ABSTRACT
This article revalidates the effectiveness of affinity chromatography, matrix sanitization, and storage procedures used in
monoclonal antibody CB.Hep-1 purification to remove and inactivate viruses after process scale-up. The scale up of the CB.Hep-1
purification process demonstrated a similar removal factor for enveloped and nonenveloped viruses compared to the initial
validation study. The HSV-1, HIV-1, and CPV viruses were sensitive to incubation with ethanol at 70% concentration (3.0–4.6
Logs). We found that 0.1N HCl is a robust chemical agent able to inactivate >6.13 Logs of nonenveloped high resistance viruses
while ethanol at 20% concentration inactivated 3.7 Logs of enveloped viruses HSV-1 and HIV-1 but was unable to inactivate
nonenveloped viruses HPV-1 and CPV.
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Monoclonal antibodies (MAbs) are employed as immunoligands in the purification processes of biopharmaceutical products.1,2 Virus transmission poses a high potential risk for patients who must be treated with these biopharmaceuticals, if MAbs come
from human or animal sources.3,4 It is necessary to validate the purification process capacity to remove and inactivate any potential viral contaminant.5,6
Regardless of extreme virus controls, several instances of biological contamination have occurred. Research on virus contamination
sources have shown that viruses can be introduced into the manufacturing process in different ways, illustrating the importance
of viral clearance studies to guarantee the biopharmaceutical product safety.7
Acronym List
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Validation of the purification method plays an essential role in establishing biological product safety, especially when there
is a high risk for the source to be contaminated with known human pathogenic viruses. Since several contamination instances
have occurred with agents whose presence was not known, validation also provides a high degree of confidence that these agents
may also be removed.8
FDA defines validation as, "Establishing documented evidence, which provides a high degree of assurance that a specific process
will consistently produce a product meeting its pre-determined specifications and quality attributes".9 The rationale is that if more effort is placed on validation at the beginning, then there will be less chance for failure
during product life.10
Validation studies for purifications proceses involve the deliberate addition of a virus to one or more purification steps
to measure the extent of its removal and inactivation capacity. It is not necessary to validate all purification steps, but
only those that could contribute to virus removal or inactivation. To prevent the deliberate introduction of viruses into
the manufacturing process, the validation studies should be done in a separate facility and in a scaled-down version of the
manufacturing process. Validation at the small scale is an efficient way to perform viral clearance validation studies.11
GENERAL PROCEDURE FOR VALIDATION
CB.Hep-1 MAb is a mouse IgG-2b, specific for HbsAg.12 This MAb is used as an immunoligand in the antigen-purification process, which is one step in the manufacturing of the Hepatitis
B vaccine for human use.13,14 The main aim of our work was to investigate if the affinity chromatography used routinely in the purification of CB.Hep-1
shows the same virus removal factor after a scale-up process. We measured the virus inactivation factor of the column sanitization
protocol using 70% ethanol and the matrix storage conditions in 20% ethanol. We also evaluated the column sanitization protocol
with 0.1 N HCl to increase the inactivation factor for high resistance non-enveloped viruses.
