OptiCHO Antibody Express Kit
Contents
1. Materials Needed 10 Follow the protocol below to thaw DG44 cells Cells are supplied in a vial containing 1 ml of cells at 1x 10 viable cells ml in 90 freezing medium and 10 DMSO Thaw DG44 cells directly into CD DG44 Medium supplemented with 8 mM L glutamine and 18 ml L Pluronic F 68 Do not thaw and grow DG44 cells in CD OptiCHO Medium Parental or untransfected DG44 cells are DHFR negative and require supplementary hypoxanthine and thymidine present in CD DG44 Medium All solutions and equipment that come in contact with the cells must be sterile Always use proper sterile technique and work in a laminar flow hood Supplement CD DG44 Medium to a final concentration of 8 mM L glutamine and 18 ml of 10 Pluronic F 68 per liter before use see page 5 Addition of antibiotics is not recommended CD DG44 Medium is light sensitive For optimal results store medium at 4 C protected from light You should have the following reagents and materials before beginning Frozen DG44 cells supplied with the kit store frozen cells in liquid nitrogen until ready to use Complete CD DG44 Medium prepared as above pre warm at 37 C before use 125 ml polycarbonate disposable sterile Erlenmeyer flasks with vented caps available from VWR West Chester PA cat no 30180 036 Orbital shaker set at 130 135 rpm in 37 C incubator with a humidified atmosphere of 8 CO Continued on next page Thawing and Subculturi2. maintain culture at 3 x 10 viable cells Be sure to monitor antibody production Because MTX selection produces a population of cells producing high amounts of protein you will have to perform clonal selection by limiting dilution page 19 prior to clone scale up Clonal Selection in Semi Solid Media Introduction To obtain a clonal cell line e derived from a single cell you will dilute your pool of stably transfected cells or your MTX amplified cells to 1 cell per well in a 96 well plate containing a semi solid cloning matrix The semi solid cloning matrix improves cloning efficiency compared to liquid culture In most cases a distinct colony will arise from each single cell which can be scaled up using the procedure on page 21 Cloning e Depending on how many clones you wish to screen before scale up you may Considerations increase the number of clonal selection plates accordingly The number of clones obtained from a 96 well plate is variable depending on the experiment e The growth and protein production of each clone are variable To increase your chances of obtaining one or more high producing clones you may use automated sorting methods such as FACS or robotic methods such as ClonePix to screen more clones Materials Needed You should have the following reagents and materials on hand before beginning e CloneMatrix 40 ml semi solid concentrate available from Genetix www genetix com e CloneXL Reagent incl
3. Cells not frozen correctly Follow the protocol on page 12 to freeze cells Incorrect thawing medium e Use pre warmed complete CD DG44 Medium supplemented with 8 mM L glutamine and 18 ml L Pluronic F 68 DO NOT USE CD OptiCHO Medium to propagate DHFR negative DG44 cells e Do not add antibiotics to media as this may negatively impact cell growth e Incubate cultures on an orbital shaker set at 130 135 rpm in a 37 C incubator with a humidified atmosphere of 8 CO Cells grow slowly Incorrect growth medium e Use pre warmed CD DG44 Medium supplemented with 8 mM L glutamine and 18 ml L Pluronic F 68 e DO NOT USE CD OptiCHO Medium to propagate DHFR negative DG44 cells Shaker not set up properly Shake on an orbital shaker at 130 135 rpm in 37 C incubator with a humidified atmosphere of 8 CO Medium is foamy Keep shaker speed at 130 135 rpm Cells too old Use healthy DG44 cells under passage 25 do not overgrow Cell culture clumpy Provide agitation of the culture a regular and frequent cell passage schedule and maintenance of cells at recommended densities 22 Continued on next page Troubleshooting continued Transfection The table below lists some potential problems and possible solutions that may help you troubleshoot your transfection experiments Problem Reason Solution Very few or no stably Improperly cu
4. The pOptiVEC TOPO TA and pcDNA 3 3 TOPO TA cloning reagents are shipped on dry ice in two separate boxes containing the cloning reagents and the One Shot TOP10 Chemically Competent E coli kits Refer to the manuals for pOptiVEC TOPO TA Cloning Kit and pcDNA 3 3 TOPO TA Kit for detailed descriptions of these kit contents Store the TOPO reagents at 20 C and the One Shot TOP10 Chemically Competent E coli kit at 80 C DG44 cells are provided as one vial shipped on dry ice containing 1 x 10 cells in 1 ml freezing medium containing 10 DMSO Important Upon receipt immediately store in liquid nitrogen TM The OptiCHO Antibody Express Kit components are shipped under various conditions as listed below Store components as indicated Item Amount Shipping Storage FreeStyle MAX Reagent 1ml Blue ice 4 C Do not Freeze OptiPro SFM 100 ml Room temperature 4 C CD DG44 Medium 1000 ml Room temperature 4 C in the dark CD OptiCHO Medium 1000 ml Room temperature 4 C in the dark Geneticin 50 mg ml 100 ml Room temperature 4 C L glutamine 200 mM 100 ml Dry ice 20 C Pluronic F 68 10 100 ml Room temperature Room temperature 1v Accessory Products Introduction Gibco Custom Media The products listed in this section may be used with the OptiCHO Antibody Express Kit For more information go to www invitrogen com or contact Technical Suppor
