Virus removal filters are widely used in biologics downstream processes to control the risk of viral contamination.
At the end of the virus removal filtration selection optimization test, a virus spiking research test will be conducted at a qualified third-party testing institution to prove and verify the virus removal capability of the selected virus removal filter.
Regular Virus Removal Validation
To better leverage the performance of the virus removal filters and save the actual production costs, generally in the process of virus removal process prefilters are introduced in series with the virus removal filters for process development and optimization, this filtering operation of the scaled-down model test not only represents the actual production process procedures, but also better improve the actual load capacity of the virus removal filters. However, during virus spiking studies, the addition of a virus removal pre-filter may adsorb or intercept virus particles, which can lead to a decrease in virus titer and interfere with the measurement of the true LRV value of the virus removal filter. Therefore, in regular virus removal validation, the standard method is to pre-filter the protein solution batch first, then spiked with virus, and then perform the virus removal filtration.
Figure 1: Traditional Validation Method Flowchart
In-line Spiking Virus Removal Validation
The Reason Why In-line Spiking Develop
Many protein samples undergo virus removal filtration after pre-filtration in batches, which can lead to increased clogging. Moreover, for unstable materials, during the period when the virus is added after batch prefiltration, clogging impurities may also re-emerge, leading to low validated capacity of the virus removal filter, and thus the validated capacity is often lower than that of the in-line prefiltration.
Inline spiking technology is an excellent solution to the problems posed by the non-serial connection of pre-filters and virus removal filters. The so-called inline spiking allows the use of inline pre-filtration and direct measurement of the virus removal capacity of the virus filters, as well as recreating the production operation and giving a true picture of the virus removal LRVs in the production loading capacity range.
Figure 2: In-line Spiking Virus Removal Validation Flowchart
Features
The "In-line Spiking" is characterized by the following features compared to the traditional model.
Increased filter loading capacity for unstable samples
Suitable for situations where proteins bind to viruses and form clogs.
More representative simulation of production operations
Requires special equipment setup
Relatively complex to operate
Figure 3: Photo of Inline Spiking Experiment
Cobetter Inline Spiking Virus Removal Validation Case Sharing
Feed solution: recombinant protein 5g/L
Relative molecular weight of protein: 70KD
Virus removal pre-filter: Viruclear PDT
Virus removal filter: Viruclear VF plus protein, etc.
Figure 4: Flux And Loading Capacity Versus Time for Virus Removal Filtration of Feed Solution for Inline And Non-series Cases
For unstable samples such as the one demonstrated in this case, the use of Inline Spiking increased the average filter throughput by three times and the loading capacity by seven times compared to the traditional non-serialized validation method. After testing by a third-party validation organization, the difference between the theoretical titer of the diluted feed solution and the actual sampling titer is very small, which proves that Inline Spiking mixes the virus and the feed solution adequately, and there is no loss of virus activity during filtration.
Cobetter engineers built a system platform to automatically regulate the virus addition, through the attenuation of the flow rate feedback to the push/pull syringe pump, to realize the automatic adjustment of the syringe pump speed in strict accordance with the ratio of virus addition, as well as the real-time and accurate control of virus addition.
Summary
Inline Spiking validation is more representative of the actual production process, and ensures the virus removal rate while increasing loading capacity, so that the validation can meet the customer's expectations.
Reference
Adith Venkiteshwaran, Mechanistic Evaluation of Virus Clear-ance by Depth Filtration, Biotechnology Progress, 2015,VOL.31