Anti-Clumping Supplement

Transcription

1 Anti-Clumping Supplement 2511 Daimler Street, Santa Ana, California USA Telephone: Fax: PN 4162 Rev.

2 Anti-Clumping Supplement Catalog # 9115 PRODUCT DESCRIPTION Anti-Clumping Supplement is designed to reduce cell aggregation for various cell types in suspension culture. This supplement is animal component and enzyme-free. This supplement is not intended to dissociate adherent cells. 2mL/L of Anti-Clumping Supplement added to HEK293 cultures Without supplement INTENDED USE For research or further manufacturing use only. QUALITY ASSURANCE All quality control test results are reported on a lot specific Certificate of Analysis that is available upon request. STORAGE INSTRUCTIONS AND STABILITY Handle using aseptic techniques to avoid contamination. Store at room temperature or 2-8 C. Do not use any bottle of medium that shows evidence of particulate matter or cloudiness. INSTRUCTIONS FOR USE Anti-Clumping Supplement is designed for use at a 1:2 1:1 dilution (1 5mL/L) range. Typically used at 2mL/L. Supplement additional amount if necessary. With supplement Note: Anti-Clumping Supplement will completely inhibit transfection via cationic liposomes or cationic polymers such as polyethylenimine (PEI). Cells must be spun down and re-suspended in media without Anti-Clumping Supplement before transfection.

3 BalanCD Transfectory CHO Product Manual BalanCD Transfectory CHO is a chemically-defined, animal derived component-free medium optimized for both transient transfection and production of CHO cells. The medium contains no hydrolysates or any other undefined components. This medium is supplied ready to use for suspension culture applications. BalanCD Transfectory CHO is formulated without L-Glutamine, antibiotics, or antimycotics Catalog # Product Size BalanCD Transfectory CHO 1L Liquid Standard BalanCD Transfectory CHO Transfectory Supplement Dry Powder Format, 1L, 1L 5mL Liquid Standard 9115 Anti-Clumping Supplement 5mL Liquid Standard Intended Use For research or further manufacturing use only. Quality Assurance All quality control test results are reported on a lot specific Certificate of Analysis which is available upon request. Storage Instructions and Precautions Liquid Medium Handle using aseptic techniques to avoid contamination. Store at 2-8 C away from light. Do not use after the assigned expiration date. Do not use any bottle of medium that shows evidence of particulate matter or cloudiness. Powder Medium Store at 2-8 C protected from atmospheric moisture. This product is very hygroscopic. Bring it to room temperature before opening and make sure to re-seal tightly after opening. The powder should be free flowing; do not use if it is caked. Do not use after the assigned expiration date.

4 Media Preparation Reconstitution from powder medium 1. Measure 1 ml/l WFI (Catalog # 939 or equivalent) into appropriate sized container. 2. Add g/l of BalanCD Transfectory CHO powder (Catalog # 94129) to water. Mix for approximately 1 minutes. Solution will be slightly cloudy. 3. Add 2.2 g/l Sodium Bicarbonate to solution. Stir for an additional 1 minutes or until solution is clear. 4. Measure ph (expected range ) and osmolality (expected range 28-31mOsm/kg). 5. Sterile filter through a.2 µm filter. 6. Store at 2-8 C for up to 1 year (protect from light). Note: Do not use if solution has precipitated or changed color. 7. Supplement with 2 ml/l of 2 mm L-Glutamine (Catalog # 9317) to reach 4 mm final concentration prior to use. Media Supplements Supplement with 2 ml/l of 2 mm L-Glutamine (Catalog # 9317) to reach 4mM final concentration prior to use. Optional Supplements o 2mL/L Anti-Clumping Supplement (Catalog # 9115): Anti-Clumping Supplement is intended for cell stock maintenance and post transfection cultures only. Cells must be spun down and re-suspended in media without Anti-Clumping Supplement before transfection. Note: Anti-Clumping Supplement will completely inhibit transfection o Poloxamers (Pluronics or Kolliphor ): BalanCD Transfectory CHO medium contains poloxamers but additional.5%-.1% can be supplemented to minimize sheer stress in the cultures. o Transfectory Supplement (Catalog # 91148): Supplement 1% of the initial culture volume one day after transfection to boost cell growth and titer. This supplement is animalcomponent-free.

