Pressure Selection of Cell Lines: A Key Approach to Obtaining High-Production Stable Cell Lines in Biotechnology. Among the numerous reagents used for pressure selection, Methotrexate (MTX) has attracted significant attention due to its unique mechanism of action and remarkable screening effectiveness. Initially used as an antimetabolite drug for the treatment of cancer and autoimmune diseases, MTX was later found in the field of cell engineering to serve as a potent selection pressure. It induces specific changes in cells to meet the needs of biopharmaceutical production and biotechnology research.

MTX has a chemical structure similar to that of folic acid and can competitively inhibit the activity of dihydrofolate reductase (DHFR). DHFR is involved in the folate metabolism process in cells and is responsible for reducing dihydrofolate to tetrahydrofolate—an essential coenzyme for the synthesis of purine and pyrimidine nucleotides in cells. MTX binds tightly to DHFR, preventing DHFR from normally catalyzing the reduction of dihydrofolate and thus blocking the production of tetrahydrofolate. This leads to insufficient raw materials for the synthesis of purine and pyrimidine nucleotides in cells, severely impairing the synthesis of DNA, RNA, and proteins.

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Under the pressure of MTX, cells initiate a series of stress responses to maintain normal metabolic functions and viability. One important response is the amplification of the gene encoding DHFR. Cells increase the copy number of the DHFR gene to enhance the expression level of DHFR, thereby overcoming the inhibitory effect of MTX on enzyme activity. This gene amplification is not a random event but an adaptive response of cells to a stressful environment. As the concentration of MTX gradually increases, the copy number of the DHFR gene in cells also increases accordingly, enhancing the cells’ tolerance to MTX.

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Meanwhile, other genes closely linked to the DHFR gene may also be co-amplified. This provides an important theoretical basis for the pressure selection of cell lines: through the action of MTX, stable cell lines can be screened that are not only resistant to MTX but also potentially highly express other desired target genes. This helps obtain cell lines capable of efficiently expressing monoclonal antibodies, recombinant proteins, and viral vaccines.

However, as a cytotoxic substance, MTX is hazardous to the human body. It can cause neurological damage, eye injury, and even poses a carcinogenic risk. Therefore, operators must exercise extreme caution when preparing and using MTX. Operations should be conducted in a well-ventilated environment, preferably in a biosafety cabinet, to reduce the spread of drug dust in the air and minimize harm to operators and the surrounding environment. Additionally, personnel involved in preparation must wear appropriate personal protective equipment to prevent MTX from coming into contact with the skin and mucous membranes and avoid inhaling drug dust.The potential hazards of MTX dry powder bring significant inconvenience to practical operations and pose potential risks to the environment and human health.

To address the potential hazards associated with MTX dry powder, Pfanstiehl has grandly launched the world’s first sterile liquid MTX that complies with both USP and BP standards simultaneously. This product meets the biopharmaceutical industry’s requirements for safety and full pharmacopoeial compliance of MTX in cell line development and commercial production. At the same time, considering the practical needs of some special application scenarios, Pfanstiehl continues to offer MTX dry powder products that comply with USP, EP, BP, JP, and ChP standards.

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Like other product lines under the Pfanstiehl brand, Pfanstiehl’s MTX (both dry powder and liquid forms) features ultra-high purity, ultra-low endotoxin levels, ultra-low microbial content, ultra-low metal residues, full pharmacopoeial compliance, and comprehensive regulatory support. These characteristics enable it to meet all requirements of biotherapeutic drugs from early-stage development to late-stage commercial launch.

Product Information of Pfanstiehl MTX

1. Pfanstiehl MTX Dry Powder

·?Product Name: Methotrexate, High Purity, Low Endotoxin, Low Metals, USP, EP, BP, JP, ChP (M-166)

·?Molecular Formula: C??H??N?O?

·?Molecular Weight: 454.44 g/mol

·?CAS Number: 59-05-2

·?Item Number: M-166

·?Packaging Specifications: 25mg, 50mg, 100mg, 500mg, 1g, 5g. Larger specifications are available upon request.

2. Pfanstiehl Sterile Liquid MTX

·?Product Name: Methotrexate Sterile Solution (25.0-27.3 mg/mL), High Purity, Low Endotoxin, Low Metals (M-166-S)

·?Molecular Formula: C??H??N?O?

·?Molecular Weight: 454.44 g/mol

·?CAS Number: 59-05-2

·?Item Number: M-166-S

·?Packaging Specification: 20mL vial, containing 500mg MTX per vial

3. Features of Pfanstiehl Sterile Liquid MTX

·?Ultra-high purity, ultra-low endotoxin levels, ultra-low microbial content, ultra-low metal residues

·?Compliance with multiple pharmacopoeias: USP, EP, BP, JP, ChP

·?GMP-grade, suitable for biopharmaceutical production

Applications of MTX

·?Cell line development

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If you are interested in Pfanstiehl’s GMP-grade MTX (dry powder and liquid forms), please contact Beijing XMJ Technology Co., Ltd.—the authorized distributor of Pfanstiehl in China—to obtain detailed product information and request a quotation.

The latest product series of Pfanstiehl Inc. is as follows. Please contact Beijing XMJ Technology Co., Ltd. to obtain free samples in 100g packaging.

Beijing XMJ?Technology Co., Ltd. is the authorized distributor of Pfanstiehl in China, providing customers with comprehensive technical support and after-sales service. For more information, please call Ximeijie's customer service hotline at 400-050-4006 or visit the website at?www.emergexcelerate.com.