Purification of Monoclonal Antibodies from Pre-existing Hybridomas owned by Customers:

CMI will provide an outline for two different services, the first being the cheaper, and the second being a more careful approach to maintaining hybridoma integrity. It should be stressed that the price difference does not influence the quality of the purified MAbs CMI will provide.

CMI expects customers to provide problem hybridomas, and CMI is proud of a track record whereby this company has salvaged four individual hybridomas that were otherwise lost. CMI offers no guarantees for this service, however.

Before commencing either service CMI, will need to know from the customer:

  • a) The parent mouse myeloma from which the hybridoma was derived, and also the Ig isotype/subclass and light chain specificity of each specific MAb.
  • b) A brief description of the culture medium in which the hybridoma is grown - and particularly if the hybridomas require culture in HAT or if any other special additives are used (e.g. interleukins, origen etc.).

CMI will expect to sign a customer provided, legally binding document, covering both confidentiality and ownership of the hybridoma cell-line and secreted products. If the customer also wishes to supply the hybridoma(s) under simple code - so much the better.

CMI will accept hybridoma cells either frozen on dry-ice, or already growing in T-25 or T-80 cm2 flasks (the latter method is preferred).

Service 1:

This is a minimum service to provide quality MAb at the lowest price. The minimum order for this service is 10.0 mg purified MAb from each hybridoma. CMI will:

1. Thaw if necessary, then expand hybridoma cells into larger volume of culture medium.

2. Titrate IgG production from each hybridoma, and further expand the culture to a volume that will provide the required MAb yield. At harvest of MAb, all viable hybridoma cells will be destroyed.

3. Isolate the MAb from culture supernatant using:

  • a) ammonium sulphate precipitation,
  • b) isolation over an SpA column using a proprietary buffer system which stabilizes the MAb, and also ensures recovery of all mouse IgG isotypes and IgG subclasses.

4. Concentrate the SpA eluted MAb to >1.0 mg/ml in PBS pH 7.2 with 0.05% sodium azide. The final MAb concentration will be determined at OD280 by applying an extinction coefficient of 1.4.

5. Ship purified MAb at ambient temperature to customer address by FedEx.

6. Invoice at $75.00 per mg for 10.0 to 50.0 mg SpA purified MAb per hybridoma.

7. The time required to perform the service is 3-6 weeks. Service 2:

This is a more detailed service recommended for customers who are likely to repeat their order in the future, or who have hybridomas that will benefit from the additional cloning step included. The minimum order for this service is 25.0 mg purified MAb from each hybridoma. With this service the end-user has input and can request modifications or variations in the service.

For service 2 the customer is encouraged to supply each hybridoma under simple code, and the customer must be prepared to supply (also under code) 50-200 ug of specific antigen relevant for each hybridoma (also under code). CMI will:

1. Thaw if necessary, then expand hybridoma cells into larger volume of culture medium.

  • 2a. Immediately clone each hybridoma by limiting dilution, and when clones have emerged (10-14 days later) check each clone for activity against specific antigen (by ELISA).
  • 2b. Briefly report on the cloning efficiency, and on the percentage of specific antibody secreting cell clones obtained.
  • 2c. Confirm Ig isotype, subclass, and light chain production, of selected, re-cloned, hybridomas.
  • 2d. Return to the customer 1 X T-80 cm2 flask containing viable, freshly cloned, hybridoma cells specific for the customer provided Ag.
  • 2e. At the customer's option, CMI will freeze 4 vials of cells from each cloned hybridoma and store these in liquid nitrogen under customer code for a period of 3 years - longer if customer requests. (There is no additional charge for this service).

3. In parallel with hybridoma cloning, CMI will titrate IgG production of each pre-cloned hybridoma, and then expand each culture to a volume that will provide the required MAb yield. At MAb harvest all viable pre-cloned hybridoma cells will be destroyed.

4. CMI will then isolate each MAb from culture supernatant by:

  • a) ammonium sulphate precipitation,
  • b) isolation over SpA using a proprietary buffer system which stabilizes the MAbs and ensures recovery of all mouse IgG isotypes and IgG subclasses.

5. Concentrate the SpA eluted MAb to > 1.0 mg/ml (or to a specific concentration as specified by customer) in PBS pH 7.2 with 0.05% sodium azide. The final MAb concentration will be determined at OD280 by applying an extinction coefficient of 1.4.

6. Purified MAbs will be shipped at ambient temperature to address provided by FedEx.

7. Invoice at $105.00 per mg for the first 25.0mg purified MAb per hybridoma. If the order exceeds 25.0 mg then the cost for additional MAb is $75.00 per mg.

The time required to perform the service is 4-7 weeks. The advantages for this service are:

i. The customer will receive back each hybridoma cell line after re-cloning. This step will help to ensure the long-term survival and specific MAb secretion of the hybridomas.

ii. The customer has the option to insure hybridoma survival by having the hybridomas stored at an independent facility (CMI) under the customer code. If the customer then wishes to repeat the order, initiation requires only an authorizing phone call.

iii. The price for repeat orders is $60.00 per mg, because Service 1 is now all that is required (i.e. the hybridoma does not require re-cloning).

Discounts for bulk MAb production:

Should a customer require purified MAb quantities significantly in excess of the minimum orders specified, CMI will be prepared to offer a further discount depending upon the quantity required.

CMI requires a contract contingency for both Service 1 and Service 2 described above as follows:

Services as offered and described above as "Purification of Monoclonal Antibodies from Pre-existing Hybridomas owned by Customers" are contingent upon the customer provided hybridoma being of good viability and having the potential to secrete a normal quantity of MAb. Should a hybridoma prove refractory to secreting MAb within the normal concentration range; should CMI be unable to rectify the problem; should the customer be unable to provide reagents or information to correct the problem, then CMI retains the right to terminate the contract at no charge to the customer.

 

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