BD Resurge User Manual
Contents
1. g Q 100 0 200 r 100 Control Process Production A BD Resurge CD1 Production BD Resurge CD2 Production Control Process VCD BD Resurge CD1 VCD BD Resurge CD2 VCD heen Days Shaker Flasks 50mL Control Process Production B BD Resurge CD1 Production BD Resurge CD2 Production Control Process VCD BD Resurge CD1 VCD BD Resurge CD2 VCD hy ened Days Bireactor 1L VCD 166 cells mL VCD 1e6 cells mL Figure 2 A B Representative data showing growth and production of CHO K1 cells fed with BD Resurge CD1 and CD2 in shaker flask 50 mL culture Fig 2A and stir tank bioreactor 1L Fig 2B Study was performed in fed batch mode using BD Resurge CD1 and CD2 on Day 3 and 5 of culture in addition to control process Data demonstrates the scalability of CD1 and CD2 from shaker flask to bioreactor BD Resurge User s Manual 11 SUMMARY AND RECOMMENDATIONS gt BD Resurge supplements CD1 CD5 are animal free protein free and chemically defined and can be used in batch and fed batch mode in cell culture processes Supplementation with BD Resurge CD supplements is scalable and has been demonstrated to perform in shake flasks bioreactors and other culture systems data not shown e Although the development of the BD Resurge supplements focused on CHO cell lines expressing monoclonal antibodies and recombinant proteins their applicatio2. Titration Batch Culture in shaker flask Step 1 Prepare shake flasks by adding an appropriate volume of reconstituted BD Resurge CD supplement 40 g L and basal medium to result in the final concentrations shown in Table 4 Table 4 Experimental Set up for testing of a BD Resurge supplement Final BD Resurge Volume of Base Volume of BD Resurge Concentration g L Medium mL 40 g L mL BD Resurge supplement 1 48 75 1 25 BD Resurge supplement 3 46 25 375 BD Resurge supplement 6 42 54 7 46 Medium only negative 0 50 0 control Note This table provides guidance for titration of one BD Resurge CD supplement similar set ups can be used for all supplements add appropriate positive control if needed The titration data from BD Resurge supplements can then be evaluated and or compared with the positive control to establish the ideal BD Resurge concentration Step 2 Prepare the seeding culture according to your standard protocol If cells are being cultured in a hydrolysate containing medium wash cells once in sterile PBS and pellet by centrifugation Prepare seeding the cell suspension by resuspending the pellet into a base media e g 3x107 cell mL Step 3 Inoculate shaker flasks with your usual seeding density e g final seeding at 3x105 cell mL Step 4 On Days 0 3 5 7 10 and on the last day of cell culture determine the viable cell density and percent viability Step 5 Adjust glucose and glutamine leve
3. and boost protein production Every cell line can offer unique challenges for devising processes that deliver desired performance One bioprocess challenge is the selection and optimization of a cell culture supplement or feed that will give desired titer growth characteristics and protein quality Therefore the avail ability of multiple chemically defined CD supplements offering a diverse performance profile can significantly improve process development timelines BD Biosciences Advanced Bioprocessing has designed and formulated a diverse set of fully chemically defined CD and animal free AF cell culture supplements BD Resurge CD1 CD5 supplements without glucose and L glutamine This set of supplements can be used in batch and fed batch processes to enhance BD Resurge User s Manual PRODUCT INFORMATION BD Resurge CD1 CD5 supplements are and structures and meet BD s stringent manufactured at the BD Biosciences Animal Origin Free Specification These Advanced Bioprocessing animal free and supplements are available as individual BD antibiotic free AF2 state of the art catalog numbers Table 1 and as a kit facility All the components are chemically BD Resurge CD PAK Table 2 defined with known molecular formulae Table 1 Product information BD Resurge CD1 CD5 Product 670011 100G EACH BD Resurge CD1 670012 1KG EACH 670013 5KG EACH 670015 100G EACH BD Resurge CD2 670016 1KG EACH 67001
