Questionnaire for Protein Production

When finished, please email reply to:Brian Dattilo

Email:

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? / FDA EMEA Other
Date Desired

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

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

a.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 / Methods / Comments / Typical Starting Specification / Desired / Requested Specification
Protein Concentration / Abs (280) or total protein assay (BCA) / TBD
Identity / N-terminal sequencing or peptide map with mass spec analysis / Matches reference standard
Identity / Isoelectric focusing / Comparable to reference std
Report banding pattern
Identity / Purity / SDS-PAGE with Western blot / MW as expected
Positive for target protein
Purity / Concentration / HPLC - Protein specific / reversed phase / AEX / ≥ 90% pure
Report impurities > 1%
Purity - Aggregates / SizeExclusionChromatography HPLC (SEC-HPLC) and/ornative PAGE / Comparable to reference
Report level of aggregates
Appearance / Visual inspection
pH / pH measurement
Sterility / Sterility test / Pass
Endotoxin / LAL - kinetic turbidimetric
Host Cell Protein / ELISA / Consistent clearance
Host Cell DNA / PCR assay for host DNA / DNA < 100 pg/dose
Potency Assay / Please describe below / TBD

Describe activity assay or any other assays that need development here, if necessary. Will these be performed by 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.):

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.)

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 qualificationlot 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)?

  1. 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)?

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 vialfor 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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