recombinant trypsin, animal origin...
TRANSCRIPT
RecombinantTrypsin,AnimalOriginFree
PRODUCTINFORMATION: BioGenomicsr-Trypsinpowder isreadytouse,animaloriginfreeoptimizedforcellcultureapplications.Itisderivedbyr-DNAtechnology.
BioGenomics r-Trypsin is a pure enzyme, which helps maximize the yield offunctionally viable cells fromculture vessels,whilepreventing the toxic effect inducedbyother non-desirable proteases. In addition, BioGenomics r-Trypsin, being a recombinantproduct, completely eliminates the riskof viruses, orotherpotential adventitious agentsfoundinanimalderivedproducts.
It is most commonly used for dissociation and disaggregation of adherent cells.When the powder Trypsin is used in the solution form, a chelating agent likeEthylenediaminetetraaceticacid(EDTA)isoftenaddedtoenhancetheenzymaticactivityofTrypsinsolution.EDTAactsbyneutralizingCalciumandMagnesiumionswhichincreasesthecelladherenceontothesurfaces.
1. Product Quality: BioGenomics r-Trypsin is manufactured under stringent GMP
environment.Therobustmanufacturingprocessensuresthefinalqualityoftheproductas per the regulatory requirements. Robust process controls ensures high qualitystandardsandconsistencyandeliminatesbatchtobatchvariability.
2. Non-animal origin: Derived as a Recombinant product from E coli, BioGenomics r-
Trypsineliminatestheriskofviruses,TSE/BSEorotherpotentialadventitiousagents.3. Enzymeinhibition:SoybeanTrypsininhibitorandotherinhibitorshavesimilarinhibitory
activityforBioGenomicsr-TrypsinsolutionastheydowithnativeTrypsin.4. High purity: BioGenomics r-Trypsin provides increased specificity and eliminates
contaminatingactivitiesfoundinlowerpurityanimalorigin(Bovine/Porcine)enzymes.
FEATURESANDBENEFITS
5. Convenience:BioGenomicsr-Trypsinisformulatedattheoptimalconcentrationcanthat
bereadilyconvertedtosolutionforuseindissociationofadherentcells.6. Shelf life: BioGenomics r-Trypsin has a proven stability period of 24monthswhen
storedat-20˚C.GelpictureshowingcomparativeSDS-PAGEprofileofBiogenomicsrecombinanttrypsinwiththatofreferencestandard.
SDS-PAGEanalysisofrecombinantBiogenomicsTrypsinperformedunderreducingconditionsfollowedbyCoomassiestaining.Lane1:Molecularweightmarker;Lane2:RecombinantBiogenomicsTrypsin;Lane3:Referencestandard.
TRYPSINIZATIONTrypsinization isusuallydonewithbuffer compositionofBioGenomics r-TrypsinatadesiredconcentrationwithadditionofchelatingagentslikeEDTAwhichisstoredat2-8˚C.Thissolutionisstablefor24monthsunderthestorageconditionsof2-8˚C.
123
StandardProtocolforTrypsinization:
1. BioGenomicsr-Trypsinsolutionshouldbethawedatroomtemperaturebeforeitsuse.Thecontainershouldbeopenedatroomtemperatureinanasepticarea,ideallyunderalaminarairflowhood.
2. Thisr-Trypsinsolutioncannowbeusedincellculturetrypsinizationprocess.
3. The excess cell culture medium should be removed by aspiration. The cell culture
monolayer is then rinsedwithaCa2+andMg2+ freesalt solution, suchasDulbecco’sPhosphateBufferedSaline,orHanks’BalancedSaltSolution.
4. Thesalt solution is further replacedwithBioGenomics r-Trypsin.Thisprocessshould
becarriedoutbyaspiratingthesaltsolutionout,followedbyarefillofBioGenomicsr-Trypsinsolutionintothevessels.Itneedstobeensuredthatther-Trypsincompletelycoversthemonolayerofthecells.
5. Incubate the vessel at 37˚C, until the cells start detaching from the surface of theculturevessel.Theprogressoftrypsinizationshallbecheckedintermittentlyatregulartimepointslike1,2or5minutesorevenmoretimepointsbasedonthecelllinebeingusedunder the invertedmicroscope. Toensureproperdetachmentof the cells, theculturevesselshallbegentlytapped.