5. e FreeStyle MAX Reagent A proprietary animal origin free formulation for high transfection efficiency of plasmid DNA into DG44 cells See page 6 for more information e CD OptiCHO Medium Defined serum free medium formulated for selection and growth of DG44 cells expressing DHFR and the recombinant protein of interest See page 7 for more information The kit also contains OptiPro serum free medium SFM for optimal DNA lipid complex formulation L glutamine provided separately for increased media stability Geneticin for stable cell line selection and the surfactant Pluronic F 68 to control shear forces in suspension culture Overview continued Advantages of the OptiCHO Antibody Express Kit TOPO TA Cloning Vector Kits TM The OptiCHO Antibody Express Kit provides the following advantages for antibody production in mammalian cells e DHFkR deficient DG44 cells derived from CHO cells Urlaub et al 1983 which provide stable and accurate glycosylation Sheeley et al 1997 Werner et al 1998 and are more likely to yield accurate glycoproteins TM e FreeStyle MAX Reagent offers high transfection efficiency of suspension CHO cells with low cytotoxicity e FreeStyle MAX Reagent CD DG44 Medium and CD OptiCHO Medium are animal origin and serum free The OptiCHO Antibody Express Kit contains the pOptiVEC TOPO TA Cloning Kit and the pcDNA 3 3 TOPO TA Cloning Kit also available separ
6. 828 6686 TM FreeStyle MAX Transfection Reagent is tested for the absence of microbial contamination using blood agar plates Sabaraud dextrose agar plates and fluid thioglycolate medium and functionally by transfection with a reporter plasmid OptiPro SFM is subjected to pH osmolality bacterial and fungal testing OptiPro SFM is performance tested using VERO cells pre adapted to serum free culture in OptiPro SFM Gibco cell culture liquid products are prepared by an aseptic process for which each step has been validated to ensure that all products meet the industry standard sterility assurance level of 10 i e product that demonstrates a contamination level of no more than 1 of 1000 units during the manufacturing process The highest level of sterility assurance equal to or greater than 10 cannot be achieved without terminal sterilization which is harmful to the performance of cell culture products References Kaufman R Wasley L Spiliotes A Gossels S Latt S Larsen G and Kay R 1985 Coamplification and coexpression of human tissue type plasminogen activator and murine dihydrofolate reductase in Chinese hamster ovary cells Mol Cell Biol 5 1750 1759 Puck T 1958 J Exp Med 108 945 Sheeley D M Merrill B M and Taylor L C 1997 Characterization of monoclonal antibody glycosylation comparison of expression systems and identification of terminal alpha linked galactos
7. Optimize codons for CHO cells 23 Appendix Technical Support Web Resources Visit the Invitrogen web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Limited Warranty 24 MSDSs Material Safety Data Sheets are available on our web site at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisf
8. caps containing 30 ml of pre warmed complete CD DG44 Medium 1 Determine viable and total cell counts 2 Using the cell density determined in Step 1 calculate the split ratio needed to seed each new shaker flask at 2 x 10 viable cells ml by dilution 3 Dilute the cells in 30 ml of pre warmed complete CD DG44 Medium to give a final cell density of 2 x 10 viable cells ml 4 Incubate flasks in a 37 C incubator containing a humidified atmosphere of 8 CO in air on an orbital shaker platform rotating at 130 135 rpm 5 Repeat Steps 1 4 as necessary to maintain or expand cells After 25 passages you should thaw a new vial of cells To maintain sufficient stocks of low passage cells e under 25 passages be sure to freeze aliquots of DG44 cells in liquid nitrogen See the next section for instructions on cryopreserving cells 11 Freezing DG44 Cells Introduction Materials Needed Preparing Freezing Medium Freezing Cells 12 You may freeze DG44 cells directly in CD DG44 Medium with 10 DMSO We recommend that you freeze cells at a density of gt 1 x 10 viable cells ml Guidelines to prepare freezing medium and to freeze cells are provided in this section You will need the following reagents and equipment before starting Complete CD DG44 Medium Tissue culture grade DMSO Reagents and equipment to determine viable and total cell counts Sterile labeled cryovials Sterile 15 ml conical tubes Aut
9. 30 ml Place flask in shaker until ready to transfect Note Do not spin cells down prior to transfection as it will decrease transfection efficiency TM Gently invert the tube of FreeStyle MAX Reagent several times to mix Do not vortex Add 18 ug of plasmid DNA 9 ug of each construct and 15 pl of FreeStyle MAX Reagent into 1 2 ml of OptiPro SFM and mix gently TM Incubate the DNA FreeStyle MAX mix for 10 minutes at room temperature to allow complexes to form Do not incubate for longer than 20 minutes Slowly add 1 2 ml of DNA FreeStyle MAX Reagent complex into the 125 ml flask containing cells while slowly swirling the flask Incubate transfected cell cultures at 37 C 8 CO on an orbital shaker platform rotating at 130 135 rpm At 48 hours post transfection pass cells into HT deficient complete CD OptiCHO Medium see page 7 Proceed to the next section Selecting for Stable Transformants 15 Selecting for Stable Transformants Introduction Geneticin Protocol Note Next Steps 16 To obtain cell lines that produce high levels of your protein you will first need to select for a pool of stably transfected cells in which the linearized pOptiVEC and pcDNA 3 3 constructs have integrated into the host cell genome You will perform two rounds of selection using CD OptiCHO Medium and CD OptiCHO Medium with 500 pg ml Geneticin Re CD OptiCHO CD OptiCHO Geneticin 48 h
10. Determine the number and viability of your cells 2 Dilute cells to 2 000 cells ml in CD OptiCHO Medium and add 500 ul of diluted cells to 100 ml of complete semi solid cloning medium 3 Gently mix the bottle and cells by inversion to evenly distribute cells throughout the matrix and let the bottle stand for about 5 minutes at room temperature 4 Using a multichannel pipettor and a sterile reservoir pipette 100 ul of diluted cells into each well of a 96 well plate to obtain 1 cell well 5 Incubate plates undisturbed without agitation at 37 C with a humidified atmosphere of 5 CO for 11 days 1 At 11 days post plating screen the wells of your plates for single clones using an inverted microscope Clones will appear as distinct round colonies in the wells either semi adherent or floating in the matrix Mark the bottom of the plate to indicate wells that contain single clones Important Screen clones visually before adding media in the next step so as not to disturb single colonies 2 Add 100 pl of CD OptiCHO Geneticin to all wells and incubate at 37 C 5 CO for 3 days This is to loosen the matrix so that clones can be transferred 3 After 3 days transfer the marked clones by pipetting gently up and down and transferring the volume to 48 well plates Wash each well with 200 pl of CD OptiCHO Medium Geneticin to make sure all cells are transferred 4 Incubate plates at 37 C 5 CO for 3 days then procee
11. Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing lifetech com 25 Product Qualification Introduction DG44 Cells CD DG44 Medium CD OptiCHO Medium FreeStyle MAX Reagent OptiPro SFM 26 This section describes the criteria used to qualify the components of the OptiCHO Antibody Express Kit DG44 cells are performance tested for viability and cell growth post recovery from cryopreservation and are screened for mycoplasma and sterility Seed Banks are screened for viruses as identified in the US Code of Federal Regulations Full 9 CFR Testing mycoplasma and sterility Species identity is conformed by isozyme and karyotype analysis CD DG44 Medium is performance tested in a growth assay using CHO S cells in a dynamic culture system Additional standard evaluations are endotoxin pH osmolality and tests for the absence of bacterial and fungal contaminants For individual lot test results and more information contact Gibco Technical Service at 1 800 828 6686 TM CD OptiCHO Medium is performance tested in a growth assay using CHO S cells in a dynamic culture system Additional standard evaluations are endotoxin pH osmolality and tests for the absence of bacterial and fungal contaminants For individual lot test results and more information contact Gibco Technical Service at 1 800
12. O S cells and FreeStyle CHO Expression Medium are available separately from Invitrogen see page v The pOptiVEC and pcDNA 3 3 plasmid constructs must be clean sterile and free from contamination with phenol and sodium chloride for transfection into cells Contaminants may kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the PureLink HiPure Plasmid Midiprep DNA Kit see page v for ordering information Note Plasmids may be linearized or circular for transient transfection For stable transfection of DG44 cells we recommend linearizing the plasmid see page 13 TM To transiently transfect FreeStyle CHO S cells use equal amounts of each pOptiVEC and pcDNA 3 3 plasmid DNA constructs containing the heavy and light chains of your antibody and follow the recommended protocol included with your CHO cells and transfection reagent After transfection culture cells for 5 7 days no medium change is required and assay for antibody production using your method of choice see below To check for production of your antibody after transient transfection you may take an aliquot of growth media and perform SDS PAGE protein specific ELISA or the bioactivity assay of choice to determine that your cells are producing your antibody of interest Thawing and Subculturing DG44 Cells Introduction Important Preparing Complete CD DG44 Medium
13. R activity and must be propagated in medium containing the purine precursors hypoxanthine and thymidine HT unless the cells are stably transfected with a vector that expresses DHER DHFR also functions as a genomic amplification marker for your gene of interest using methotrexate MTX selection Kaufman et al 1985 Tanaka et al 2002 See page 18 for more details on genomic amplification using MTX The DG44 cell line exhibits the following characteristics e Adapted to serum free suspension growth in CD DG44 Medium containing hypoxanthine and thymidine TM e Demonstrates high transfection efficiencies with FreeStyle MAX Reagent e Suspension cultures may be transfected in CD DG44 Medium As with other human cell lines when working with DG44 cells handle as potentially biohazardous material under at least Biosafety Level 2 containment CD DG44 Medium Introduction Features of CD DG44 Medium Making Complete CD DG44 Medium Growth Characteristics of DG44 Cells in CD DG44 Note CD DG44 Medium is a defined serum free medium containing hypoxanthine and thymidine for high density suspension culture of untransfected DG44 cells The medium contains no human or animal origin components CD DG44 Medium has the following features e Chemically defined containing no proteins or peptide components of animal plant or synthetic origin and no undefined hydrolysates or lysates e Supplemented with hypoxanthine and thy
14. ansfecting DG44 Cells with FreeStyle MAX Reagent Introduction Plasmid Preparation Linearizing the Plasmids You will use FreeStyle MAX Reagent to transfect suspension DG44 cells with the combination of pOptiVEC TOPO and peDNA 3 3 TOPO constructs that gave you the highest yield of antibody from page 9 TM TM The pOptiVEC and pcDNA 3 3 plasmid constructs must be clean sterile and free from contamination with phenol and sodium chloride for transfection into DG44 cells Contaminants may kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the PureLink HiPure Plasmid Midiprep DNA Kit see page v for ordering information Prior to using the OptiCHO Antibody Express Kit to transfect DG44 cells with your pOptiVEC and pcDNA 3 3 constructs you may linearize the plasmids While linearizing your vectors may not improve transfection efficiency it increases the chance that the vectors will integrate into the host cell genome without disrupting the gene of interest or other elements required for expression in mammalian cells e We suggest using Pvu I which cuts once in the ampicillin resistance gene on each plasmid Other unique restriction sites are possible Complete restriction maps of pOptiVEC TOPO and pcDNA 3 3 TOPO are available at www invitrogen com Be sure that your inserts do not contain the restriction en
