Downstream Processing: A Revalidation Study of Viral Clearance in the Purification of Monoclonal Antibody CB.Hep-1 - - BioPharm International


Downstream Processing: A Revalidation Study of Viral Clearance in the Purification of Monoclonal Antibody CB.Hep-1


Figure 3. Comparison of two validation studies of virus removal factor of the Protein A–Sepharose affinity chromatography. Bars show percentage of the applied virus removed by chromatography. All experiments were done in triplicate.
When the two studies were compared, the results demonstrated no significant differences in the removal factors of these model viruses. These results corroborate that the scale-up of the MAb CB.Hep-1 purification process does not modify the capacity of Protein A–Sepharose to remove the HSV-1 (p = 0.0808), HIV-1 (p = 0.6625), HPV-2 (p = 0.1904), and CPV (p = 0.0765). As expected, affinity chromatography showed higher removal capacity for enveloped and large viruses (Figure 3). The percentage reductions were 97.55%±2.42 (HSV-1) and 90.29%±10.46 (HIV-1). Small and nonenveloped viruses were removed in lower percentages: 47.42%±1.11 (HPV-2) and 62.79%±5.24 (Figure 3), which appears to occur because the small size of these viruses affects their interaction with and penetration into the matrix, thus retarding the elution of virus particles.

Affinity column (20%) carryover studies showed virus presence in the product after subsequent runs.22 Thus, matrix sanitization and storage protocol effectiveness demonstration is also mandatory for viral clearance studies. Robust virus clearance studies are defined as those that have been shown to work accurately under a variety of conditions such as pH or ionic strength of column buffers. Virus inactivation may be achieved by a number of physical (heat, radiation, and sonication) or chemical (detergents, alcohol, solvents, acids, bases, glutaraldehyde, and B-propiolactone) methods.3,23 It is also important to consider that enveloped RNA and DNA viruses have low resistance to physical–chemical agents, because solvents like ethanol destroy their envelopes, which are composed of proteins and lipids from the cells. Conversely, the lack of an envelope makes a virus quite resistant to these agents.

Figure 4. Virus inactivation factors of the Protein A–Sepharose affinity column sanitization and storage protocols. Bars represent the average and the confidence limit of the inactivation factor. All experiments were carried out in triplicate.
Our results also confirmed the expected results for the enveloped viruses and for the HPV-2. The viral inactivation study performed in this work demonstrated 3.0–4.6 Logs of inactivation for enveloped viruses HVS-1 and HIV-1, respectively, in 70% ethanol and greater than 3.7 Logs in just 2 h in 20% ethanol for both viruses (Figure 4). It is consistent that the nonenveloped virus HPV-2 retains its infection capacity in both ethanol concentrations.

The exception to our theory was that the 70% ethanol was able to inactivate 3.97 Logs of the high-resistance CPV, thereby demonstrating that the lack of an envelope is not a necessary for these kinds of viruses to be able to resist the chemical agents (organic solvents). We hypothesize that the proteins involved in the virus infectivity mechanism of the CPV suffer some kind of chemical modification that blocks its capacity to infect the host cells. The 20% ethanol was totally inefficient for inactivating (<1.3 Logs) the CPV in 72 h (Figure 4).

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