Monoclonal Antibody Development
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Offering the Industry’s Best Tools to Refine Clone Selection
Phase 1 Project Initiation
- Hybridoma Initiation Checklist: To develop the best application specific antibody, communication is paramount. The Hybridoma Initiation Checklist is the customer’s first opportunity to provide MBS with the details necessary to strategize an optimal approach.
- Technical Meeting: Open access to MBS technical resources and personnel begins with a conference call with the Hybridoma Development team to customize the project for success.
Phase 2 : Immunization
With a success rate of over 85%, the MBS Rapid Immunization Protocol enables a fusion on day 28 of the project. This protocol ensures the fastest possible outcome for monoclonal antibody development.
- Immunization, Day 0: 3-5 BALB/c or AJ mice are immunized with antigen. 2-3 mgs of antigen is required. 1 mg of additional counter-screening reagents required, if applicable.
- Titer Determination, Day 20: Titer is determined by indirect ELISA Fusion Boost, Day 24: A mouse (or mice) with adequate titer (1:31K) is selected for fusion and boosted on day 24. The fusion occurs on day 28 post immunization.
- If mice do not reach titer by day 20, additional boosts may be administered until titer is reached.
Important Immunogen Factors To Consider
Phase 3: Fusion featuring MultiPure Technology
- Fusion, Day 28: The fused mouse spleen is plated on 10-20 plates (96 wells each). Fusion size is variable and is at the discretion of the customer.
- Primary Screening, Day 38-42: Fusion product supernatants are screened by indirect ELISA (primary screen) against the immunogen, along with counter-screens (if necessary).
- Scale Up and Cryopreservation, approx. 2 weeks: Depending on the amount of antibody required for customer evaluation, selected positive fusion products are scaled up to generate 3-5 mLs of supernatant, or alternatively 15 mLs of overgrown supernatant. Supernatant from the 15 mL volume samples can be purified using the MBS developed MultiPure technology. Two vials of each selected fusion product are cryopreserved.
- Secondary Screening,: Selected fusion products are screened a second time after scale up, by ELISA, to confirm stability and ensure that they are still actively secreting antibody. A small percentage of fusion products can lose productivity during the scale up phase and the secondary screen is important to confirm productivity before selecting fusion products for subcloning.
- Once 15ml supernatant samples are available, the customer can opt to use our novel MultiPure system to generate purified antibody. Doing so allows early insight into antibody performance, relative affinities, and even matched pair capabilities. With the use of MultiPure, subcloning choices can be made with real, final application data saving both time and money.
- A fusion product(s) is selected for subcloning.
Phase 4: Subcloning
- Subcloning,: The selected fusion product(s) is subcloned by limiting dilution.
- Subclone Screening: Screening by indirect ELISA is performed against the immunogen and counter-screening agents (if necessary).
- Subclone Scale Up: Selected clones can be scaled up to 3-5 or 15mLs of supernatant and shipped to the customer for evaluation. 15 mL samples can optionally be purified by MultiPure technology to generate concentrated, purified samples of known concentration for evaluation. Two vials of each selected clone are cryopreserved at this stage.
Phase 5: Antibody Production
Our premium monoclonal antibody development service is complimented by being a trusted partner in cGMP bulk antibody production.
- In Vivo Production: Antibody can be produced in vivo via mouse ascites. Ascites production typically takes 6 weeks with an additional 1week added for purification. A typical cell line will produce 3-5 mLs of ascites per mouse with an antibody concentration of 3-5 mg/mL.
- In Vitro Production: In vitro production is performed in roller bottles. A 1 liter production takes approximately 3-4 weeks with an additional 1 week for purification. Production levels typically range from 20-50 mg/L depending on the cell line.
- Yields are cell line dependent and are not guaranteed.