15. ansfer to 12 well plates using the same procedure 3 Proceed with clones that continually produce the highest amount of your antibody at the 6 well stage 4 Once cells are cultured in a 125 ml flask incubate cells at 37 C 8 COs with shaking at 130 135 rpm Note If MTX selection has been performed include the same amount of MTX i e 200 800 nM in the media for shake flask growth Prior to optimizing antibody production cell stocks should be frozen down Antibody production experiments can then be optimized using different culture conditions or scale up can be continued to meet your bioproduction needs 21 Troubleshooting Culturing DG44 Cells The table below lists some potential problems and possible solutions that may help you troubleshoot your cell culture experiment Problem Reason Solution No viable cells after thawing original vial Cells not stored correctly Order new cell stock and store in liquid nitrogen Keep in liquid nitrogen until thawing Incorrect thawing medium or method e Use pre warmed CD DG44 Medium supplemented with 8 mM L glutamine and 18 ml L Pluronic F 68 DO NOT USE CD OptiCHO Medium to propagate DHFR negative DG44 cells Do not add antibiotics to media as this may negatively impact cell growth Incubate cultures on an orbital shaker set at 130 135 rpm in a 37 C incubator with a humidified atmosphere of 8 CO No viable cells after thawing stocks
16. ately see page v for details The pOptiVEC TOPO TA Cloning Kit contains the pOptiVEC TOPO vector a TOPO adapted bicistronic plasmid that allows rapid cloning of a PCR product containing a mammalian secretion signal and the gene of interest downstream of the CMV promoter In the pOptiVEC TOPO vector the transcription of the gene of interest is separated from the dihydrofolate reductase DHFR auxotrophic selection marker by an internal ribosome entry site IRES allowing transcription of the gene of interest and the selection marker on the same mRNA The pcDNA 3 3 TOPO TA Cloning Kit contains the pe DNA 3 3 TOPO vector a TOPO adapted plasmid that allows rapid cloning of a PCR product containing a mammalian secretion signal and the gene of interest downstream of the CMV promoter The pcDNA 3 3 TOPO contains a neomycin resistance gene slowing selection using Geneticin Continued on next page Overview continued Experimental Important Refer to the pOptiVEC TOPO TA Cloning Kit and pcDNA 3 3 Outline TOPO TA Cloning Kit manuals included with the kit for detailed information on cloning the heavy and light chains of your antibody of interest into these vectors prior to Step 1 below This manual provides instructions and guidelines to perform the following steps Step Action 1 Transiently transfect CHO cells or stably transfect DG44 cells with pOptiVEC TOPO and pcDNA 3 3 TOPO plasmid constr
17. d to the next section Clone Scale Up Clone Scale Up Introduction Note Materials Needed Protocol Next Steps After isolating clones in semi solid media and moving single cell colonies to 48 well plates previous section you will slowly scale up the volume of cells by allowing them to become nearly confluent and then moving the cells into the next larger volume i e 24 well 12 well and 6 well plates then to 125 ml flasks The total clone scale up process from a 96 well plate to a 125 ml flask will take about 5 6 weeks depending on the growth rate of each clone You will also need to monitor the antibody production of each clone using your specific method of choice see page 9 You will need the following reagents and materials before beginning e Single cell derived clones in 48 well plates from step 4 previous page e Complete CD OptiCHO Medium Geneticin e Sterile tissue culture dishes 24 well 12 well and 6 well and sterile 125 ml polycarbonate flasks e Non shaking incubator set at 37 C humidified atmosphere at 5 CO e Shaking incubator set at 37 C humidified atmosphere at 8 CO shaking at 130 135 rpm e Assay for determining antibody production e MTX optional see Note below 1 When individual clones are nearly confluent in 48 well plates transfer cells by pipetting up and down gently into 24 well tissue culture plates and add new medium 2 When each clone becomes nearly confluent tr
18. e Anal Biochem 247 102 110 Tanaka H Tapscott S Trask B and Yao M C 2002 Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells PNAS 99 8772 8777 Urlaub G Kas E Carothers A and Chasin L 1983 Deletion of the Diploid Dihydrofolate reductase Locus from Cultured Mammalain Cells Cell 33 405 412 Werner R G Noe W Kopp K and Schluter M 1998 Appropriate mammalian expression systems for biopharmaceuticals Arzneimittelforschung 48 870 880 Pluronic is a registered trademark of BASF Corporation 2007 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 21 Notes 28 8 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
19. e Selection 1 Round of MTX Clone Selection Scale Up Clones Selection for Ab Production Points to Consider When choosing a workflow from the options above you should consider the Protocols amount of antibody you wish to produce your available resources and the amount of time it will take to obtain your clonal high producing cell lines Because MTX selection produces a polyclonal population you must always perform clone selection prior to scale up Additional cloning media supplements and other products may be purchased separately from Invitrogen page v e To perform 1 round of genomic amplification using MTX selection to obtain a population of cells expressing high levels of your antibody see page 18 e To perform limiting dilution to obtain single clones expressing high levels of your antibody see page 19 e To scale up your clones for antibody production see page 21 17 Genomic Amplification by MTX Selection Introduction Preparing 1 mM MTX Preparing Media with MTX Protocol Next Steps 18 Methotrexate MTX is a folic acid antagonist that is actively transported into cells by the folate transporter In the cell it is converted to a high molecular weight polyglutamate metabolite by folylpolyglutamate synthase which binds to DHER and inhibits its activity If MTX is present in the medium cells compensate by increasing the DHER copy number in the genome to overcome inhibition by MTX Since the gene of in