4. 7 5KG EACH 670018 100G EACH BD Resurge CD3 670019 1KG EACH 670020 5KG EACH 670021 100G EACH BD Resurge CD4 670022 1KG EACH 670023 5KG EACH 670024 100G EACH BD Resurge CD5 670025 1KG EACH 670026 5KG EACH Table 2 Product information BD Resurge CD PAK Product BD Resurge CD1 100G EACH BD Resurge CD2 100G EACH Kit Cat BD Resurge CD3 100G EACH 670030 BD Resurge CD4 100G EACH BD Resurge CD5 100G EACH BD Resurge User s Manual RECONSTITUTION PROCEDURE Reconstitution at 40 g L BD Resurge CD1 CD5 supplements are supplied as powders and are recommended to be hydrated at 400 1 using the following instructions e Weigh 40 grams of BD Resurge CD supplement powder e Fill a clean 1L beaker with approximately 900 mL room temperature 25 C water for injection WFI or equivalent e Add the powder to the beaker and mix for a minimum of 30 minutes e Once completely dissolved bring volume to 1L with WFI or equivalent e Measure and record pH and osmolality e Filter sterilize the concentrated solution through a 0 2um filter membrane and use as needed gt Store solution at 2 8 C protected from light Use within six months of reconstitution or the expiration date of the original powder stock whichever is earliest Reconstitution at 100 g L More concentrated stock solutions of BD Resurge CD supplements can be prepared using the following instructions for making a 1000 1 stock solution e Weigh 100 grams of BD Resur
5. BD Resurge CD1 CD5 CD PAK without Glucose and L Glutamine SUPPLEW ATION a F User Manual Helping all people live healthy lives 11 Introduction Product Information Reconstitution Procedure Specifications of Reconstituted Supplements Storage and Shelf Life Testing Procedures 6 1 Testing Procedure 6 2 BD Resurge Titration Batch Culture in shaker flask 6 3 BD Resurge CD1 CD5 supplements Fed Batch Culture 6 4 BD Resurge CD1 CD5 supplements Bioreactor Compatibility Studies Representative Data 8 1 Representative Data 8 2 Performance Data Summary and Recommendations Troubleshooting References BD Resurge User s Manual Table of Contents recombinant protein production of CHO cells while retaining suitable product quality Optimal results with BD Resurge supplements can be attained by varying supplement concentrations and cell culture feeding regimen These supplements have been tested on a variety of host CHO cells and in various culture systems including shaker flasks and bench scale bioreactors As a single supplement may not be ideal for enhancing performance across multiple cell lines conditions a wider range of CD supplement formulations could be more effective for process development INTRODUCTION Cell based production of active pharma ceutical ingredients generally requires supplementation of cell culture media to promote cell growth
6. ces death receptor and mitochondrial apoptotic pathways Biotechnol Lett 2006 28 18 1445 1452 Kaufmann H Mazur X Fussenegger M Bailey JE Influence of low temperature on productivity proteome and protein phosphorylation of CHO cells Biotechnol Bioeng 1999 63 5 573 582 Kumar N Gammell P Clynes M Proliferation control strategies to improve productivity and survival during CHO based production culture A summary of recent methods employed and the effects of proliferation control in product secreting CHO cell lines Cytotechnology 2007 53 1 3 33 46 Sandadi S Ensari 5 Kearns B Heuristic optimization of antibody production by Chinese hamster ovary cells Biotechnol Prog 2005 21 5 1537 1542 BD BD Biosciences Advanced Bioprocessing 800 638 8663 www bdbiosciences com advbio BD BD Logo and all other trademarks are property of Becton Dickinson and Company 2013 BD 223623 September 2013 USA NG WwW
7. fore supplementation with BD Resurge supplement s BD Resurge User s Manual COMPATIBILITY STUDIES For troubleshooting any compatibility of the supplement on basal medium issues experienced while using BD characteristics A supplement would Resurge supplements with a basal be considered compatible with the medium in cell culture processes it basal medium if the following charac is important to determine the effect teristics are observed Table 5 Table 5 Required Compatibility Characteristics of BD Resurge Media Supplements BD Resurge User s Manual Production ug mL Production ug mL DATA entative Data REPRESENTATIVE Repres BD Resurge CD1 CD5 supplements can be used in batch and fed batch modes in cell culture processes Figure 1 A E demonstrates the performance of BD Resurge CD1 CD5 in batch and fed batch mode in shaker flasks 400 t 0 300 200 j r 0 100 r 50 0 l 2 5 7 9 Days BD Resurge CD2 E Batch Production Fed Batch Production E Batch VCD E Fed Batch VCD D 800 t 700 t 500 400 T 300 t 200 100 3 5 7 10 Days BD Resurge CD4 Batch Production WE Fed Batch Production Batch VCD Fed Batch VCD Figure 1 A E Growth and mAb production of various host CHO cells cultured in their respective optimal medium with BD Resurge CD Supplements in batch and fed batch modes Fed batch mode cultures we
