Yes, it is. Please contact us and give us at least 1 month for schedule adjustment.

Order Flow

1. Getting an estimate

Please contact us freely for any questions via web meeting or emails.

We accept test cells cryopreserved in vials. If you are are shipping them in a dry shipper, please be prepared to make return arrangements. For details, please inquire at the time of request.

2. Submitting the order after the quotation confirmation

Please contact us before shipping test cells in advance. We generally prepare for the reception and sometimes need to answer to the customs once you ship them.

3. Receiving the study report

Delivery date depends on a number of test cells, delivery type of test cells (vial, flask or carnoy fixed) and a number of metaphase analyzed. Please contact us for more details.

Ex)
10 weeks for 1 test cell as a cryopreserved stock with 100-cell analysis
12 weeks for 2 test cells as cryopreserved stocks with 100-cell analysis

■ Partner Company

In the following country, please consult your local dealer.

Korea:B2Bio,Inc

Click here to download PDF documents
http://www.b2bio.co.kr/(Go to B2bio’s page.)