20. ied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchan
21. invitrogen OptiCHO Antibody Express Kit For transfection of DG44 cells and development of stable cell lines for antibody production Catalog no 12762 019 Version B 4 November 2010 25 1009 Table of Contents Table Of Conti EE ee iii Kit Contents and Storage unica nda aloe ln iv IS A OS v Introduction VERERSUERERERSRPORNEEESSRELENES EL SEEPRVEPEDEEDEPNPETRR SELL EIEPELEDELEPEEELLSELEELPRUELEEEBESEDEPEPREL TEILTE 1 EE 1 DGM Cll 2222 es A A A 4 ED DE44 E RT EE 5 Freestyle MAX Reagent o lA AA AA ld AA eg 6 EC EE 7 A een 8 Testing Constructs by Transfection EEN 8 Thawing and Subculturing DG44 Cells EEN 10 Preezing DG Cell inaccesible ide cbc 12 Transfecting DG44 Cells with FreeStyle MAX Reagent csiicsusssesssesvacessssnsouisseaviesascssncorsiesireaasnanssaasdeionensase 13 Selecting for Stable Transtormante een 16 Choosing a WOrKUOW ee eer ee NEE A AA A sha AAA AA AA Soe 17 Genomic Amplification by MTX Selection EE 18 Clonal Selection in Semi Solid Media ENEE 19 Clone Scale iii A A diia 21 TFOUDICSNOOUING BT 22 ele le u uns uni dd 24 RE E EE 24 Purchaser Nota a ds 25 Product Qualification ae Ra dit ete eh 26 Eltere tee EE ee 27 Notes ui adicta ideada nidad 28 111 Kit Contents and Storage Shipping Storage TOPO TA Cloning Reagents DG44 cells OptiCHO Antibody Express Kit Components The components of the OptiCHO Antibody Express Kit are shipped and should be stored as listed below
22. it is designed for easy cloning and expression of recombinant antibodies in dihydrofolate reductase DHFR deficient Chinese hamster ovary CHO derived DG44 cells in suspension culture The OptiCHO Antibody Express Kit provides reagents for cloning the gene encoding the heavy and light chains of your antibody high efficiency transfection of the DNA constructs into DG44 cells and generation of stable cell lines producing your antibody of interest TM The OptiCHO Antibody Express Kit includes the following major components e pOptiVEC TOPO TA Cloning Kit A TOPO adapted bicistronic plasmid and reagents for cloning a PCR product containing a mammalian secretion signal and the heavy and light chains separately of your antibody of interest See the next page for more information e pcDNA 3 3 TOPO TA Cloning Kit A TOPO adapted plasmid and reagents for cloning a PCR product containing a mammalian secretion signal and the heavy and light chains separately of your antibody of interest See the next page for more information es DG44 cells DHFR negative CHO derived cells adapted to high density serum free suspension culture in CD DG44 Medium that are capable of producing high levels of secreted recombinant protein See page 4 for more information e CD DG44 Medium Defined serum free medium supplemented with hypoxanthine and thymidine to allow growth of DHFR negative DG44 cells See page 5 for more information TM
23. ltured cells e Exactly follow procedures as outlined in transfected cells Thawing and Subculturing Cells page obtained 11 e Thaw a new batch of early passage cells e Do not add antibiotics during transfection Cells not passed 24 hours before Approximately 24 hours before transfection transfection pass cells at 3 x 10 cells ml FreeStyle Max Reagent e Store at 4 C Do not freeze handled incorrectly e Mix gently by inversion Do not vortex Used poor quality expression Do not use mini prep plasmid DNA for construct plasmid DNA Oe transfection Prepare midiprep plasmid DNA plasmid DNA from a mini prep with low endotoxin contamination DNA contaminated Sterilize DNA using a 0 22 pm filter Protein The table below lists some potential problems and possible solutions that may Expression help you troubleshoot your antibody expression levels Problem Reason Solution No or low antibody detected in the supernatant after transient or stable transfection PCR primer does not contain Kozak translation initiation sequence Add a Kozak consensus site to the forward PCR primer resynthesize your DNA and re clone See the appropriate manual for each TOPO TA Cloning Kit for details Premature stop codons Remove stop codons by your method of choice Improper or ineffective secretion signal Replace secretion signal Use endogenous secretion signal if possible Codons not optimized for mammalian cells
24. midine HT for growth of DHFR negative cells e Formulated without L glutamine to avoid problems associated with L glutamine degradation including ammonia accumulation e Formulated without Pluronic F 68 e Formulated without phenol red to minimize potential estrogen like effects e CD DG44 Medium requires supplementation with L glutamine Aseptically add L glutamine to a final concentration of 8 mM to the medium before use e CD DG44 Medium requires the addition of a surfactant to protect against shear forces in suspension culture Aseptically add 18 ml L of Pluronic F 68 to the medium before use Caution Pluronic F 68 is an irritant to the eyes respiratory system and skin Review the Material Safety Data Sheet MSDS before use e Store complete CD DG44 Medium at 4 C protected from light Typically DG44 cells cultured in CD DG44 Medium have a doubling time in the range of 16 22 hours doubling time can exceed 22 hours during the first few passages after the cells have been thawed Do not allow DG44 cells to reach a cell density above 1 x 10 cells ml before transfection as this will result in a decrease of transfection efficiency Individual culturing and passaging techniques coupled with cellular heterogeneity inherent within the DG44 cell population may result in experimental variability FreeStyle MAX Reagent FreeStyle MAX Reagent OptiPro SFM FreeStyle MAX Reagent is a proprietary animal origin free f