8. ge CD supplement powder e Fill a clean 1L beaker with approximately 700 mL room temperature water for injection WFI or equivalent e Add the powder to the beaker and mix until all the powder is in suspension e Measure and record pH and osmolality e Adjust the pH to between pH 9 0 and pH 10 0 using 5N or 6N NaOH e Mix for a minimum of 30 minutes e Once completely dissolved adjust pH to pH 8 0 0 2 or desired pH using 5N or 6N HCI e Bring volume to 1L with WFI or equivalent e Measure and record pH and osmolality Note BD Resurge reconstituted at 100 g L may not meet specifications defined in Table 3 e Filter sterilize the concentrated solution through a 0 2um filter membrane and use as needed gt Store solution at 2 8 C protected from light Use within one month of reconstitution or the expiration date of the original powder stock whichever is earliest e Continue to monitor for precipitation and color change BD Resurge User s Manual SPECIFICATIONS OF RECONSTITUTED SUPPLEMENTS Reconstituted BD Resurge CD supplements at 40g L should have osmolality and pH as illustrated in Table 3 below Table 3 Osmolality and pH of BD Resurge CD1 CD5 at 40g L BD Resurge Specification Specification mOsm Kg pH 50 6 8 0 6 6 5 0 8 0 8 6 E DA 360 440 360 440 415 475 400 460 400 460 Product Name BD Resurge Supplements CD 1 BD Resurge Supplements CD 2 BD Resurge Supplements CD 3 BD Resur
9. ge Supplements CD 4 BD Resurge Supplements CD 5 STORAGE AND SHELF LIFE BD Resurge CD1 CD5 supplements powder and reconstituted stock solutions should be stored at 2 8 C Refer to the product label for the expiration date of each powder supplement when stored at 2 8 C Stock solutions of BD Resurge CD supplements should be protected from light and should be used within six months of reconstitution or the expiration date of the original powder stock whichever is earliest Higher concentrations of BD Resurge CD supplements will have the same chemical stability but possibly different physical stability due to possibility of precipitation BD Resurge User s Manual TESTING PROCEDURES Testing Procedure BD Resurge CD1 CD5 supplements canbe supplement s that are most optimal for reconstituted and or supplemented based your system For a batch culture process an on your specific culture or process require initial titration using each BD Resurge CD ments The procedures in this manual are supplement is recommended For this test recommended as starting points each of the supplement s can be used at a final supplemental concentration of 19 L When using the BD Resurge CD Supple 3g L and 6g L on Day 0 or Day 2 of the ment PAK for the first time it is recom mended that all five supplements be concurrently evaluated to determine the culture Glucose and glutamine should be maintained as per cell line requirements BD Resurge
10. ls as appropriate throughout the experiment Step 6 Determine protein titer on various days of culture including the last the day to determine cumulative protein production BD Resurge User s Manual 6 TESTING PROCEDURES cont BD Resurge CD1 CD5 supplements Fed Batch Culture For a fed batch process BD Resurge CD Supplements can be used at a final supplemental concentration of 2 4g L per day for multiple days starting on Day 0 or Day 2 to mid growth phase The culture should be started at a reduced volume to accommodate the anticipated feeding volume Glucose and glutamine should be maintained as per cell line requirements BD Resurge CD1 CD5 supplements Bioreactor Select two or three optimal concentrations of BD Resurge CD Supplements from the shaker flask batch culture study for the bioreactor batch study Monitor and control glucose and glutamine levels as defined for the control process Monitor process variables as well as metabolites including lactate and ammonia to assess cell culture performance For fed batch processes in bioreactors we recommended testing a minimum of two feeding schemes that show promising results at the shaker flask level Further optimization of the feeding scheme should be done based on metabolite levels and the bioreactor system Note If using a cell line for the first time in a bioreactor system we recommend optimizing the bioreactor parameters for the specific cell line and medium be
