Millipore has described how tangential flow filtration (TFF) membranes can be used for upstream purification of the influenza egg/cell-culture-based vaccine process.
Influenza cell-culture-based processes are being developed as a new method to meet industry demands and supplement seasonal egg-based influenza vaccine supplies.
This new technology has the potential to substantially increase vaccine production capacities, especially as new pandemic influenza emerge.
In cell-culture-based processes, a recombinant fermentation technique based on mammalian cell culture is used to produce highly purified antigen-specific subunits.
This reliable and robust method offers distinct advantages over egg-based manufacturing methods, including eliminating the need for embryonated chicken eggs, combining and automating upstream and downstream processes, reducing potential for contamination by viable and non-viable particulates, eliminating the six-month lead time for egg supply logistics, faster processing, and higher batch volume with higher initial purity.
The effectiveness of egg-based upstream purification expertise supports the actual development of robust, consistent, scalable and cost-effective upstream TFF cell-based processes.
Millipore conducted a study to show how the development of purification platforms for influenza vaccine is dependent on the specific contaminant removal requirements.
The objective of the upstream influenza clarification step is to create a robust process that supplies a purified integral active influenza virus.
The major difference between egg-based and cell-culture-based processes is the type of contaminant to be removed: ovalbumin (50kD) for the egg-based process, and host cell protein (HCP) and DNA, for the cell-culture-based process.
The upstream clarification success criteria are: quality - maintaining virus activity and control and/or limiting aggregation, desegregation; and purity, yield and contaminant removal.
Additional specific diafiltration objectives are determined based on a defined filtration schematic.
The decision tree is a guideline to select appropriate open ultrafiltration (UF) membranes to perform the first UF step of both egg- and cell-based process.
Selection is based on purity.
When benzonase treatment and inactivation are performed prior to the first UF step, diafiltration with phosphate buffer saline (PBS) could be performed for benzonase and desactivation removal.
When sucrose gradient ultracentrifugation and inactivation are performed prior to the first UF step, diafiltration with PBS buffer could be performed for sucrose and desactivation agent removal.
Open ultrafiltration membranes are the membrane of choice for influenza virus purification.
Selection of the screen depends on the viscosity and fouling properties of the influenza fluid, final targeted volumetric concentration factor (VCF), and type and percentage of contaminants to be removed.
A standard UF system can be a solution of choice for classical TFF influenza purification.
If high contaminant level removal is required, an optimised set-up to achieve a control of the transmembrane pressure (TMP) would be necessary.
This is achieved by controlling permeate pressure, which limits fouling the membrane, and therefore meet success criteria.
The choice of operating conditions may vary depending on the main result to be obtained at the end of the purification step.
In this study, two cases were examined.
In case one, the key critical factor is contaminant removal.
Given that the success criteria are focused on contaminant removal as well as yield, retention and purity in both egg-based and cell-based flu processes, optimal process results are obtained with a huge limitation of the TMP value at both laboratory and production scale.
In case two, the key critical factor is high flux and high antigen concentration.
Because the success criteria are focused on yield, retention, purity and flux for a large-volume process in both egg-based and cell-based flu processes, optimal process results are obtained after determining the highest TMP possible to have the best compromise between flux, energy consumption and production system sizing.
The important criteria to be reached during the UF/DF step will have an impact not only on managing the purification process step but also on the membrane area required for large scale.
Development trials are typically performed at a small scale, in which only a few litres of product are processed on 0.1m membrane area.
However, when scaling up and increasing batch size, membrane area linearly increases.
For the influenza vaccine purification process, the upstream influenza clarification step separation objective is to create a robust process that is able to supply a purified integral active influenza virus.
While the major difference between egg-based and cell-culture-based processes is the type of contaminants to be removed (egg ovalbumin for egg-based process versus HCP and DNA for cell-based process), there are also differences in terms of success criteria to be achieved.
The various requirements for a process, including yield, flux, and contaminant removal, will determine the regulation method to be applied for the influenza vaccine purification process.
This will consequently have an impact on scale-up data for different batch volumes.
A complete optimisation process helps find the best compromise among the success criteria.
