Attachment 1 Statement of Work.pdf

Statement of Work

Hybridoma Production

GENERAL INFORMATION

1.0 Scope of Work. The United States Department of Agriculture (USDA) National Center for Agricultural Utilization Research in Peoria, Il has a need for Hybridoma Production.

The contractor will facilitate the development of cell lines that produce antibodies directed against fungal toxins (mycotoxins). The development is a process, involving as many as three (3) Stages, with the products from each Stage evaluated by Agricultural Research

Service (ARS). At the completion of each Stage the Government will have the option to proceed to the next Stage.

1.1 Stage 1. The contractor will administer compounds to mice to induce an immune response and provide ARS with samples of sera obtained from the mice. The project involves three (3) groups of mice, each receiving a different compound (hapten-protein conjugate, or immunogen’). The antisera will be evaluated for the presence of antibodies that bind the target compounds by ARS.

1.2 Stage 2. The contractor will perform splenocyte fusions on mice selected based upon the results of Stage 1. The cultures will be evaluated for the presence of antibodies that bind to protein conjugates supplied by ARS. Cultures that bind successfully will be stabilized and samples of their culture supernatant fluid sent to ARS for further evaluation.

1.3 Stage 3. The contractor will clone fusion products, obtained in Stage 2, in order to obtain immortal cell lines that are monoclonal. In this work, the cell lines, which contain a mixture of different cell types, will be cloned to isolate cell lines that produce single types of antibodies (monoclonal antibodies). Culture fluid from selected monoclonal cell lines will be sent to ARS for further evaluation.

2.0 Background.

2.1 Description of Stage 1. This Stage of the project involves three (3) groups of mice, each receiving a different compound (hapten-protein conjugate, or ‘immunogen’).

Each immunogen will be administered to a separate group of mice to induce immune responses. The sera from the mice will be shipped to ARS for evaluation. The contractor will provide all of the services related to obtaining the animals, their maintenance, administration of the compounds, obtaining samples of sera from each animal, limited testing of the sera, and shipping of the antisera to ARS.

This portion of the contract will be successfully completed if the antisera bind to their corresponding immunogens (that is, there are antibodies present that bind to the immunogens given to the mice). Because mycotoxins are small molecules it is common for antisera that recognize the immunogen to have poor recognition for the associated mycotoxin. It is the responsibility of ARS to test the antisera for recognition to the associated mycotoxins. Based on the results from this testing ARS will determine whether to exercise the option of proceeding with Stage 2. The contractor will maintain the mice in an immunized state while such testing is conducted by ARS. Testing will be conducted by ARS within a timeframe such that it does not confer upon the contractor an undue burden with regard to maintenance of the animals (i.e. less than 3 weeks following receipt of the antisera).

2.2 Description of Stage 2. A single group of mice, selected among the three (3) groups present in Stage 1 will be selected for Stage 2. Within the selected group, ARS will provide to the contractor a ranked list of the mice having the most favorable responses. The contractor will fuse spleen cells from the selected mice with an

'immortal' cell line. The products (hybridoma fusions) will then be evaluated two (2) ways. One of these (a non-competitive binding assay) will be conducted by the contractor. Hybridomas capable of producing antibodies that bind to the immunogen will then be stabilized so that they can be evaluated further (in competitive assays) by

USDA- ARS. Samples of supernatant solutions of the hybridoma cultures will be shipped to USDA-ARS-NCAUR to determine if their performance characteristics are sufficient to proceed further.

This portion of the contract will be successfully completed if supernatant solutions from hybridoma fusion products bind to the associated immunogen. Because mycotoxins are small molecules it is common for hybridoma fusion products that recognize the immunogen to have poor recognition for the associated mycotoxin. It is the responsibility of ARS to test the hybridoma fusion products for recognition to the associated mycotoxins. Based on the results from this testing ARS will determine whether to exercise the option of proceeding with Stage 3. The contractor will maintain the hybridoma fusion products in a fashion that they determine facilitates stability (e.g. stored frozen, maintained in continuous culture, or other) while such testing is conducted by ARS. Testing will be conducted by ARS within a timeframe such that it does not confer upon the contractor an undue burden with regard to maintenance of the hybridoma fusion products (i.e. less than 4 weeks following receipt of the fusion products).

2.3 Description of Stage 3. In the preceding Stage mixtures of cell lines that produce antibodies will have been developed. These mixtures of cell lines (hybridoma fusion products) will be used as the basis from which to draw individual cell lines that produce individual types of antibodies (“clones”). That is, each cloned cell line will produce one type of antibody (a monoclonal antibody). Attaining clonality generally requires multiple rounds of cloning by a process known as "limiting dilution" in order to separate mixtures of cells into those that are potential clones. After an initial round of testing the desired cells will then be subjected to another round of cloning. The desired cells from the second round will then be re-tested. Additional rounds may be necessary to obtain cell lines that are stable (i.e. do not lose either the ability to grow or produce antibodies). The number of cell lines is then further reduced by selecting for growth in a specific medium. At this Stage small amounts of each of the monoclonal antibodies are produced by inducing, and collecting, ascites fluid in mice.

