Questionnaire for Plasmid DNA Production Services
When finished, please reply to:
Kari Thostenson
Email: Thostenson@waisman.wisc.edu
Fax: 608.263.5725
Today’s Date
Principal Investigator or Client
Company or Institution
Product Name
Services Requested
Assay Development
Cell Banking
cGMP Manufacture
Aseptic Fill
Quantity of plasmid desired (mg or g)
What agency will this product be regulated by?
Date Desired
FDA
EMEA
Other
1. How did you hear about Waisman Biomanufacturing?
2. Product and Intended Use
a. Gene Product
Please briefly describe the identity of your gene product.
b. What is the plasmid backbone?
pUC
pBR322
Other
c. This product is intended for use in (check all that apply):
Research only and not for use in animals or humans
Animal /tox studies
Human clinical trials:
Phase I
Phase II
Phase III
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Questionnaire for Plasmid DNA Production Services
d. If used in human clinical trials, this product will be used for:
Ex-vivo applications (cell transfection/culture)
Direct injection
e. What indication is this product for?
3. Safety Information
a. Is this product a select agent?
Yes
No
b. What biosafety level?
BSL-1
BSL-2
BSL-3
4. Manufacturing Information
a. Vector Information
Please describe your vector including overall size of plasmid, identity and size of gene, source of
gene, promoters, enhancers, selection marker, etc. Include a copy of the vector map if
available. Note - β-lactam antibiotics may not be used for cGMP production.
b. Product Quality - please indicate desired product quality
(*Specifications represent a recommended starting point for new plasmid projects. Results are typically much better
for standard pUC-based plasmids with transgenes that don't cause cell toxicity. Typically specifications are set broad
initially and then narrowed as plasmid characteristics in the WB process are established. Tighter specifications may be
requested. However, additional process qualification trials and process development work may be required. An
estimate of the additional work (time, materials) may be
included in the initial budget estimate if tighter specifications are requested.)
Use Now
Desired
Test
Typical Starting
Methods / Comments
Requested
Visual
pH
Identity
Plasmid Size
DNA
concentration
A260/280 ratio
A260/230 ratio
% of
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Specification
Clear/colorless
Conforms to the
reference
Conforms to the
reference
Specification
pH meter
Restriction Map
“Uncut” plasmid
OD 260nm
1.7 – 2.0
2.0-2.2
> 75%
OD 260/280nm
OD 260/230nm
HPLC
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Questionnaire for Plasmid DNA Production Services
Test
Typical Starting
Specification
supercoiled*
Residual RNA*
< 5%
Endotoxin
Sterility*
E.coli DNA*
Residual Protein
Potency
<20 EU/mg
Sterile
< 5%
< 5%
TBD
Methods / Comments
Use Now
Desired
Requested
Specification
HPLC or gel
electrophoresis
LAL
USP
Quantitative PCR
BCA
Bioactivity Assay
(performed by Customer
or can be transferred to
Waisman)
Describe activity assay or any other assays that need development here, if necessary. Will these be
performed by Customer or transferred to Waisman?
c. What will you be providing as starting material?
cGMP Master Cell Bank
Research Cell Bank
Plasmid DNA
Other
d. Waisman recommends using either DH5-α or DH-10B strains of E. coli for producing plasmid
DNA. Please indicate if your cell bank is different or would prefer using an alternative bacterium
or strain of E. coli.
e. If applicable:
Please describe cell bank you are providing (e.g. bacterial strain, cGMP, research,
growth/expression, purity, etc.).
Please describe testing on plasmid (e.g. fully sequenced (GLP/GMP-grade?), insert sequenced,
copy number, etc.). Was this plasmid previously produced by your group or others?
Please describe knowledge of fermentation plasmid productivity (scale, mg/L).
Please provide any information regarding plasmid stability in fermentation.
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Questionnaire for Plasmid DNA Production Services
5. Master Cell Bank
a. If not provided, how large of a bank is needed (200-300 vial is typical)?
b. Cell Bank Testing – The Table below lists standard master cell bank testing.
Please indicate any additional tests you would like below. Please note that nonstandard testing
may incur extra cost for development or sourcing.
Test
Description
Growth
Characteristics
Report cell counts and growth characteristics, final O.D. @ 600
nm
Culture purity
Plate out culture, gram stain, examine morphology and for
presence of bacterial or fungal contaminants
Identity
Tests conducted to confirm correct host genotype
Bacteriophage
Test for the presence of bacteriophage (this must be done
prior to entry into the cleanroom)
Antibiotic
Resistance
Demonstrate kanamycin resistance to verify retention of
plasmid; demonstrate no colonies in Amp
Restriction
Digest/AGE
Verify correct plasmid. Perform prior to sending out for
sequencing
Plasmid Sequence
Sequence entire plasmid for FDA submission
6. Fill / Finish
If fill / finish is required, how many vials and at what DNA concentration?
Waisman uses WFI or TE as a standard plasmid solution. Is there a specific formulation for your
plasmid, or is WFI or TE acceptable?
7. Please provide any other information or clarification
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