Questionnaire for Protein Production
When finished, please reply to:
Kari Thostenson, MPA
Email: Thostenson@waisman.wisc.edu
Fax: 608.263.5725
Today’s Date
Principal Investigator or Client
Company or Institution
Product Name
Services Requested
Process Development
Assay Development
Cell Banking
cGMP Manufacture
Aseptic Fill
Quantity of protein 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. Please briefly describe the identity of your protein:
b. How is it produced?
Recombinant (microbial)
Recombinant (mammalian)
Native
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 Protein Production
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. Raw Materials
Please list any specialized raw materials or vendors for your existing production process. Please
note any special testing that is required for raw materials (e.g. cell culture testing of serum
lots).
5. Manufacturing Information
a. Please describe any unique structural elements that impact manufacturing (e.g. disulfide
bridges, homo-multimerization, etc.).
b. Product Quality
Table 1 lists commonly used release specifications. Please check criteria you currently use or
would like to use and fill in required specification if possible.
Table 1
Test
Typical Starting
Specification
Methods / Comments
Protein Concentration
TBD ±5%
Identity
Matches reference
standard
Identity
Comparable to reference
std
Report banding pattern
MW as expected
Positive for target protein
≥ 90% pure
Report impurities > 1%
Comparable to reference
Identity / Purity
Purity / Concentration
Purity - Aggregates
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Desired
Abs (280) or total protein
assay (BCA)
N-terminal sequencing or
peptide map with mass spec
analysis
Isoelectric focusing
SDS-PAGE with Western blot
HPLC - Protein specific /
reversed phase / AEX
Size Exclusion
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Requested
Specification
Questionnaire for Protein Production
Test
Typical Starting
Specification
Methods / Comments
Report level of aggregates
Appearance
pH
Sterility
Endotoxin
Host Cell Protein
Varies
Varies
Pass
< 5.0 EU/kg/dose
Consistent clearance
Host Cell DNA
DNA < 100 pg/dose
RNA – consistent
clearance
All animals survive test
period, no weight loss
TBD
General safety test
Potency Assay
Use Now
Desired
Requested
Specification
Chromatography HPLC (SECHPLC) and/or native PAGE
Visual inspection
pH measurement
Sterility test
LAL - kinetic turbidometric
ELISA
PCR assay for host DNA
21CFR610.11 – 7 day test in
mice and guinea pigs
Please describe below
Describe activity assay or any other assays that need development here, if necessary. Will these be
performed b Customer or transferred to Waisman?
c. Do you have an executed batch record for this process?
Yes
No
d. What will you be providing as starting material?
cGMP Master Cell Bank
Research Cell Bank
Plasmid DNA
Other
e. If applicable:
Please describe cell bank (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?
f. Outline general production steps (use attachments if needed)
Cell Culture / Fermentation:
Harvest (centrifugation, microfiltration, etc.):
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Purification / Chromatography:
g. Please indicate any critical in-process assay requirements (attachments if available).
6. Master Cell Bank
a. If not provided, do you need a Master Cell Bank?
Yes
No
If yes, how large of a bank (200-300 vial is typical)?
b. If provided, has your cell line and/or tissue source undergone appropriate testing for
adventitious agents? (Note: we cannot accept cell lines that have not undergone required
testing for adventitious agents.)
7. Plasmids
a. If your system employs plasmid DNA, please provide general information including restriction
map, selectable markers, transgene(s), and regulatory components (promoter, other 5' and 3'
inserts).
b. Has the entire plasmid and insert been sequenced & characterized?
Yes
No
8. Process Development
a. What stage is process development needed (fermentation / cell culture, purification, etc)?
b. What improvements are you targeting (yield, activity, purity, scalability, etc)?
9. Cell Culture / Fermentation
a. What is the overall protein yield from your current process? What is the specific yield—e.g.
milligrams per liter fermentation.
b. Is there a need for special agents (antibiotics, other induction agents) in the production
process? (Use of kanamycin as a selection agent is preferred over ampicillin.)
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Questionnaire for Protein Production
c. Are there any special nutrients required for cell growth and/or maintenance?
d. Are there special assays that need to be performed during fermentation / cell culture?
10. Protein Recovery & Purification
a. What assays will be performed on process intermediates during the purification process?
b. Should the purification process include recovery from both media and cells?
c. Are there any special steps required before starting downstream processing? Cell lysis, RNA or
host DNA removal, etc.
11. Quality Control
a. Earlier in this document (Table 1) were common assays that are used for release testing of
proteins. Please check which assays you’d like done and indicate your proposed acceptance
criteria (Note: acceptance criteria should be based on at least one qualification lot and ideally
three qualification lots). Typical acceptance criteria is listed, please indicate if you have
different requirements. Also, please indicate which (if any) assays you would like qualified (for
Phase 1-2, assay qualification is usually limited to assays that effect labeling such as
concentration or potency).
b. What assays are used to verify proper protein folding and/or function? Is a validated potency
assay available? Are there any special product assays available (e.g. titer by HPLC)?
c. Can you provide us with assays for measuring the identity and concentration of the active
ingredient (ELISA, Western Blot, etc)?
12. Bulk Storage
a. What are your packaging and storage specifications for final purified bulk product?
13. Dispensing (optional)
a. What is the total number of vials or other containers required (note: include enough excess for
long term storage, sampling, and stability testing)?
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b. Does the vehicle/adjuvant require any special preparation?
c. Do you require a placebo fill (how many, if so)?
d. What are the specifications for fill volume (please note how much material you need to extract
from the vial for your intended use)
e. Are you aware of any problems with material compatibility— containers, tubing, glass, filters,
etc.?
f. Do you have information on the preferred storage conditions and stability of the product in
bulk form, in process, and in final state?
14. Please provide any other information or clarification
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