Making a Master or Working Cell Bank

Bioman 2009

July 27 to July 30Rochester, NY

Presenter: Dana M. Hopkins

Wm. Davies, Jr. Career & Technical HS

Master Cell Bank (MCB)

Established from a single cloneRepresents a cell reserve “Frozen in Time”

--Preserves characteristics

--Prevents contamination and deteriorationProduced in accordance with regulatory

standards (21CFR 610)

Cell line characterization

Testing objectives of cell line

--Confirm identity (expression construct)

--Confirm purity (contamination)

--Confirm genetic stability (coding region)Quality assurance established from master

bank to end-of-production/post production cells (EPC/PPC)

Safety testing of cell banks

Eliminates/minimize adventitious agents to the biopharmaceutical

--bacteria

--mycoplasma

--fungi

--viruses

Source of Contaminants

Cell Substrate• Endogenous viruses• Exogenous microbial contaminants• Source material screening

-Human (HIV, HBV, HCV, CJD, etc)

-Animal (TSE sources, species-specific viruses.

Contaminants (cont.)

Raw Materials

*Cell culture reagents (animal and non- animal)

Environment

• Water

• Air

• Human/Technicians

Regulatory Documents

CBER/FDA: Points to consider in cell line characterization

CBER/FDA: Points to consider in manufacturing and testing

European Pharmacopeia (EP)US Pharmacopeia (USP)Japanese Pharmacopeia (JP)

Validated In-house Guidelines

Species Identity: Confirmed by iso-enzyme analysis and cross-species contamination

Species Banding Pattern: Confirmed by agarose gel electrophoresis

DNA Fingerprinting: Karyology

Purity Testing

Sterility:

--Bulk harvest/cell banks tested for bacterial & fungal contamination.

Mycoplasma:

--Two methods recommended by inoculation in broth and agar.

Purity Testing

Adventitious viruses

--Invitro assay with indicator cell lines

--Invivo assay with embryonic chicken eggsRetrovirus (rodent cell lines):

--XC plaque assay

--S+L- focus assay

Purity Testing

Transmission Electron Microscopy (TEM)Reverse transcriptase assay:

--Product enhanced RT (PERT)

--Unique enzyme in retroviruses

--PCR sensitive to cDNA enzyme

Building the Master Cell BankTransformation or Transfection

• Introduce Foreign Gene that expresses Protein Product: (bacterial transformation)

pick one pick one

Screen for expression of foreign gene

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oo o Bacteria

Lawno

Grow Cells to 90% Confluence

Images Courtesy of Corning Inc

Cell Preparation for Freezing• Check cell line for stability

and contaminations.• Refeed cells to ensure log

phase of growth.• Label cryotubes with cell

line, cells/vial, date, MCB• Count cells with a

hemocytometer. Use trypan blue for viability.

Freezing Media 60 ml DMEM, 40 ml FCS, 1 ml

Penicillin-StreptomycinFilter through 0.2u filter

Aliquot in 15ml conical tubes for long storage at -80, or refrigerator for shorter periods of time.

For 100% DMSO:4ml pH adjusted medium (DMEM)1ml sterile 100% DMSO0.1ml versene (prevents clumping of cells)

Filter through 0.2u filter

Cell Prep. Cont.Transfer cell suspension to centrifuge tubes.Centrifuge at 1000rpm fo 5 minutes, 2-8oC.Siphon off all the medium.Slowly add chilled freezing medium to yield

1 x 107 cells/ml.Resuspend cells by gently pipetting.Place tubes on ice. Dispense cells, 1 ml/vial.Parafilm cryovials and place in -80 freezer.

Mammalian Cells Trypsinize adherent cells and pellet For each 100 mm dish, resuspend pellet in half the

volume of freezing medium. (if freezing 10 vials, add 5 mLs media)

20% DMSO made fresh daily Add dropwise DMSO to 10% final vol. Final suspension: DMEM with 20% FCS, 10%

DMSO, cells from 100 mm dish. Chill cells on ice in centrifuge tube before

dispensing in pre-chilled, 1 mL vials

Thawing Mammal CellsWorking Cell Bank

• Points to Consider: Cells should be thawed rapidly and then diluted

slowly into warm growth medium. Transfer one 1ml vial to 50ml centrifuge tube.

Slowly (dropwise) add 10ml warm medium. Pelleting DMSO cells may harm fragile cells. DMSO is toxic to cells. Change media quickly. DMSO is OSHA sensitive. Replace with glycerol

if at all possible

Schrieber’s Protocol1) Thaw vial quickly in 370C water.2) Transfer cells to sterile, 15ml centrifuge tube3) Add FBS in 1 minute increments:

50ul/1min;100ul/1min; 200ul/1min; 400ul/1min; 800ul/1min

4) Centrifuge for 5 minutes at 1000rpm5) Aspirate supernatant, resuspend in 5-6ml warm

media.7) Transfer to T-25 flask, incubate at 370C/5% CO2

8) After 24 hours, check viability, remove/add 5-6 ml fresh warm media.

References

1. Shama, B., “Manufacturing of Low Molecular Drugs”, Contocor, Raritan, NJ, 2005.

2. Blackwell, JV, “Mycoplasma-Recent developments in Detecting and Preventing Bioreactor Contaminants”, ISPE annual meeting, Scottsdale, AZ, Nov. 2005

3. Cooper, J., ECACC, “A cell banking process for the provision of cryo-preserved, “assay ready” cells for drug discovery programs. 16 July, 2009.

4. http: www.newlab: Cell line characterization.

5. http://cerhb.ufl.edu/pdf/edcenter/cellbanking.