Sub-samples from selected clones (i.e. the antibody producing cell lines) and the mouse ascites (i.e. the associated antibodies) will be shipped to USDA-ARS-

NCAUR.

This portion of the contract will be successfully completed if USDA-ARS receives between one (1) and ten (10) cell lines that produce antibodies which bind to the associated immunogen, as well as small samples of mouse ascites fluid from each.

For the purposes of this contract, it is not essential that the antibodies recognize the target mycotoxins, as this aspect will have been screened for by USDA-ARS-

NCAUR at the end of Stage 2.

CONTRACTOR REQUIREMENTS

3.0 Technical Requirements/Tasks.

3.1 Stage 1. The contractor will administer three hapten-protein conjugates

(immunogens), provided by ARS, to two groups of eight-week-old mice. Each group will consist of 10 female Balb/C mice provided and maintained by the contractor at their facility. The sera will not be used for development of therapies, and therefore may be produced using standards applicable with Research Protocols, and not the more stringent standards associated with human diagnostic use. All animal procedures must meet the requirements established by the U.S. National Institutes of

Health-Office of Laboratory Animal Welfare, for the care and use of experimental animals.

ARS will provide two immunogens for testing. The immunogens will consist of low molecular weight fungal toxins (mycotoxins) covalently linked to serum proteins

(bovine serum albumin: BSA, or chicken egg albumin: OVA). The immunogens will be administered according to the following protocol:

3.1.1 A primary subcutaneous immunization consisting of 100 micrograms of immunogen prepared in an adjuvant of the contractor’s choice (Freund’s complete adjuvant, or other). The immunogens will be provided to the contractor as 2 milligram/milliliter solutions either frozen, or shipped freeze-dried in appropriate buffer (phosphate buffered saline).

3.1.2 A secondary immunization of 50 micrograms of immunogen in adjuvant

(incomplete Freund’s or equivalent) given 28 days after the primary immunization.

3.1.3 A third immunization (booster), given 28 days after the secondary immunization, also with 50 micrograms per mouse.

3.1.4 Two weeks following the third immunization, small volumes of serum (circa

0.1 to 0.2 milliliter) will be collected from each mouse and shipped to USDA-

ARS-NCAUR for evaluation.

3.1.5 The mice will continue to be maintained for a period of up to 1 month following the shipment, in order for the sera to be evaluated by scientists at

USDA-ARS-NCAUR. This period is necessary in order for it to be determined whether the option for the next Stage (Stage 2) will be exercised.

3.1.6 Modifications to the above protocol may be acceptable if approved by the government (i.e. ARS PI) before their implementation.

3.1.7 The contractor may also, at their discretion, conduct quality control assays to ensure the procedures are meeting internal expectations. For example, the contractor may decide to conduct non-competitive enzyme-linked immunosorbent assays (non- competitive ELISAs) to ensure that the mice have generated an immune response to the immunogen.

3.2 Stage 2. At the option of USDA-ARS one of the three groups of mice from Stage 1 will be selected and will proceed to Stage 2. Within the selected group USDA-ARS-

NCAUR will provide a ranked list of the responses of the antisera from the mice. This is essential in order to enhance the chances of obtaining antibodies that recognize the mycotoxin (hapten), rather than the immunogen alone. Based upon this information the contractor will perform hybridoma fusions (aka splenocyte fusions). The contractor will conduct splenocyte fusions on as few as one, or as many as three of the mice (with the mice selected by USDA-ARS as those most likely to yield successful products). All animal procedures must meet the requirements established by the U.S. National Institutes of Health-Office of Laboratory Animal Welfare, for the care and use of experimental animals.

USDA-ARS will provide the antigen required by the contractor to use in their initial evaluations of the hybridoma culture supernatant solutions. The antigens will consist of low molecular weight fungal toxins (mycotoxins) covalently linked to a serum proteins (bovine serum albumin: BSA, or chicken egg albumin: OVA). The contractor will evaluate the hybridoma culture supernatant solutions using a format known as a non-competitive enzyme-linked immunosorbent assay (non-competitive

ELISA). In this type of assay, positive responses signify that antibodies are present that bind the antigen. Supernatant solutions (of a volume greater than 0.1 mL of each) from positive culture will be shipped to USDA-ARS- NCAUR (c/o Chris Maragos) for evaluation in more extensive, competitive, immunoassay formats.