25. ng DG44 Cells continued Thawing Procedure Determining Cell Density and Viability Subculturing Cells Important To thaw and establish cells 1 Remove the cryovial of cells from the liquid nitrogen and thaw quickly lt 1 minute in a 37 C water bath Decontaminate the outside of the vial with 70 ethanol Gently break up any clumps with a sterile pipette tip and aseptically transfer the entire contents of the cryovial into a disposable sterile polycarbonate 125 ml Erlenmeyer shaker flask containing 30 ml of pre warmed complete CD DG44 Medium Incubate cells in a 37 C incubator containing a humidified atmosphere of 8 CO in air on an orbital shaker platform rotating at 130 135 rpm After 24 48 hours in culture determine the cell density and viability using the protocol described below Once the culture has reached gt 1 2 x 10 viable cells ml expand cultures using the subculturing protocol below Follow the procedure below to determine viable and total cell counts 1 2 3 Transfer a small aliquot of the cell suspension to a microcentrifuge tube Determine viability using trypan blue dye exclusion Determine cell density electronically using a Coulter Counter or manually using a hemacytometer and an inverted microscope Passage the cells once the culture has reached gt 1 2 x 10 viable cells ml When passaging DG44 cells use disposable sterile polycarbonate 125 ml Erlenmeyer shaker flasks with vented
26. omated or manual controlled rate freezing apparatus Prepare freezing medium immediately before use 1 In a sterile conical centrifuge tube mix together the following reagents for every 1 ml of freezing medium needed Complete CD DG44 Medium 0 9 ml DMSO 0 1 ml Filter sterilize the freezing medium through a 0 22 um filter and place the tube on ice until use Discard any remaining freezing medium after use Grow the desired quantity of DG44 cells in shaker flasks harvesting when the cell density reaches 1 x 10 viable cells ml Transfer cells to a sterile conical centrifuge tube Determine the viable and total cell counts and calculate the volume of freezing medium required to yield a final cell density of 1 x 10 viable cells ml Centrifuge cells at 1 200 rpm for 5 minutes at room temperature and carefully aspirate the medium Resuspend the cells in the pre determined volume of chilled freezing medium Place cryovials in a microcentrifuge rack and aliquot 1 ml of the cell suspension into each cryovial Freeze cells in an automated or manual controlled rate freezing apparatus following standard procedures For ideal cryopreservation the freezing rate should be a decrease of 1 C per minute Transfer frozen vials to liquid nitrogen for long term storage Note You may check the viability and recovery of frozen cells 24 hours after storing cryovials in liquid nitrogen by following the thawing procedure on page 11 Tr
27. ormulation for the highly efficient transfection of plasmid DNA into eukaryotic cells FreeStyle MAX Reagent is specifically formulated to achieve the highest expression levels and lowest cytotoxicity in DG44 cells and other suspension cell lines including FreeStyle CHO S and FreeStyle 293 F cells Store FreeStyle MAX Reagent at 4 C Do not freeze FreeStyle MAX Reagent is also available separately from Invitrogen see page v for more information OptiPro SFM is included with the OptiCHO Antibody Express Kit to facilitate optimal formation of DNA lipid complexes OptiPro SFM is a serum free medium that is devoid of any components of animal or human origin OptiPro SFM has an ultra low protein concentration of 7 5 pg ml Store OptiPro SFM at 4 C OptiPro SFM is available separately from Invitrogen see page v for ordering information CD OptiCHO Introduction Important Features of the Medium Making Complete CD OptiCHO Media Medium CD OptiCHO Medium is a defined serum free medium for selection and high density suspension culture of stably transfected DG44 cells expressing DHER and the neomycin resistance gene You will perform two rounds of selection on your transfected cells one with CD OptiCHO medium and one with CD OptiCHO and 500 pg ml Geneticin as detailed on page 16 Do not use CD OptiCHO Medium or CD OptiCHO Medium Geneticin to propagate unt
28. ote below 5 When culture reaches gt 70 viability maintain culture at 3 x 10 viable cells During the selection rounds cell viability may drop dramatically to lt 10 due to the death of untransfected and transiently transfected cells To promote optimal growth of stably transfected cells maintain cultures as described in Steps 4 5 When you have a pool of stably transfected cells you should cryopreserve several aliquots of the pool using the procedure on page 12 Depending on your antibody production needs refer to the next section Choosing a Workflow to determine the next steps Choosing a Workflow Introduction At this stage you will have a population of stably transfected DG44 cells expressing your antibody at various levels For most bioproduction applications several clonally derived cell lines producing your antibody are desirable for screening However the growth and optimization of each clone is dependent upon the integration locus of the plasmid the response to amplification using MTX and the nature of the protein Depending on your protein production needs the time and effort required to generate clonal high producing cell lines is also variable Several common pathways from stable pool to clone scale up are outlined below 1 month 2 3 months 3 months 1 Round of MTX Scale Up Clones Selection EE KSE for Ab Production Stably Transfected Population 2 3 months 1 month 2 3 months 3 months Clon
29. ours post 2 weeks until 2 weeks until transfection viability gt 90 viability gt 90 Geneticin blocks protein synthesis in mammalian cells by interfering with ribosomal function It is an aminoglycoside similar in structure to neomycin gentamycin and kanamycin Expression in mammalian cells of the bacterial aminoglycoside phosphotransferase gene APH derived from Tn5 results in detoxification of Geneticin Southern and Berg 1982 Calculate the concentration based on the amount of active drug Cells will divide once or twice in the presence of lethal doses of Geneticin so the effects of the drug take several days to become apparent Complete selection can take up to 2 weeks of growth in selective medium Transfected DG44 cells should be passaged in complete CD OptiCHO Medium for the first round of selection and in complete CD OptiCHO Medium with 500 pg ml Geneticin for the second round of selection To passage cells 1 Determine viable and total cell counts using your preferred method 2 Dilute the cells in pre warmed complete CD OptiCHO Medium to give a final cell density of 3 x 10 viable cells ml 3 Incubate flasks in a 37 C incubator containing a humidified atmosphere of 8 CO on an orbital shaker platform rotating at 130 135 rpm 4 Spin down cells remove medium by aspiration and add fresh medium to a final volume of 30 ml every 2 days for 10 14 days until cell viability increases to gt 70 see N
