Our purification process was designed taking into consideration a combination of two main principles: saline precipitation
to reduce ascites volume and Protein A affinity chromatography. We also introduced incubation of the IgG for one hour at low
pH into this process to increase the viral safety of the process as well as several filtration steps to get the required sterility.14
The conditions used in this purification process provided a purity level in the purified MAb higher than the 95 percent measured
by SDS-PAGE and HPLC procedures. In this work, the homogeneity and identity of the purified MAbs were checked by isoelectrofocusing,
isotyping, amino acid light-chain composition, and ELISA. The isoelectrocfocusing pattern resulted in the presence of four
majorities bands for a total of eight bands, which corresponded with an isoelectric point in the range of 6.75 to 7.04 (data
Several toxic effects have been associated with the presence of Staphylococcal Protein A.15 The Protein A leakage was determined by means of a specific ELISA, which permitted quantification with high sensitivity
and without unspecific reactions to the levels of Protein A in pure samples of the IgG. The level of this contaminant was
less than 10 ppm for all batches.16 It represents a very low amount of Protein A and it is not a problem at all because it is drastically reduced during the
immobilization of the MAb and also during the antigen downstream process, where the Protein A level was undetectable.
Due to its oncogenic properties, mouse DNA level is an important point to consider. The regulatory agencies are very strict
in this area. Today, the acceptable level is less than 100 pg of residual cellular DNA per human dose. One of the advantages
of the production from ascitic fluid is the relatively low DNA level in the downstream starting material. In our process,
the mouse DNA present in the purified CB.Hep-1 did not exceed 7 pg/mgIgG.
MAb Linked to Sepharose CL-4B
Tests and acceptance criteria for the monoclonal antibody linked to a solid support include the following:
- Surface density of MAb (mg MAb/g of resin)
- Specific binding capacity
- Integrity of solid support
With our process, we obtain the following results: purity on the rHBsAg eluted higher than 85 percent,17 which demonstrated the high purification power and the consistency of this immunochromatographic step. Another aspect of
notable importance for the purification of molecules for human use is the level of antibody leakage of the antigen from the
matrix. The MAb present in the final pharmaceutical preparation could have several potential negative effects. One could be
stimulation of undesired immune responses (HAMA response) and neutralization of the antigen. In this case, the ligand leakage
from the matrixes did not exceed 0.005% of the rHBsAg eluted. This represents a very low amount of IgG, especially when we
consider that this is the first chromatographic step of the rHBsAg downstream process and that the dose utilized with other
mouse MAb is very low.
Saving all of the crucial points mentioned earlier, the problem resides now in potential viral contamination. In this sense
there have been some negative experiences in the biopharmaceutical industry in the last years, that is why several controls
have been recommended by different guide-lines.18 The MAP test is another recommended assay for determining adventitious agents. We have monitored the MCB, the ascites, and
the purified antibodies for several years during which time no viruses have been found.11
The validation of virus removal may be performed during one of the steps in the reagent manufacture, or during one of the
steps of reagent preparation, such as linking the reagent to a column.2, 14 Validation study provides a high level of assurance that the final product will be free of contaminants. These studies should
be done spiking the chromatographic steps with high titer infectious viruses. Several model viruses were used to challenge
this chromatographic process. The purification process showed a high clearance factor of all viruses studied.
We have demonstrated that the manufacturing process can consistently produce reasonably pure and active MAb reagent. Because
our final product is the inmunogel that constitutes a biological reagent inside the process of purification of the surface
antigen of Hepatitis B, its characterization and consistency have been efficiently demonstrated. Quality control tests are
carried out routinely on each batch of purified bulk product according to the Guide to GMP.