In vitro – Bioreactor Concentrated Supernatant

The bioreactor system is an excellent animal-free alternative for custom bulk antibody production. One bioreactor flask can produce as much antibody as ascites from 10 to 12 mice. In addition, monoclonal antibody production in bioreactors contains no contaminating host mouse immunoglobulin.

Step 1: Receipt and Expansion
Materials needed from client for custom bioreactor production: 1 x 106 – 1 x 107 hybridoma cells, viable, shipped on dry ice. Upon receipt, the cell line is transferred to a quarantine area where it is grown in culture, tested for mycoplasma and expanded to generate cells ready for inoculation.
1 week
Step 2: Inoculation
The membrane compartment is inoculated with 20 to 50 million cells, depending on the size of bioreactor, and incubated for a week.
1 week
Step 3: Production Harvest
Concentrated cell culture rich with antibodies is harvested twice per week after proper cell density is established.
7 weeks
Step 4: Filtration
The cells are removed from the harvest by centrifugation and filtration and the antibody-containing supernatant is ready for use or for purification.
IPA offers bioreactor production in 1000mL sized flasks.
Specific mAb concentration will range from 0.2 –1mg/mL depending on the cell line.
Approximately
9 weeks

In vivo – Ascites fluid

Ascites fluid is often an ideal medium for the reliable production of custom monoclonal antibodies. Clients can take advantage of IPA’s full range of small and large scale custom ascites production services in diverse mouse and rat strains. Traditionally, ascites fluid containing antibody is tapped from each mouse up to three times.

Step 1: Receipt and Expansion
Materials needed from client for custom ascites production: 5 x 106 hybridoma cells, viable, shipped on dry ice. Upon receipt, the cell line is transferred to a quarantine area where it is grown in culture, tested for mycoplasma and expanded to create cells ready for inoculation.
1 week
Step 2: Inoculation
Monoclonal antibodies are produced in bulk by implanting hybridoma cells into the peritoneal cavity of a primed mouse. Custom ascites production is initiated under GMP guidelines.
1 week
Step 3: Production Harvest
Ascites is collected from the mouse abdominal cavities and held on wet ice. It is then centrifuged to remove cellular and lipid material and filtered to clarify. The clarified ascites is placed into sterile containers, labeled, and immediately frozen.
5 weeks
Yield:
5 mice typically provide 20mL on average, with specific antibody concentration ranging from 1mg/mL to 10mg/mL.
Approximately
7 weeks

What are the advantages and disadvantages between Bioreactor generated antibody and Ascites generated antibody?

Generation Method Advantages Disadvantages

In vitro – Bioreactor

Low variation in quality between batches of antibody Lower level of specific antibody – Range: 0.2 – 1mg/mL
No contamination with murine pathogens Bioreactors are more costly than mice and labour intensive
Absence of contaminating host mouse IgGs Use of fetal bovine serum in media
Can produce large volumes in weeks Potential for change in DNA in the hybridoma. Prolonged culture of hybridoma cell line not recommended
100% monoclonal in a well defined media Bioreactor flasks can become easily contaminated with improper care

In vivo – Ascites

High level of specific antibody – Range: 1 – 20 mg/mL Animal welfare concerns
Relatively low costs of production and not labour intensive Variability in the capacity of hybridomas to elicit ascites
Most (but not all) hybridoma cell lines grow after intraperitoneal administration Potential for change of DNA in the hybridoma
Contamination with host mouse immunoglobulin  (10-20%)

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