30. ransfected or parental DG44 cells DG44 cells are DHFR deficient and require supplementary hypoxanthine and thymidine HT e Only cells that have an active DHFR enzyme or have been transfected with pOptiVEC TOPO can be propagated in CD OptiCHO Medium e Only cells that have an active DHFR enzyme or have been transfected with pOptiVEC TOPO and pcDNA 3 3 TOPO constructs can be propagated in CD OptiCHO Medium Geneticin CD OptiCHO Medium has the following features e Chemically defined containing no proteins or peptide components of animal plant or synthetic origin and no undefined hydrolysates or lysates e Formulated without L glutamine to avoid problems associated with L glutamine degradation including ammonia accumulation e Formulated without phenol red to minimize potential for estrogen like effects of phenol red TM e CDOptiCHO Medium requires supplementation with L glutamine Aseptically add L glutamine to a final concentration of 8 mM to the medium before use e For Geneticin selection aseptically add Geneticin to CD OptiCHO Medium at a concentration of 500 pg ml e Store complete media at 4 C protected from light Methods Testing Constructs by Transfection Introduction Prior to making stable transfectants in DG44 cells You may perform transient transfections of CHO cells see Recommendation next page with pOptiVEC TOPO and pcDNA 3 3 TOPO plasmid constructs to dete
31. rmine which vector combination gives optimal antibody yield using your detection method of choice You may also perform several stable transfections of DG44 cells with various Note combinations of pOptiVEC TOPO and pcDNA 3 3 TOPO plasmid constructs See page 15 for protocol 1 TOPO clone mammalian secretion signal SS heavy chain and SS light chain separately into both into pOptiVEC TOPO and pcDNA3 3 TOPO Ej Heavy _ pOptivEC peDNA3 3 OptiVEC pcDNA3 3 Heavy Chain Light Chain PSRS Light Chain j Heavy Chain 2 Transiently transfect each set of clones into CHO cells OR perform stable transfections in DG44 cells 3 Wait 5 7 days for transient trans G fection in CHO cells OR Select cells using CD OptiCHO HT Geneticin for stable transfection in DG44 YYYYY E YYYYY 5 Choose the vector combination that gives the highest antibody production level and proceed to stable transfection in DG44 cells OR Determine which stably transfected DG44 pool gives highest antibody production level and proceed to clone selection and or amplification Continued on next page Testing Constructs by Transfection Continued Plasmid Preparation General Guidelines for Transient Transfection Antibody Production We recommend FreeStyle CHO S cells and transfection with FreeStyle MAX Reagent for transient transfection Both FreeStyle CH
32. s in a 30 ml volume we recommend using the following optimized conditions Final transfection volume 30 ml Number of cells to transfect total of 1 5 x 10 cells cell density at time of transfection should be 5 x 10 cells ml Amount of each plasmid DNA 9 ug each total 18 ug FreeStyle MAX Reagent 15 ul Note Further optimization of culture volume or transfection conditions is not necessary for stable cell line production Continued on next page Transfecting DG44 Cells with FreeStyle MAX Reagent continued Transfection Procedure Follow the procedure below to transfect DG4 cells in a 30 ml volume We TM recommend including negative controls no DNA no FreeStyle MAX Reagent in your experiment to help you evaluate your results 1 10 At 48 hours before transfection pass DG44 cells at 3x 10 cells ml in complete CD DG44 Medium Place the flask s on an orbital shaker platform rotating at 130 135 rpm at 37 C 8 CO At 24 hours before transfection pass DG44 cells at 3 x 10 cells ml in complete CD DG44 Medium Place the flask s on an orbital shaker platform rotating at 130 135 rpm at 37 C 8 CO On the day of transfection perform a viable cell count To ensure optimal transfection results viability of cells must be over 95 For each transfection or control transfer 1 5 x 107 viable DG44 cells to a new 125 ml flask Add pre warmed complete CD DG44 Medium to a final volume of
33. t see page 24 Item Amount Catalog no pOptiVEC TOPO TA Cloning Kit 1 kit 12744 017 pcDNA 3 3 TOPO TA Cloning Kit 1 kit K8300 01 One Shot TOP10 Chemically Competent E coli 10 reactions C4040 10 20 reactions C4040 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 FreeStyle MAX Reagent 1 ml 16447 100 OptiPro SFM 100 ml 12309 050 1000 ml 12309 019 L glutamine 200 mM liquid 100 ml 25030 081 Pluronic F 68 10 100 ml 24040 032 Trypan Blue Stain 100 ml 15250 061 CD DG44 Medium 1000 ml 12610 010 CD OptiCHO Medium 1000 ml 12681 011 DG44 Cells 1 x 107 cells 12609 012 CD OptiCHO Cloning Medium 2x 500 ml 07 0040DJ Through our Gibco custom media services we can develop cloning or growth media formulations specifically suited to your cells We can provide the best nutrient media delivery scheme for your recombinant cell line optimizing a Gibco medium or one in the public domain or your own formulation All final media manufacturing is performed in our ISO 9001 certified OSR cGMP compliant facilities and held to the same high standards as our own Gibco catalog products ensuring scalability robustness and compliance For more information go to www invitrogen com or contact Technical Support see page 24 vi Overview OptiCHO Antibody Express Kit Components of the OptiCHO Antibody Express Kit Introduction TM The OptiCHO Antibody Express K
34. tability or fitness for a particular purpose Purchaser Notification Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 296 DG44 Cells The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the prod