5 Culture Conditions Medium: BalanCD Transfectory CHO supplemented with 4mM L- Glutamine (Catalog # 9317) Culture vessels: Vented Baffled Polycarbonate Shake Flasks (Corning # or equivalents) Culture volume: 3-4mL in 125mL flasks, 6-8mL in 25mL flasks Seeding density: 2-3 x1 5 cells/ml Incubator setting: 37 C, 5% CO 2, humidified, 14 rpm Cell Recovery and Adaptation 1. Supplement BalanCD Transfectory CHO medium (Catalog # 91147) with 4mM of L-Glutamine (Catalog # 9317) and 2mL/L of Anti-Clumping Supplement (Catalog # 9115). Aseptically transfer appropriate volume (3mL) of supplemented media into a 125mL shake flask and prewarm in the incubator. 2. Thaw frozen vial rapidly in a 37 C water bath. 3. Transfer the cells to a centrifuge tube containing 1mL of cold BalanCD Transfectory CHO medium. 4. Centrifuge for 5 minutes at 2g and decant the supernatant without disturbing the cell pellet. 5. Re-suspend the cell pellet in the medium and transfer the cell suspension to a culture flask with pre-warmed BalanCD Transfectory CHO medium (step 1) to achieve an initial cell density of 3 x 1 5 cells/ml. Incubate cultures for 3-4 days. 6. Cells can be directly adapted into BalanCD Transfectory CHO medium. After a minimum of three passages in BalanCD Transfectory CHO, viable cell density and percent viability should reach above 1.5x1 6 cells/ml and 9% within 4 days, if they are successfully adapted. 7. If severe cell aggregation is observed, increase amount of Anti-Clumping Supplement to 2-4mL/L. 8. If cells grow slowly (less than 1.5x1 6 cells/ml within 4days) with viability below 9%, continue passaging with the following recommendations. o Increase seeding density to.5-1x1 6 cells/ml. o Spin down and re-suspend cells into fresh medium at each passage. o Sequential adaption at ratios of 1:1, 1:2, 1:4, and :1 of the original medium and BalanCD Transfectory CHO medium

6 Subculture 1. Add 4mM of L-Glutamine (Catalog # 9317) to BalanCD Transfectory CHO medium (Catalog # 91147). 2. Subculture cell stocks every two to four days to keep cells in early logarithmic growth phase with a seeding density of 2-3 x1 5 cells/ml. Viable cell density and percent viability should reach above 2x1 6 cells/ml and 9% within 4 days. Note: It is strongly recommended to keep the cultures under 3x1 6 cells/ml to achieve highest transfection efficiencies 3. Anti-Clumping Supplement may not be necessary at low cell densities (<3x1 6 cells/ml), but if cell aggregation is observed, supplement 2mL/L of Anti-Clumping Supplement. o Note: Recommended at a concentration between 5x and 1x (1mL/L 5mL/L) depending on degree of clumping. Anti-Clumping Supplement will completely inhibit transfection. Cells must be spun down and resuspended in media without Anti-Clumping Supplement before transfection. Cryopreservation 1. Prepare required volume of freezing medium: 9% cold BalanCD Transfectory CHO + 1% DMSO. Keep in 4 C until use. 2. Centrifuge appropriate quantity of healthy cells for 5 minutes at 2g and decant the supernatant without disturbing the cell pellet. 3. Re-suspend cells in cold freezing medium to reach 1x1 7 viable cells/ml or desired cell density. 4. Aliquot 1mL or desired volume into sterile cryovials. 5. Gradually lower the temperature of the vials to -8 C at a rate of -1 C/minute. 6. Store vials in liquid nitrogen vapor phase.

7 Transient Transfection Example Method and Data The following PEI-mediated transient transfection protocol can be used as a general guideline to begin optimization work. Since optimal transfection parameters may vary from different culture applications, optimization of transfection parameters is highly encouraged. BalanCD Transfectory CHO medium is also compatible with other transfection methods using cationic liposomes or electroporation. Culture condition used in this example Cells: CHO-3E7 cells (Canadian National Research Council L-11992) Medium: BalanCD Transfectory CHO + 4 mm L-Glutamine Culture vessels: Corning 125mL Erlenmeyer shake flask (Corning # or equivalent) Working Volume: 3mL Seeding density: 2 x1 5 cells/ml Rotation speed: 14 rpm Incubator: 37 C, 5% CO 2, humidified Cell Counts: Beckman Coulter, ViCell XR Cell Viability Analyzer Protein Concentration: Octet QKe system (fortébio) PEI Transfection o Transfection VCD at 3x1 6 cells/ml o DNA to PEI ratio at 1:5 DNA: 1µg/mL in final culture volume, 4:4:15:5 Hc:Lc:Akt:GFP PEI (PEIpro, PolyPlus #115-1 or equivalent): 5µg/mL in final culture volume Note: It is recommended the amount of DNA and PEI be optimized for each application One day post-transfection o Temperature shift to 32 C o Transfectory Supplement: 1% working volume o Anti-Clumping Supplement: 2mL/L Procedure 1. Measure VCD of cell stock and calculate required volume. 2. Centrifuge cells for 5 minutes at 2g and decant the supernatant without disturbing the cell pellet. 3. Re-suspend the cell pellet in the medium and transfer the cell suspension to a culture flask with 27mL of BalanCD Transfectory CHO medium (with 4mM L-Glutamine) to achieve an initial cell density of 2 x 1 5 cells/ml. 4. Incubate cultures for 3 days. VCD should be 2.5-3x1 6 cells/ml. Note: Cells should be in the early logarithmic growth phase for optimal transfection and transfection density should be optimized for each culture application.

