recombinant trypsin, animal origin...

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Recombinant Trypsin, Animal Origin Free PRODUCT INFORMATION: BioGenomics r-Trypsin powder is ready to use, animal origin free optimized for cell culture applications. It is derived by r-DNA technology. BioGenomics r-Trypsin is a pure enzyme, which helps maximize the yield of functionally viable cells from culture vessels, while preventing the toxic effect induced by other non-desirable proteases. In addition, BioGenomics r-Trypsin, being a recombinant product, completely eliminates the risk of viruses, or other potential adventitious agents found in animal derived products. 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 like Ethylenediaminetetraacetic acid (EDTA) is often added to enhance the enzymatic activity of Trypsin solution. EDTA acts by neutralizing Calcium and Magnesium ions which increases the cell adherence onto the surfaces. 1. Product Quality: BioGenomics r-Trypsin is manufactured under stringent GMP environment. The robust manufacturing process ensures the final quality of the product as per the regulatory requirements. Robust process controls ensures high quality standards and consistency and eliminates batch to batch variability. 2. Non-animal origin: Derived as a Recombinant product from E coli, BioGenomics r- Trypsin eliminates the risk of viruses, TSE/BSE or other potential adventitious agents. 3. Enzyme inhibition: Soybean Trypsin inhibitor and other inhibitors have similar inhibitory activity for BioGenomics r-Trypsin solution as they do with native Trypsin. 4. High purity: BioGenomics r-Trypsin provides increased specificity and eliminates contaminating activities found in lower purity animal origin (Bovine/Porcine) enzymes. FEATURES AND BENEFITS

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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.

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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.