35. terest is integrated into the same genetic locus as DHFR the gene of interest is amplified as well leading to increased production of the protein of interest Kaufman et al 1985 Tanaka et al 2002 MTX as methotrexate hydrate is available from Sigma 10 mg catalog number A6770 MTX is toxic to the skin eyes and respiratory system Wear suitable protective clothing gloves and eye and face protection when working with MTX Refer to the product MSDS for complete precautions To prepare a 1 mM MIX stock solution 1 Dissolve 10 mg MTX in 22 ml of PBS 2 Filter sterilize the solution through a 0 22 um filter 3 Store in 250 ul aliquots at 20 C To make complete CD OptiCHO Medium with MTX you will need to use complete CD OptiCHO Medium 500 pg ml Geneticin Using the sterile 1 mM MTX stock solution prepared as described above prepare media with 500 nM MTX Note You can very the amount of MTX from 200 800 nM if desired however our data show that additional rounds of MTX amplification do not dramatically increase antibody production For each clone spin down cells and aspirate old medium 2 Seed cells at a density of 2 5 x 10 cells ml in 30 ml of media containing MTX in 125 ml flasks 3 Incubate flasks at 37 C 8 CO with shaking at 130 135 rpm 4 Spin down cells remove medium by aspiration and add fresh medium containing MTX to 30 ml every 2 days for 14 21 days 5 When cell viability is gt 70
36. uct or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carls bad California 92008 Phone 760 603 7200 or e mail outlicensing lifetech com These cells are sold under license from Lawrence and Gail Urlaub Chasin for research purposes only and no license for commercial use is included Requests for licenses for commercial manufacture or use should be directed to Lawrence Chasin at 212 854 4645 or at lac2 columbia edu or to
37. ucts to determine which vector combination gives optimal antibody yield Thaw and propagate DG44 cells in CD DG44 Medium Transfect your pOptiVEC TOPO and pcDNA 3 3 TOPO plasmid constructs into DG44 cells using FreeStyle MAX Reagent 4 Select for a pool of stably transfected cells by performing two rounds of selection using CD OptiCHO Medium and CD OptiCHO Medium with Geneticin 5 Perform MTX amplification and clonal selection using limiting dilution on semi solid media 6 Scale up your high producing clonal cell line to suit your bioproduction needs DG44 Cells Introduction Parental Cell Line DHFR Characteristics of DG44 Cells The DG44 cell line is a dihydrofolate reductase DHFR deficient cell line derived from suspension Chinese hamster ovary CHO S cells Urlaub et al 1983 DG44 cells are adapted to suspension culture in CD DG44 Medium Frozen cells are supplied at 1x 10 cells ml and may be thawed directly into CD DG44 Medium see Thawing and Subculturing Cells page 8 The CHO S cell line is a stable aneuploid cell line established from the ovary of an adult Chinese hamster Puck 1958 CHO cells are commonly used cell lines for transfection expression and large scale production of recombinant proteins DHER catalyzes the reduction of 5 6 dihydrofolate to 5 6 7 8 tetrahydrofolate which is essential for DNA synthesis CHO derived DG44 cells lack DHE
38. uded with CloneMatrix e 2x cloning medium of choice such as CD OptiCHO Cloning Medium 2x available from Invitrogen as a stock custom medium see page v e Growth medium complete CD OptiCHO Medium e 200 mM L glutamine e Geneticin e Cell culture incubator 37 C humidified atmosphere of 5 CO e Reagents and equipment to determine viable and total cell counts e Multichannel pipettor sterile reservoir and sterile pipette tips e Sterile tissue culture grade 96 well plates and 48 well plates with covers Continued on next page 19 Clonal Selection in Semi Solid Media Continued Semi Solid Cloning Medium Plating Cells Screening for Single Cell Clones 20 The following recipe is sufficient to make 100 ml of complete medium for clonal selection which is sufficient for approximately nine 9 96 well plates 1 On Day 1 one day prior to plating cells for clonal selection thaw the CloneMatrix and Clone XL Reagent overnight at 4 C 2 On Day 2 aseptically add the following reagents to the CloneMatrix CD OptiCHO Cloning Medium 2x 50ml CloneXL Reagent 2 ml 200 mM L glutamine 4 ml Geneticin 1 ml of 50 mg ml 500 pg ml final volume CD OptiCHO Medium to a final volume of 100 ml 3 Replace the cap and mix by shaking vigorously for 10 seconds and let stand undisturbed for 20 minutes to eliminate large air bubbles Small air bubbles may persist but these will not cause adverse effects 1
39. zyme site you use to linearize the vector Note If an appropriate linearization site is not present you may transfect the circular plasmid Transfection efficiency will not be affected e After digestion precipitate the DNA resuspend pellet in sterile water and re quantify using your method of choice Continued on next page 13 Transfecting DG44 Cells with FreeStyle MAX Reagent continued ar EC gt Now Materials Needed Optimal Transfection Conditions 14 Calculate the number of DG44 cells that you will need for your transfection experiment and expand cells accordingly Make sure that the cells are healthy and greater than 95 viable before proceeding to transfection You should have the following reagents and equipment before beginning Suspension DG44 cells cultured in complete CD DG44 Medium at 5 x 10 cells ml Purified linearized pOptiVEC TOPO and pcDNA 3 3 TOPO plasmids DNA containing your heavy and light chains of interest as determined from page 9 FreeStyle MAX Reagent supplied with the kit store at 4 C until use OptiPro SFM supplied with the kit pre warmed to room temperature Disposable sterile 125 ml polycarbonate Erlenmeyer flasks Orbital shaker in 37 C incubator with a humidified atmosphere of 8 CO Reagents and equipment to determine viable and total cell counts e g Trypan Blue hemacytometer Coulter Counter To transfect suspension DG44 cell
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