6. When all the adherent cells become suspended and the cell morphology appears
rounded under the microscope, it shall mark the successful completion of thetrypsinizationprocessandproveitsefficiency.
7. Uponthecompletionofthetrypsinizationprocess,thecellculturesuspensionshallbedilutedwithmediacontaininganappropriateconcentrationofserum.Suspendedcellstendtoformclumpsandtheseshallbebrokenupbygentlypipettingthecellculturemedium.Thiscellculturesuspensionshallnowbetransferredtoacentrifugetubeforfurtherprocessing.
8. Centrifugationofthecellculturesuspensionshallbecarriedoutat100xgfor5to10
minutes.Atthisstage,theTrypsinactivityshallbeinhibitedbyadditionofSoybeanTrypsininhibitor.Thecentrifugationstepshallbecarriedoutmultipletimestogetridoftrypsinsolutionbyre-suspendingthecellpelletwithfreshcellculturemedium.
9. Checkingthecellviabilityandcellcountingaftercentrifugationshallbedoneandthe
cell pellet shall be further sub cultured into fresh culture vessel with cell culturemediumasperthestandardoperatingprocedureoftheenduser.
Note:1. Different cell lines require different times for the process of dissociation from the
surface.Major factors affecting the process duration depends on the cell type, celldensity, potency of Trypsin, serum concentration in growthmedium and time sincelast subculture. Excessive exposure of the cell culture with respect to Trypsinconcentrationandreactiontimeshallbeavoidedtopreventcellulardamage.
2. Whenever there is a use of serum in cell culture medium it is important to use
SoybeanTrypsininhibitorin1:1ratiotoneutralizetheovertactionofTrypsin.
ExtensivestudieshavebeencarriedoutonBioGenomicsr-Trypsinwithvariouscelllinestostudytheeffectoftrypsinization.Theresultofourstudiesondifferentcelllinesisashighlightedbelow:
CellLine OptimumDissociationTime
Vero 10–15minutesMDCK 15–20minutesCHOK1 1–2minutesMRC5 4–5minutes
SUMMARYBioGenomicshas conductedextensive, comparative cell dissociation studieswithmultiplecelllinesforserumfreeandserumsupplementedadherentcellcultures.ThestudyhasbeencarriedoutspecificallyfocusingonTrypsinofanimaloriginincomparisonwithBioGenomicsr-Trypsin.BioGenomicsstandstoendorsetheenhancedperformanceof itsproductbasedonthestudiesconducted.BioGenomicsr-Trypsinhasgivenexceptionalresultsvis-à-viscellviability greater than 95% under all our typical experimental conditions of 37°C for 10minutes for CHO cell line, in addition to other widely used mammalian cell lines.BioGenomicshasgeneratedextensivedataondifferentcelllineslikeVero,CHO,andMRC5etc. Kineticsofdissociationhasalwaysbeenbetter forBioGenomics r-Trypsinunderourexperimentalconditions.Theviabilityofdissociatedandsubsequentlyharvestedcellshavealwaysbeengreaterthan90%(Figure:2).
Figure:1:ThebelowgraphshowsthecomparisonofBioGenomicsr-TrypsinwithAnimalOriginTrypsinavailableinthemarket.
Adherentculturesofmultiplecell lineswereusedtoevaluatetheutilityofBioGenomicsr-Trypsinforuse incellculture. Inthisexperiment,BioGenomicsr-Trypsinandanimalorigintrypsinpowderswereusedfortrypsinizationexperiment.Thecellviabilityexceeded90%incomparisontoAnimalOrigintrypsin.(Figure:2).Figure:2:ThegraphshowscomparisonoftheviabilityofcellsofvariousdifferentcelllinesafterdissociationwithBioGenomicsr-TrypsinandAnimalOriginTrypsin.