8 5. In a separate tube, prepare 3mL of Polyplex (1µg/mL DNA and 5µg/mL PEI) add appropriate volume of DNA and polyethylenimine stocks in BalanCD Transfectory CHO medium. 6. Incubate the mixture at room temperature for 7 minutes and add the entire amount (3mL) to the flask containing 27mL of culture. 7. One day post-transfection, adjust the temperature to 32 C and add 6uL of Anti-Clumping Supplement (2mL/L, Catalog # 9115) to the flask containing 3mL of culture. 8. To achieve higher cell growth and titer, add 3mL of Transfectory Supplement (1% of culture volume, Catalog # 91148) one day after transfection. 9. Monitor and maintain glucose level at a 2-8g/L range throughout the culture. Example Data Viable Cell Density (x1 6 cells/ml) Day Post Transfection BalanCD Transfectory CHO with 1% Transfectory Supplement (VCD) BalanCD Transfectory CHO (VCD) BalanCD Transfectory CHO with 1% Transfectory Supplement (% Viability) BalanCD Transfectory CHO (%Viability) % Viability Protein Concentration (mg/l) Days Post Transfection BalanCD Transfectory CHO with 1% Transfectory Supplement BalanCD Transfectory CHO Figure 1. PEI-mediated transient transfection in shake flasks - cell growth and protein production. 215 Irvine Scientific. All rights reserved. BalanCD and Irvine Scientific are registered trademarks of Irvine Scientific Sales Company, Inc. Transfectory is a trademark of Irvine Scientific Sales Company, Inc. All other trademarks are the property of their respective owners. Irvine Scientific 2511 Daimler Street, Santa Ana, California USA Telephone: Fax: Irvine Scientific P/N 164ICC Rev.1

9 BalanCD TRANSFECTORY CHO SYSTEM Rapid, scalable, high yield protein production Day 14 Antibody Concentration (mg/l) Choose the protocol that suits your needs Improve workflow Ensure process consistency BalanCD BalanCD Transfectory CHO Transfectory CHO + Supplement Medium A The BalanCD Transfectory CHO system of medium and supplements is a scalable platform that produces milligram to gram-scale yields of recombinant proteins using a simplified, affordable protocol. The system supports small-and large-scale transient transfection in CHO cells enabling scientists to work with CHO cells from early phase discovery through to production. Figure 1. The BalanCD Transfectory CHO system supports sustained, balanced growth resulting in high protein yields. Improved productivity and growth compared to other commercially available media Viable cell density (x1 6 cells/ml) 1 Viable cell density Percent viability Post-transfection cell viability Protein production (mg/l) Antibody production Days post transfection Days post transfection Days post transfection BalanCD Transfectory CHO + Supplement BalanCD Transfectory CHO Medium A Medium B Medium C Figure 2. CHO-3E7 Cells were cultured in duplicate 125mL Erlenmeyer flasks with a 3mL working volume. PEI-mediated transfection was performed on day 3 to produce a biosimilar antibody. Transfectory Supplement was added to one of the BalanCD Transfectory CHO test conditions one day after transfection. Cultures in Medium B were terminated on day 7 due to poor transfection. Cultures in Medium C were terminated on day 9 due to poor viability. All data shown are the average of duplicate cultures.

10 Choose the protocol that suits your needs Minimize risk of adventitious agents The chemically-defined, animal component-free medium produces protein levels equivalent to, or better than, commercially available media containing hydrolysates. Maximize protein yield When the priority is protein yield, add Transfectory Supplement, a concentrated hydrolysate supplement that increases cell growth and protein expression level while maintaining viability and efficiency. Improve workflow Avoid switching from HEK293 to CHO cells during development phases Rapidly achieve high protein yields to save time and development costs compared to conventional stable cell line development Eliminate the need to exchange medium before or after transfection using the simplified, scalable protocol Compatible with most commercially available and affordable transfection reagents such as polyethylenimine (PEI) Ensure process consistency The formulation of BalanCD Transfectory CHO is based on the BalanCD CHO platform of growth media and feeds to provide process consistency through all phases of development, and at all production scales. Ordering information PRODUCT CATALOG # SIZE* BalanCD Transfectory CHO liquid L liquid BalanCD Transfectory CHO powder L dry powder Transfectory Supplement mL Anti-Clumping Supplement mL *Custom sizes and packaging available on request Corporate Headquarters 183 E Warner, Santa Ana, CA 9275, USA Tel: getinfo@irvinesci.com Irvine Scientific 158ICC Rev.1 All rights reserved. Transfectory and BalanCD are trademarks of Irvine Scientific Sales Company Inc.