The contractor will continue to maintain the hybridoma cell cultures at their facility while the evaluation at USDA-ARS-NCAUR takes place. If stabilized, the hybridoma cultures may be stored by the contractor under conditions conducive to long-term storage and revival (i.e. storage under liquid nitrogen, or continued passage of the cells, or other techniques as deemed appropriate by the contractor). This step is necessary in order to determine whether the project should continue onto the next potential Stage.

Modifications to the above protocols may be acceptable if approved by the

The contractor may also, at their discretion, conduct quality control assays to ensure the procedures are meeting internal expectations. For example, the contractor may decide to conduct additional non-competitive ELISAs to ensure that the cell lines have retained the ability to bind to the protein conjugates.

3.3 Stage 3. At the option of USDA-ARS the project may proceed to the third, and final, Stage. If this option is exercised, the contractor will use as starting material hybridoma products (aka fusion products) developed previously in conjunction with

USDA-ARS-NCAUR in Stage 2. The contractor will select at least six positive wells from which to proceed with cloning. Cell lines will be cloned by the limiting dilution method and tested in non-competitive enzyme-linked immunosorbent assay (ELISA).

The ELISA test used will be based upon materials ('coating antigens') supplied by

USDA-ARS-NCAUR. The antigens will consist of low molecular weight fungal toxins (mycotoxins) covalently linked to serum proteins (bovine serum albumin:

BSA, or chicken egg albumin: OVA). Cell lines that are positive will be expanded and subjected to another round of cloning. Prior to final cloning, the initial positives will be stored frozen (as backup). Supernatant solutions from the second round of cloning will be tested by ELISA, and clones will be selected that exhibit stability with regards to the ability to grow in culture and produce antibodies.

3.3.1 A minimum of one and a maximum of 10 clones will be selected for further expansion to produce small amounts of antibody for further evaluation by

USDA-ARS- NCAUR. Samples of culture fluid or ascites fluid from the selected clones will be shipped to USDA-ARS-NCAUR on dry-ice, for further evaluation.

3.3.2 Stabilized cell lines may be stored by the contractor under conditions conducive to long-term storage and revival (i.e. storage under liquid nitrogen, or other as deemed appropriate by the contractor).

3.3.3 Modifications to the above protocols may be acceptable if approved by the

3.3.4 The contractor may also, at their discretion, conduct quality control assays to ensure the procedures are meeting internal expectations. For example, the contractor may decide to conduct additional non-competitive ELISAs to ensure that the cell lines have retained the ability to bind to the protein conjugates.

4.0 Government Furnished. ARS will provide the contractor with the following materials:

4.1 Stage 1.

4.1.1 Three hapten-protein conjugates (immunogens) to use in immunization of mice. Each immunogen will be provided at a concentration of 2 mg/mL and will be provided either frozen (shipped to the contractor on dry-ice), or freeze-dried (shipped to the contractor at ambient temperature). The amounts provided for immunization will be 5 mg of each conjugate. Additional amounts may be available upon request.

4.1.2 Additional conjugates to facilitate screening by non-competitive ELISA. ARS will provide small amounts of additional conjugates (up to 3 mg each, more if available) for this purpose.

4.1.3 ARS will use government-furnished facilities to evaluate the sera using competitive and non- competitive immunoassays (competitive and non-competitive ELISAs). Sera will be evaluated based upon titer, and ability to cross-react with the hapten or its analogs.

4.2 Stage 2. ARS will provide the contractor with the following materials:

4.2.1 If the option to proceed to Stage 2 is exercised, a summary of the results of the evaluation by USDA-ARS from Stage 1 will be provided to the contractor to facilitate selection of mice for the splenocyte fusions.

4.2.2 Sufficient hapten-protein conjugate (immobilized test antigen) for use in evaluating the hybridoma fusion products. The conjugate will be provided at a concentration of 2 mg/mL and will be provided either frozen (shipped to the contractor on dry-ice), or freeze-dried (shipped to the contractor at ambient temperature). The amounts provided for immunization will be 3 mg of each conjugate. Additional amounts may be available upon request.

4.2.3 ARS will use government-furnished facilities to evaluate the hybridoma fusion products using non-competitive and competitive ELISAs. Cultures will be evaluated based upon titer, and ability to cross-react with the hapten

(mycotoxin) or its analogs. If the option to proceed to Stage 3 is excercised, a summary of the results of such evaluation will be provided to the contractor.

4.3 Stage 3. ARS will provide the contractor with the following materials:

4.3.1 If the option to proceed to Stage 3 is exercised, a summary of the results of the evaluation by USDA-ARS from Stage 2 will be provided to the contractor to facilitate selection of hybridoma fusion products for cloning.

4.3.2 Sufficient hapten-protein conjugate (coating antigen) for use in evaluating the hybridoma fusion products. The conjugates will be provided at a concentration of 2 mg/mL and will be provided freeze-dried (shipped to the contractor at ambient temperature). The amounts provided will be 2 mg of each conjugate. Additional amounts may be available upon request.

