Jun 132014

Production of MAb

Fig.1 Production of MAb

Large Scale Production Of MAbs:

Commercially, on large scale, MAbs are produced by two methods.

(a) Ascites production in mice

(b) In-vitro fermentation

The production method is summarized in & 2b.

a) Ascites Production In Mice:

The first monoclonal antibodies approved by FDA for therapeutic use OKTS, is produced by ascitic technology19.

In this method hybridoma cells are injected into peritoneal cavity of histocompatible mice. The mice are pretreated by i.p. injection of Pristane to irritate the peritoneal cavity which facilitates the growth of ascitic tumor. The fluid produced may contain the high concentration of secreted MAbs, 2 to 20 μg / ml and 2 to 6 ml or more can be harvested per mouse. Comparison of different MAb production22,23 methods is shown inTable 1.

Drawbacks of this method are:

1. It is very costly, very difficult and not reliable.

2. Product may get contaminated with mouse immunoglobulins and also with other mouse proteins.

3. Viruses can be introduced as contaminants.

4. Antibody yield is often less as compared to other methods.

b) In-Vitro Fermentation:

In this method, the cells are grown and gradually moved to larger and larger culture ensuring exponential growth. Typical antibody levels in the culture supernatant ranges from 5-50 μg/ml depending on the individual clone and on cell density. When more production of antibody is required 1-litre cultures in roller bottles are used. Required cells are removed from rest of media by centrifugation or filtration, generally followed by ultra filtration step for concentrating the filtrate by up to 20 folds.

Advantages of this method are:

(1) As serum required in culture media is reduced, it is cost effective.

(2) There will not be any contamination with mouse immunoglobulin.

But the major drawback is that of contamination of final product with serum or protein based growth factors.

Table 1: Comparison of different MAb production methods.

Production system


Volume (ml)

Concentration (mg/ml)

Production time (weeks)


Ascites (in vivo)

20-250 mg


< 20


Stir growth




Dialysis membrane

< 50 mg




Roller bottles

< 2 gm




Hollow fiber

0.15-30 gm





2-100 gm

< 2000 lit




 MAb Production

Fig. 2a:  MAb Production (Flowchart)

 Freeze Dried MAb Production

Fig. 2b:  Freeze Dried MAb Production (Flowchart)

i) Purification:

Contamination, during production process, such as protein, nucleic acid, endotoxins, immunoglobulin and adventitious agent can be removed by purification method. The purification methods such as precipitation with ammonium sulphate, zone electrophoresis, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration and affinity chromatography are used19.

· Affinity chromatography is often used for initial purification.

· Ion exchange chromatography is used for removing endotoxins and DNA.

· Gel filtration chromatography can remove both high and low molecular form of monoclonal antibodies and it is usually used as the final polishing step.

j) Characterization:

The final determination of monoclonality requires biochemical and biophysical characterization of the immunoglobulin. It is also characterized immunochemically to define its affinity for antigen, its immunoglobulin subclass, the epitopes for which it is specific and the effective number of binding site that it possesses19.

k) Final Processing:

Depending upon the intended application, the antibody may be conjugated to specific radionuclide or toxin. Then the stabilizing agent is added, and the product is filled into final container under inert gas or other specialized conditions.  Lyophillization is frequently applied to get freeze dried product.

Antigenicity Of Murine MAb:

The main problem for mouse MAb is that, human body recognizes it as a foreign agent and produces antibodies against such mouse MAb. The induced human anti-mouse antibodies (HAMA) quickly reduce the effectiveness of mouse MAb and also their interaction may lead to allergic reactions.

To overcome the problem, Human MAbs can be used. Though difficult, this is possible by fusion of EBV (Epstein Barr Virus) transformed human B-lymphocyte with appropriate fusion partners21. EBV is a lymphotrophic DNA herpes virus which is capable of converting normal B-lymphocytes of human and/or mouse into cancer cell having proliferating capacity in vitro. But the presence of EBV as contaminant can pose a problem of producing cancer24.

Even the human-human hybridomas producing MAbs have been produced 25,26. Olsson and Kaplan in the year 1980 produced first human-human myeloma (SKO-007), against the hapten 2, 4-dinitrophenyl (DNP) 19.

The routine production of human MAbs is prevented due to following reason:-

  • Sources of antibody producing cells27.
  • Reliable methods for lymphocytes immortalization.
  • Stability28 and antibody producing capacity.
  • Administration of some antigens to humans could endanger their health29.
  • Recovery of B-lymphocytes from the spleen of human is impracticable.
  • The fusion of human lymphocytes with human lymphoblastoid cell lines is a very inefficient process.
  • Low production yield of human monoclonal antibody.

Hence, other alternatives methods come forth.

Advantages Of MAbs:

  • Pure one molecular species with high specificity for a particular antigenic target.
  • Anti-serum titer values are high.
  • Antibodies with high avaidity can be produced.
  • In vitro and in vivo production is possible.
  • Radiolabelling and fluorescent conjugation of monoclonal antibody are easy.

Disadvantages Of MAbs:

  • Initial cost involved in the technique is high. However, continuous production is somewhat economical.
  • Methods are time consuming.
  • Antigenicity of Murine MAb.
  • MAbs have comparatively less complement fixing ability than that of convectional antiserum.
  • MAbs are highly selective for a particular single antigenic determinant. This renders them incapable of distinguish between different molecules, cells bearing the chemical structure or determinants except one against which it is targeted.
  • The high antibody avidity (energy of binding to an antigen) of MAbs is advantageous for immunoassay but some property is undesirable for purification process.

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