BioGenomicsr-Trypsinisprovenforitsstability.TrypsinizationefficiencyofBioGenomicsr-Trypsinremainsunchangedoveraperiodof24months.(Figure:3)
0
1
2
3
4
5
6
7
Animal Origin Trypsin (0.125%)
BG-rTrypsin (0.125%)
Animal Origin Trypsin (0.25%)
BG-rTrypsin (0.25%)
4.75.4 5.2
6.3
CellNumber(Cells/ml)X10^6
ComparisonofActivityofAnimalOriginTrypsinandBioGenomicsr-Trypsin
0 20 40 60 80 100
Verocellline
CHOK1Cellline
MDCKcellline
%Viability
ViabilityofDissociatedcells
Trypsinformulation
Figure: 3: Graph showing the Trypsinization efficiency of BioGenomics r-Trypsin onvariousdifferentcelllines.
STORAGE&STABILITYBioGenomicsr-Trypsinissuppliedas lyophilizedpowderwhichisstablefor24monthsat-200C. The solution made out of lyophilized r-Trypsin is also proven to be stable for 24monthsat2-80C.BioGenomicsr-Trypsininitssolutionformremainsstableforaperiodof6monthsatambienttemperaturewhenstoredunderundersterileconditions.
RECOMBINANTTRYPSIN:KEYQUALITYPARAMETERS:
APPEARANCE Whiteoralmostwhitepowder
SOLUBILITY Solublein0.001NHCland20MmTris,Ph8.0ataconcentrationof5mg/Ml.(ForenzymepreparationforInsulinproductionandcellculture)
AnAmountequivalentto500000USPTrypsinunits,solublein10Mlofwaterandin10mLofsalineTS
ACTIVITYPERMILLIGRAMOFSPECTROSCOPICPROTEIN(1mg/Mlsolutionofrecombinanttrypsinhasabsorbanceof1.47unitsat280nm)
Notlessthan2500USPunits/mg
ACTIVITYPERMILLIGRAMOFTOTAL Notlessthan2500USPunits/mg
0 6 12 18 24VeroCellLine 10 10 11 11 11
CHOK1CellLine 1.5 2 2.5 2 3
MDCKCellLine 15 15 15 16 16
0
4
8
12
16
20
Time(m
inutes)
Months
TwoYearsTrypsinizationDataForBioGenomicsr-TrypsinFormulation
POWDER
SDS-PAGE
IDENTIFICATIONBYSDS-PAGE Theelectrophoreticmobilityofthesampleshouldcorrespondtotheelectrophoreticmobilityofreferencestandard.
IMPURITIESWITHMOLECULARMASSESHIGHERTHANTHATOFTRYPSINBYSDS-PAGE
Notmorethan5%
RATIOOFTOTALSPECTROSCOPICPROTEINCONTENTPERMILLIGRAMOFPOWDER
Notlessthan0.5
RESIDUEONIGNITION Notmorethan2.5%
LOSSONDRYING Notmorethan5%
MICROBIOLOGYTEST
a. Escherichiacolib. Staphylococcusaureusc. Pseudomonasaeruginosad. Salmonella
ShallbeabsentShallbeabsentShallbeabsentShallbeabsent
REFERENCES:
1. TrypsinUSPmonographNF2. “Onthemechanismofactionofproteolytic inhibitors: IV.Effectof8Mureaonthe
stability of trypsin in trypsin-lnhibitor complexes". Archives of Biochemistry andBiophysics140
3. Voet&Voet(1995).Biochemisty(2nded.).JohnWiley&Sons.pp.396–4004. “Complete disassociation of adult pancreas into viable single ce lls through cold
trypsin-EDTA digestion.” Journal of ZhejiangUniversity - Science B (Biomedicine&Biotechnology)
5. “FacileTrypsin Immobilization inPolymericMembranes forRapid, EfficientProteinDigestion.” Anal Chem. 2010 December 15; 82(24): 10045–10051.doi:10.1021/ac101857j
6. “CultureofAnimalCell”-AManualofBasicTechniquebyR.IanFreshney7. “CellDissociationwithtrypsin”-SigmaAldrich8. “Dissociationofcellmonolayersusingtrypsinsolutions”-Corningcellgro.9. “TrypanBlueexclusion testof cell viability” -WarrenStrober;CurrentProtocols in
Immunology.