Integration of Large-Scale Chromatography with Nanofiltration for an Ovine Polyclonal Product - Pharmaceutical Technology

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Integration of Large-Scale Chromatography with Nanofiltration for an Ovine Polyclonal Product
The authors examine the challenges of integrating a large-scale chromatography and nanofiltration process for purification of a polyclonal antibody.


Pharmaceutical Technology
Volume 33, Issue 1, pp. 62-70

Spiking studies

As a next step, non-GLP spiking studies using PPV and the same scaled-down model were performed at Catalent Pharma Solutions to verify the PP7 data obtained. As required by regulatory guidance, the scaled down model used during the GLP validation run should reflect process conditions as closely as possible unless a justification to deviate from this can be provided (7, 8).

Consequently, the volume to be filtered using the scaled-down model is derived from the required filtration volume at process-scale. This volume was initially established by testing the virus filter without a virus spike to determine the flow rate and capacity. It has been reported that virus spike preparations can lead to significant fouling of nanofiltration membranes in spiking studies, and properties of the virus spike itself are an often-overlooked source of contaminants that can affect filter performance (9). Potential contaminants in virus stocks may include serum proteins, host-cell proteins, host-cell DNA, and lipids (10, 11).

To determine the effect of the virus spike on product filterability, nonpurified and purified spike preparations of PPV were filtered using the same scaled-down model. The purified virus was prepared using a Q-Sepharose chromatography step.


Figure 3: Filtration characteristics of purified versus nonpurified PPV at 1% spike. (Figure 3 courtesy of the authors.)
Figure 3 outlines the filtration characteristics of the Fab product using Virosart CPV, comparing a 1% virus spike of chromatography-purified PPV with a 1% spike of non-purified PPV. A nonspiked control is also shown. The purified PPV samples gave a better flow rate and capacity than nonpurified virus, which demonstrated that contaminants within the viral spike were affecting filter performance. As a result of these studies, a Q-chromatography purified PPV preparation was used for subsequent validation of the nanofiltration step to achieve the required scaled-down volume of product (400 L/m2 ).


Table III: Non-GLP PPV test results for Virosart CPV.
A series of scaled-down runs was then performed using purified PPV at different concentrations, to determine the viral spike concentration that would give the best log reduction without affecting filter performance. Table III outlines PPV retention data for three runs using different spike concentrations of purified PPV.

All studies used a test pressure of 2 bar (30 psi) and filter lot number 0650773R50Z3/2. For all runs, a scaled-down filtrate volume equivalent to 400 L/m2 was obtained to reflect the full-scale filter capacity requirements.

All three viral spike concentrations resulted in acceptable viral reduction. A range of 0.5–1% was therefore selected for validation runs to maximize the flow rate and capacity of the filter, and to minimize the amount of virus required.


Table IV: Overall GLP spiking study results for Virosart CPV.
To validate the nanofiltration step, GLP runs were performed using the following panel of viruses as model viruses: PPV, murine leukemia virus (MuLV), bovine viral diarrhea virus (BVDV), and reovirus type 3 (Reo 3) virus. The overall GLP spiking study results are outlined in Table IV.

All studies used a viral spike concentration of 0.5%, a test pressure of 2 bar (30 psi), and filter lot number 0650773R50Z3/2. For all runs, a scaled-down filtrate volume equivalent to 400 L/m2 was obtained, to reflect the full-scale filter capacity requirements.


Table V: Results from GLP robustness spiking study for Virosart CPV.
GLP studies also included robustness studies using PPV as the model virus. The robustness studies incorporated anticipated worst-case protein concentration based on laboratory data for the SP Sepharose process, and worst-case pressure. Results are outlined in Table V.

All studies used filter lot number 0650773R50Z3/2. For all runs, a scaled-down filtrate volume equivalent to 400 L/m2 was obtained, to reflect the full-scale filter capacity requirements.


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