Design of Highly Retentive and Robust Virus Filters through Fundamental Understanding
Nigel Jackson1, Engin Ayturk2, Stanley Kidd2
1. Pall Europe Ltd, Portsmouth UK; 2. Pall Corporation, Port Washington, USA
INTRODUCTION
COST EFFICIENT VIRUS FILTER DESIGN
Understanding of the fundamental mechanisms governing performance of a membrane is key to realising the design of a high
performance next generation product. This study demonstrates the original internal polarization model derived for first generation
virus removal filters and its effective utilization to develop highly retentive and robust next generation virus filters.
MATERIALS AND METHODS
Retention experiments were carried out using PP7 bacteriophage (model parvovirus) derived from ATTC samples and stored in
tryptone soya broth (TSB). Titres were determined using a plaque assay method. PP7 was fluorescently labelled using an Alexa
Fluor® 488 protein labeling kit (Invitrogen, OR, USA) after dialysis into PBS. Confocal microscopy images were generated using
a Leica SP5 confocal microscope (Leica Microsystems GmbH, Germany). XY Images were stacked in the Z-plane to generate a
3-D image of the membrane using ImageJ software (NIH, US). Full details of all methods are published[1]. All other test solutions
and filtration conditions are as described.
The lack of model fit for single-layer can be explained by hindered convection of the virus through narrower pores in the Pegasus
SV4 membrane.
A revised model is shown in Figure 2 taking into account a hindered convection value equivalent to 20L.m-1 delay, based on the
relatively slow hindered velocity (Equation 4)[3].
uH = hindered velocity, uP = pore velocity
Equation 4:
KS = convective resistance coefficient
KS
uH = uP
2K T
(2
)
K S , KT ,
= f(
)
KT = diffusive resistance coefficient
Φ = partition coefficient, λ = dvirus / dpore
In order to generate a significantly high hindrance, a virus to
pore ratio of 96% is required.
Figure 3
Highly Retentive Pegasus SV4
8
Further work is ongoing to evaluate the impact of the tortuous
path a virus must take through a filter and the likelihood
hindrance from the bulk convective flow.
Figure 1 demonstrates the location of phage within the depth of the Ultipor VF DV20 membrane. Experiments carried out at
different phage loading levels demonstrated that the distribution of the phage throughout the membrane did not change, however
the intensity of the signal did. This led to the formulation of the internal polarization (IP) theory[1] which describes an accumulation
of phage in the reservoir zone causing an increased challenge to the rejection zone and an apparent reduction in retention
(Equations 1-3).
Nevertheless, the new filter design approach successfully
predicted highly retentive performance of the dual layer
Pegasus SV4 (Figure 3) for both PP7 bacteriophage and PPV
mammalian virus at high throughput.
6
LRV
INTERNAL POLARIZATION
4
0
Figure 1
Confocal microscope image of Ultipor VF DV20 membrane representing a small cross section of membrane but the full membrane
depth. Upstream (layer main), downstream (layer inset).
PPV Mammalian Virus
PP7 Bacteriophage
2
0
100
200
300
Throughput (L.m-2)
PRESSURE INTERRUPTIONS
FLOW
RESE
RVOIR
RESERVOIR
ZONE
Robust performance of Pegasus SV4 after a 10 minute pressure interruption preceding a 10% buffer flush for both IVIG and
BSA solutions is shown in Figure 4.
REJECTION
ZONE
Overall, Pegasus SV4 demonstrates very robust performance in the presence of pressure interruptions, which is expected due
to the deviation from the standard IP model and the lack of virus mobility. Parameters in the diffusion driven phenomena of
pressure interruptions[4], are summarized in Table 1.
It should be noted that such phenomena are a theoretical risk for all virus filters, no matter how robust, and best practice requires
process control of pressure interruptions to maximise virus safety.