5.0 Deliverables / Schedule.

5.1 Stage 1 Deliverable. Deliverables will include samples of sera from mice, collected two weeks following the third immunization. The sera may be provided as undiluted sera (i.e. blood with clot removed), or as diluted sera. If provided as diluted sera, the extent of dilution should be noted and described in the shipment. Sera should not be provided at dilutions greater than 1:10 (i.e. one volume of serum in 9 volumes of buffer). Sera should be stabilized with an appropriate preservative before shipment, or immediately frozen after collection and shipped frozen, on dry-ice. The goal is to prevent microbiological or chemical degradation of the samples during shipment.

Volumes shipped should be no less than 0.1 milliliter of serum (or up to 1:10 dilution) for each mouse. Shipment should be as soon as is practical following serum collection, but no later than two weeks following serum collection.

Because there will be three groups with 10 mice per group, the expectation is for ARS to receive a total of 30 samples of serum. Allowances will be made for fewer samples if a small number of animals perish before sera can be collected, or if there is excessive difficulty in obtaining sera from some of the animals

Shipment should be made to:

USDA-ARS-National Center for Agricultural Utilization Research (NCAUR)

1815 N. University Street

Peoria, IL, USA 61604 c/o Chris Maragos

5.2 Stage 2 Deliverables. Deliverables will include samples of culture fluid from the hybridoma supernatants. Culture supernatant solutions may be provided undiluted or diluted, as long as the extent of any dilution is specified and described in the shipment. Culture supernatant solutions should not be provided at dilutions greater than 1:10 (i.e. one volume of supernatant solution in 9 volumes of buffer), and should be stabilized with an appropriate preservative before shipment. The goal is to prevent microbiological or chemical degradation of the samples during shipment. Volumes shipped should be no less than 0.1 milliliter of undiluted supernatant solution (or 1 mL if diluted 1:10). Shipment should be as soon as is practical following generation of the fusion products.

1815 N. University Street

5.3 Stage 3 Deliverables. Deliverables will include sub-samples of the cell lines cloned in

Stage 3, and samples of mouse ascites fluid generated from those cell lines. Volumes of the ascites fluid will be in the range of 5 to 15 mL for each culture. In order to prevent microbiological or chemical degradation, the mouse ascites samples should be stabilized with an appropriate preservative before shipment.

1815 N. University Street

6.0 Contractor’s Key Personnel.

6.1 Education level of Bachelor of Science with emphasis on biological sciences

(molecular biology, biochemistry, toxicology, pharmacology, animal husbandry, or other degree directly related to hybridoma production)

6.2 The contractor shall have a minimum of ten (10) years of direct experience managing hybridoma production programs.

6.3 Provide evidence of previous contracts to perform hybridoma production-related services.

7.0 Quote Submission. Quotes submitted in response to this solicitation shall include a Price

Quote and a Technical Quote. The Government will evaluate quotes in accordance with the evaluation criteria set forth in Section 8.0 of this RFQ.

7.1 Offerors shall submit their quotes electronically via email to debora.wells@usda.gov.

The following information shall be included:

7.1.1 Price (to include shipping)

7.1.2 Technical Specifications

7.1.2.1 Ten (10) years of direct full-time experience managing commercial hybridoma production programs.

7.1.2.2 Provide evidence of previous contracts to perform hybridoma production-related services.

7.1.3 Completed copy of FAR Provision 52.212-3.

7.2 Offerors are responsible for ensuring and verifying their offer and all required quote documents are received by the date designated in block 8 of the SF-144

8.0 Evaluation Factors.

8.1 Factor 1 – Price. This is a Lowest Price Technically Acceptable (LPTA) source selection. Award will be made on the basis of the lowest evaluated price meeting or exceeding the acceptability standards for the non-cost factors. The lowest priced mailto:debora.wells@usda.gov quote will first be evaluated for technical acceptability. If determined not technically acceptable, it will be removed from competition and the next lowest priced quote will be evaluated for technical acceptability. This will continue until the lowest price quote is determined technically acceptable. Once the lowest priced quote is determined technically acceptable, then a determination of responsibility will be completed. If found responsible, evaluations will be closed and award will be made.

8.2 Factor 2 – Technical. Offerors technical quote shall include all documents at the time of submission. The absence of such evidence will adversely influence the evaluation of the quote. All documents shall be submitted in accordance with 10 pages maximum, Page Size: 8½” x 11” and size 12 font.

8.2.1 Experience. Offeror shall provide resume documenting ten (10) years of direct full-time experience managing commercial Dairy nutritional programs.

8.2.2 References. Offeror shall provide evidence of previous contracts to perform hybridoma production-related services.

9.0 Data Rights. The contractor will have no rights to data generated by ARS or other government agencies related to this contract.