Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

EXAMPLE BATCH RECORD

Bill of Materials
Item Description

Vendor/Cat#

RM #

Lot #

Expiration
Date

DMEM
Fetal Bovine Serum
100X Glutamax
HEK293 Cell Bank (2x106
cells/vial, P20)
50ml Conical Tubes
T75 Flasks
T175 Flasks

Non-RM Materials
Item Description

Vendor/Cat #

Pipets
Trypan Blue

Ancillary Equipment
Item Description

Equipment ID #

Page 1 of 11

Lot#

Quantity

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

BSC
Waterbath
Centrifuge
Incubator
Refridgerator

Estimated Quantities of Materials Required:

Item Description

Quantity

DMEM
Fetal Bovine Serum
100X Glutamax
Pipets

Manufacturing Room:

Mfg Rm Release Ref: SOP-XXX-XXX Log # _______________

Date/Time Begin:

Signature:

Step 1: Date/Time _________________ Room ____________

Step #

Step Description

1.0

Preparation of Media

Operator
Initials

1.1 Thaw 1 (one) x 100ml bottle of Fetal Bovine Serum

(RM-XXX, lot ____) .
Page 2 of 11

Verifier
Initials

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

Thaw bottle in a water bath set at 37 3 C

Waterbath ID# __________ Temp ______ C
Time in water bath ________________
Time out of water bath _____________
1.2 Sanitize the BSC with Cavicide followed by 70% IPA.
Sanitize and transfer required materials into the
BSC per SOP-XXX-XXX. Follow aseptic technique for
all steps performed within BSC.

____________ ____________

____________

1.3 Prepare three (3) individual bottles of Complete

Medium (DMEM, 10% FBS, 1X Glutamax)
Into each 500 ml of DMEM Medium (RM-XXX, lot
____ ) add:

____________ ____________

55 ml FBS (RM-XXX, lot ____ )

5.6 ml 100x Glutamax (RM-XXX, lot ____ )
Volume FBS added _______ ml/bottle
Volume 100X Glutamax added _______ ml/bottle
# of bottles of Complete Medium prepared
_______

Step #

Operator
Initials

Step Description

1.4 Label bottles: Complete Medium per SOP-XXX-XXX.

Include 45 day expiration date. Place example of
label below. If not immediately using, store
Complete Medium in refrigerator at 4C 2C.

Page 3 of 11

Verifier
Initials

____________ ____________

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

Fridge ID # _______________ Temp ________

Date/time to fridge _____________________

1.5 End Date/Time ___________________________

____________ ____________

Step 2: Date/Time _________________ Room _______________

Estimated Materials Required:
Item Description

Quantity

HEK293 Cell Bank (2x10e6

cells/vial, P20)
Complete Medium (step 1.4)
50ml Conical Tubes
T75 Flasks
T175 Flasks
Pipets
Trypan Blue

Step #

Operator
Initials

Step Description

Page 4 of 11

Verifier
Initials

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

2.0

Thaw/Seed of HEK Cell Bank (Passage 20)

2.1 Obtain two (2) vials of HEK Cell Bank @ 2x106
cells/vial (RM-XXX, lot _____ ) from the liquid N2
freezer and place on dry ice.
2.2 Sanitize the BSC with Cavicide followed by 70% IPA.
Sanitize and transfer required materials into the
BSC per SOP-XXX-XXX. Follow aseptic technique for
all steps performed within BSC.
2.3 Pipet 18 ml of Complete Medium (prepared in step
1.4) into a 50ml conical tube (RM-XXX, lot ____)
2.4 Thaw two (2) vials of cells in a water bath set at 37
+ 3 C with constant swirling until a small amount
of ice remains. Remove vial from water bath and
continue thaw at room temperature until
completely thawed.

____________ ____________

____________

____________ ____________

____________ ____________

Water Bath ID# __________ Temp ____ oC

2.5 Remove label from vial of cells and place
representative label below.

2.6 Using a pipet, transfer thawed cells to 50 ml conical

(step 2.3) adding drop wise while carefully swirling
to distribute cells in medium.

Page 5 of 11

____________

____________

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

2.7 Centrifuge at 200g x 5 minutes at 18 3oC.

Centrifuge ID# __________ Speed _____ g _______
rpm

____________ ____________

Time ________ Temp ______ C

2.8 Aspirate supernatant and re-suspend pellet in 2 ml
Complete Medium (prepared step 1.4).

____________ ____________

Volume Complete Medium added ______ ml

2.9 Prepare dilution for cell count (suggestion 1:4
dilution):

____________ ____________

_____L cells + _____ L trypan blue =____ L

total
_______ L total _______ L cells = _______
dilution factor
Step #

Operator
Initials

Step Description

2.10 Record counts from four diagonal 1 mm2 squares

(#of viable cells / # of dead cells.)
A) _____/b _____ C) _____/d)_____
E)_____/f_____ G)_____/h_____
a). Average Live Cells = A+C+E+G 4 = ________
b.) Average Dead Cells = b+d+f+h 4 = ________
c). Avg. Live _______ + Avg. Dead _______ = Total
Cells _________
d). Avg. Live Cells ________ Total Cells ________

Page 6 of 11

Verifier
Initials

____________ ____________

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

= ____% Viable Cells

2.11 Calculate total number of viable cells/ml as follows:
____________ ____________
______ Avg live cells (step 2.10.a) X ____ Dilution
factor (step 2.9) X 104 = __________ cells/ml
2.12 Calculate total number of viable cells as follows:

Step #

_________ cells/ml (step 2.11) X _______resuspension volume (step 2.8) = _________ total
viable cells

____________ ____________

Step Description

Operator
Initials

Verifier
Initials

2.13 Intention is to seed 1x T175 flasks at 2x10e4

0.5x10e3cells/cm2. Calculate size and number of
flasks to seed in this seeding density range.
3.5x10e6 cells/T175 flask __________ cells/ml
(step 2.11) = _______ml cell suspension per flask
Based on total re-suspension volume (step 2.8)
_______ml, determine size and number of flasks to
seed. Record information below.
Flask
Type

# Flasks

Seed
Density

Volume Cell
Suspension

T75
T175
Page 7 of 11

____________ ____________

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

2.14 Pipet Complete Medium (step 1.4) into each flask

required (T75 use 20ml, T175 use 40ml).
T75 (RM-XXX, lot _____ )
T175 (RM-XXX, lot ______ )
Record volume Complete Medium added per flask.
T75 _________ ml/flask

____________ ____________

T175 ________ ml/flask

Step #

Operator
Initials

Step Description

2.15 Add calculated volume of cell suspension (step

2.13) to each flask.

Verifier
Initials

____________ ____________

T75 _________ ml/flask

T175 ________ ml/flask
2.16 Label and date seeded flasks per SOP-XXX-XXX.
Place representative label below.
2.17 Place all flasks into incubator at 37 2 C, 5 1%
CO2

____________ ____________

____________ ____________

Incubator ID# _______ Temp _________ %CO2

______
Date/time ______________________
2.18 End Date/Time _______________________
____________ ____________

Page 8 of 11

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

Operator Signature

Date

Verifier Signature

Date

Operator Comments:

Deviations Notes:
Step #

Description

Page 9 of 11

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

Page 10 of 11

Document Title: Manufacture of HEK Master Cell Bank

Company: Cells R Us
Reference Project Code: MATC-HEK-MCB
Lot Number:
Document Approval Date:

QA Review/Approval Notes:

Manufacturing Management Approval

Date

Quality Assurance Management Approval

Date

Page 11 of 11