Process Development and Spiking Studies for Virus Filtration of r-hFSH - This study on a recombinant human follicle stimulating hormone demonstrates the use of virus filters to reduce the risk of cont

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Process Development and Spiking Studies for Virus Filtration of r-hFSH
This study on a recombinant human follicle stimulating hormone demonstrates the use of virus filters to reduce the risk of contamination.


BioPharm International
pp. 24-30

RESULTS

Hydraulic performance study results


Figure 2: Vpro hydraulic performance study.
Figure 2 summarizes the results of the initial hydraulic performance study with the unspiked feed.

Figure 2A shows the V max plot, from which the Vmax, Qi is obtained and subsequently Amin is calculated from equation 1. The minimum area requirement was calculated to be 0.04 m2 for a 50 L batch to be processed in 2 h (with the unspiked feed and without adding any safety factors)—this is equivalent to a process throughput of 1250 liters/m2 (The max throughput or Vmax for the process is 5922 L/m2).

Figure 2B shows the hydraulic flow decay as a function of throughput—projection from the Vmax model is also shown in the figure and it indicates that the VPro virus filter is only marginally plugged in the application.

Virus spiking study results


Table I: Virus removal by Vpro virus filter.
The virus titers of the four viruses under test remained stable when incubated with the starting material next to the process. The enveloped MuLV and PRV showed a reduction factor of at least 5.37 log10 and 6.01 log10 respectively. For the non-enveloped virus Reo-3 and MVM the reduction factors were at least 6.43 and 6.32, respectively (see Table I). Very low residual infectivity was found in fraction B and C of MVM run1. Two and four out of 384 analyzed wells, respectively, were positive. No residual infectivity was found in any fraction of MVM run 2.

DISCUSSION

The Vpro hydraulic performance study depicted a very high throughput of 1250 Liters/m2 for the protein solution. The high flux and throughputs that were validated during the spiking study permits the design of an economical virus filtration step with a relatively small virus filter area for a given batch size (in the current situation, an area of 0.04 m2 is required to filter a batch of 50 L in 2h). The virus filtration step will be located in downstream purification after a final chromatography step and prior to the final sterile filtration step. Adsorptive prefiltration (Virus prefilter, Merck Millipore) was also evaluated (data not shown) to determine whether such prefiltration would help to increase or further optimize the throughput of the VPro virus filter. Because the protein concentration was quite low and the hydraulic performance study revealed a relatively high throughput and flux for VPro filter alone, the prefiltration option was not pursued further.

With predefined throughput of 387 liters/m2 (120 mL of spiked feed solution filtered through a 3.1 cm2 Vpro micro device), high and robust LRVs (>5-6) were obtained. The current work may possibly be among the earliest of studies that demonstrate the feasibility and robustness of using virus filters to reduce the risk of virus contamination in a recombinant human follicle stimulating hormone purification process. With the data obtained in the scaledown, studies for scaleup were successfully implemented for scaleup for several manufacturing batches.


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