11. n can certainly be investigated on other host cell lines gt This manual provides guideline for usage of BD Resurge supplements CD1 CD5 however each cell line would need further optimization for optimal performance For further questions contact BD Technical services at 1 800 638 8663 BD Resurge User s Manual TROUBLESHOOTING Table 6 Trouble Shooting Guidelines e Use a beaker to reconstitute small amounts of BD Resurge supplements e Increase the mixing speed to maximize the vortex e Reconstitute with room temperature 25 C water in an environment controlled at 25 C gt Perform compatibility test to identify the correct concentration of BD Resurge supplements to use 5 Contact BD Technical Services for trouble shooting unwanted chemical reactions e Identify contaminant and root cause of contamination e Retest e Contact BD Technical Services for trouble shooting unwanted chemical reactions gt Perform compatibility test to identify the optimum concentration of BD Resurge to use for your application e Perform BD Resurge supplements titration study to determine appropriate concentration range e Re check calculations for amount of BD Resurge added e Perform compatibility test to identify the optimum concentration of BD Resurge to use for your application e Reduce other supplementation levels e Try alternate feeding plan with reduced amount of other feeds amino acids hydrolysates e
12. re fed on day 0 2 and 5 for CD1 and 2 day 0 5 and 7 for CD3 and day 0 3 5 and 7 for CD 4 amp 5 CHOK1 cells producing mAb were used for CD1 and 2 while CHO GS cells were used for CD3 4 and 5 VCD 166 cells ml VCD 166 cells ml Production ug mL Production ug mL Production ug mL A 10 350 300 g T lt 250 am yey 200 2 4t 150 O t 100 Za 0 0 t 0 0 2 5 7 9 Days BD Resurge 1 EE Batch Production Fed Batch Production Batch VCD E Fed Batch VCD 5 1200 al 1000 T 0 600 oO 400 a gt 200 0 0 0 5 7 9 12 15 Days BD Resurge CD3 Batch Production Fed Batch Production Batch VCD Fed Batch VCD E 5 1200 4 1000 800 0 3 j Co D 600 2 O 400 5 14 200 0 t 0 0 3 5 7 10 Days BD Resurge 5 Fed Batch Production E Fed Batch VCD E Batch Production Batch VCD BD Resurge User s Manual 10 REPRESENTATIVE DATA cont Performance Data Performance of BD Resurge CD supplements is scalable to bioreactors BD Resurge CD supplements produced a 2 3 fold increase in protein production over media control in bench top bioreactors in batch or fed batch mode Figures 2A B 400 300 Protein Production ug mL 500 400 any 56 bie Ss 300 no 5 B 200
13. tc e Try various days of cell culture supple mentation including Day 0 supplemen tation to determine optimal feeding conditions 12 Low vortex due to slow mixing speed or wrong reconstitution vessel Temperature of the water or room is unusually low Possible reaction between components resulting in undissolved products Concentration of some components above solubility limits Possible contamination Possible reaction between components or degradation of the components High concentration of BD Resurge used High levels of BD Resurge High osmolality due to use of other additives like amino acid feeds hydrolysates feeds etc Over supplementation if using other feed additives amino acids hydrolysates etc together with BD Resurge Timing of supplementation requires further optimization BD Resurge supplements are either not dissolving or taking excessively long time to completely dissolve Medium is cloudy or a precipitate is observed following addition of BD Resurge supplements Color of medium containing BD Resurge supple ments changes during storage High osmolality and pH after addition to medium Low cell growth and low cell viability BD Resurge User s Manual 13 TROUBLESHOOTING Table 6 Trouble Shooting Guidelines BD Resurge User s Manual 1 14 REFERENCES Yeo JH Lo JC Nissom PM Wong VV Glutamine or glucose starvation in hybridoma cultures indu
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