Equation 1:
(
C1 x
1 e
CF S
C1 = first layer filtrate concentration
Equation 2:
SV
VR
)
(
C2 x
=
1 e
CF
S
2
Process controls are crucial for the smooth transition from protein to buffer in order to minimise the impact on retention
(particularly for recombinant proteins or monoclonal antibodies). An example of such process control is as shown in Figure 5
(using an Allegro™ MVP System).
SV
VR
)
x V (
e
VR
2
SV
VR
C2 = second layer filtrate concentration
)
Figure 4
Effect on pool LRV of a 10 minute pause and 10%
buffer flush for 4% IVIG and 0.1% BSA
IVIG
IV
VIG
BSA
BS
SA
0
CF = feed concentration
x = fraction of viruses in free solution
Equation 3:
()
V
n log
VR
V = volume filtered
ΔLRV
ΔLRV
LRV n log xS + log( n!) S = sieving coefficient of the rejection zone
VR = volume of reservoir zone
n = number of layers
The IP model (Eqns. 1-3) describes the theoretical change in filtrate virus concentrations and LRV, which is not seen for larger
viruses, nor is it universal for all feed solutions.
The polarization effect is strongest in the initial aliquots when the amount of virus retained in the membrane is low compared to
the input concentration. This is followed by a stable LRV asymptote at higher throughputs.
Theoretically a similar membrane with even higher intrinsic retention (lower xS) would generate a higher LRV at the stable
asymptotic region and a very highly retentive filter.
NEW VIRUS FILTER DESIGN
Pegasus SV4 maximises the benefits of first generation virus
filters by maintaining the strong resistance to flux decay but
increasing the flow and the retention performance (Figure 2).
Figure 2
Relative PP7 filtrate concentration vs Throughput for single
/ dual layer Pegasus SV4
1.E-04
Improved single layer intrinsic retention leads to a stable
and high LRV performance combined with strong flux and
resistance to flux decay.
1.E-05
Cfiltrate / C feed
Achieved good agreement between the IP model fit and stable
high retention of the dual layer membrane.
Developed a revised model to better explain single-layer
performance (Equation 4).
Figure 5
Process Control of Pressure Interruption
Using an Automated Virus Filter System
-0.5
-1
Table 1
Key Parameters that can Affect this Phenomenon Include:
Fixed Parameters
Virus Size
Membrane Pore Size/Distribution
Low-Impact Parameters
Feed Viscosity
Process Temperature
Virus Input Concentration
Throughput
High-Impact Parameters
Time (duration of flow pause or pressure
interruption)
Magnitude of Pressure Reduction
(reduction in convectice flow)
• Fixed by membrane choice and virus type
• Minimal variation possible within a given process
• Affects both input viral load and number of difused
viruses – low impact on process titer reduction
• Potential to vary by orders of magnitude for a
given process
• Can be reduced to minimize pressure interruption
using proper process controls (see Pall’s position
risk mitigation strategy) see section 2
CONCLUSIONS
1.E-06
By understanding the fundamental mechanism behind previous generations of virus filters it was possible to drive improvements
in virus filter development and generate a highly retentive and robust virus filter. For best practice and to maximise virus safety a
robust virus filter should still be combined with a robust and controlled process.
1.E-07
0
50
100
Throughput (L.m-2)
150
Dual Layer
Dual Layer IP Model
Single Layer
Single Layer IP Model
Single Layer IP Model with Hindered Convection
*BSA challenge solution prepared as per PDA guidelines[2].
REFERENCES
[1] Jackson N, Bakhshayeshi M, Zydney AL, Mehta A, van Reis R, Kuriyel R (2014) Biotechnol Prog 30(4):856-63
[2] PDA Virus Filter Task Force (2005) Technical report 41. PDA J Pharm Sci Technol. 59(S-2).
[3] Bungay, Brenner (1973) Int J Multiphase Flow, 1:25-26.
[4] Pall publication USD2997: Pressure Interruptions (Stop/Start) During Virus Filtration: Assuring Virus Safety Using Robust
Process Technology and An Appropriate Risk Mitigation Strategy.
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