The recovery of fluids retained in a flowpath downstream of a sterilizing grade filter installed in a closed single-use system (SUS) can be achieved using a number of methods. Under aseptic conditions, many of these can be challenging to accomplish and can add risk to the process.
We evaluated two single-use system recovery methods, applicable to closed processing, and that remove the need for a blow-down procedure downstream of a final sterilizing grade filter in this technical study.
We quantitated fluid recovery downstream of a 20 cm² Mini Kleenpak™ 0.2 µm filter (KM5 format) in an SUS configuration used for the sterile filtration of a batch volume of 0.2 L.
The data confirms that up to 15% (30.9 mL) of the filtered 0.2 L batch volume may be lost without a recovery procedure for fluid entrained downstream of a sterilizing grade filter. Both methods reduced fluid loss to only 2% of the sterile-filtered batch volume (4.2 to 4.3 mL).
Objective
This study is designed to quantitate the product hold-up volume downstream of the Mini Kleenpak KM5 filter after filtration and to determine the recoverable product volume downstream of the final filter when using the Downstream Biocontainer method and the Secondary Tube method.
Background
For this study, we used two SUS models based on the design of a small volume filling system using redundant filtration (Fig 1). The test models were simplified to include components from the final Supor™ grade EKV filter in Mini Kleenpak 20 format (product code KM5EKVP2G) to the 500 mL collection biocontainer.
This study simulated a small batch with volumes between 0.2 and 0.3 L to represent a worst-case scenario where losses may be significant to the overall process yield.
Fig 1. Redundant filtration assembly using Mini Kleenpak format sterilizing grade filters.
Materials and methods
Test Model A (Fig 2) was used to evaluate the Downstream Biocontainer recovery method.
Test Model B (Fig 3) was used to evaluate the Secondary Tube recovery method.
Fig 2. SUS Model A for the evaluation of the Downstream Biocontainer recovery method.
Fig 3. SUS Model B for the evaluation of the Secondary Tube recovery method.
For each model, the dry weights of the filter (“H” values in Table 2) and SUS model (“E” values in Table 1) were recorded before adding the inlet tube and the biocontainer(s).
Following this, for Model A and Model B, a gravimetric method was used for the determination of the downstream hold-up volume in the filtrate flow path between the filter and the inlet of the 500 mL collection biocontainers. For each model, the inlet tube was installed in a beaker containing colored water (density = 1 g/mL) and the filled beaker was placed on a balance (Fig 4). A peristaltic pump was used to fill the models with water, up to the point where the water was reaching the filter inlet, permitting air to be introduced into the downstream biocontainer.
The pump was stopped, and the weight of the beaker was registered at this point (A values in Table 1). The pump was then restarted to fill the downstream part of the model assemblies up to the point where the water was reaching the inlet of the 500 mL collection biocontainer.
The weight of the beaker was registered again at this point (B values in Table 1).
Fig 4. Filling and determination of downstream hold-up volume for SUS Model A.
The weight difference of the colored water in the beaker before and after filling the filtrate tube manifold (C values in Table 1) therefore corresponds to the downstream hold-up volume of the model assemblies before the recovery (Fig 4).
Once the volume downstream of Filter 2 had been filled, the models were inverted in a free-draining orientation (Fig 5).
Recovery of fluid downstream of Filter 2 for each model was then achieved with the opening of the pinch valves (Fig 5). The final system weight (D values in Table 1) was recorded for each test model after removal of the inlet tube and draining of the upstream part of the filter.
Fig 5. Filtrate recovery with SUS Model A in free-draining orientation.
Following application of the recovery method with each model, the nonrecoverable downstream volume was calculated for each model by subtracting the dry weight of the model without the biocontainer(s) from the corresponding post-recovery weight;
Nonrecoverable product volume = (Post-recovery weight) – (Dry weight)
The recovered product volume (G values in Table 1) was calculated by subtracting the nonrecoverable product volume from the downstream hold-up volume.
Finally, Filter 2 filter was removed from the SUS model and weighed for the determination of the residual water in the filter, post-recovery (Table 2).
Results
The results for both test models are presented in Table 1 and Table 2.
Table 1. Test results for both test Model A and B
|
SUS |
Beaker weight before filling DS volume, |
Beaker weight after filling DS volume, |
DS hold-up volume before recovery, |
Post-recovery weight w/o biocontainers, |
Dry model weight w/o biocontainers, |
Non recoverable DS volume, |
Recovered product volume, |
|
Model A |
3974.4 |
3945.5 |
30.9 |
140.5 |
136.2 |
4.3 |
26.6 |
|
Model B |
527.2 |
496.5 |
30.7 |
141.3 |
137.1 |
4.2 |
26.5 |
DS = Downstream
Table 2. Test results (contd.)
|
SUS |
Filter dry weight, |
Filter wet weight post-recovery, |
Residual water in the filter post-recovery, |
|
Model A |
23 |
25.8 |
2.8 |
|
Model B |
23 |
26.2 |
3.2 |
Conclusions
Based on the results of the study, without applying any recovery method, up to 30.9 mL of nonrecoverable filtered fluid could result, representing 15% loss in the 0.2 L batch volume.
With the given system design, by applying the Downstream Biocontainer method, we recovered 26.6 mL (up to 13% of the 0.2 L filtered batch volume) and by applying the Secondary Tube method, we recovered 26.5 mL (13% of the filtered batch volume) confirming comparability of both methods.
In both cases, air displacement during the filling of the systems permitted enough air in the region of the of the system downstream of the filter, to recover the filtrate held up after filtration. With both scenarios the eventual product hold-up (i.e., nonrecoverable volume of sterile-filtered fluid) was comparable and limited to only 2% of the filtered batch volume. Most of this volume was entrained in the membrane and the downstream part of Filter 2. This nonrecoverable hold-up volume in the filter capsule is independent of the applied recovery method and dependent on the filter area and the design of the filter capsule.
In this study, we inverted the system before filling to enable the gravimetric determination of the downstream volume. In a real application, you can install the system in a free-draining position from the start, without needing to invert the system.
FAQ
How much product can be lost downstream of a sterilizing grade filter without a recovery method?
This study demonstrated that up to 30.9 mL, equivalent to approximately 15% of a 0.2 L batch volume, can remain trapped downstream of a sterilizing grade filter when no recovery method is used.
What causes product hold-up volume downstream of a sterilizing grade filter?
Product hold-up volume occurs when liquid remains entrained within the filter capsule, tubing, connectors, and other downstream components of a single-use filtration assembly after filtration is complete.
What recovery methods were evaluated in this study?
The study evaluated two recovery approaches suitable for closed processing in single-use systems:
- Downstream Biocontainer method
- Secondary Tube method
Both methods eliminated the need for a downstream blow-down procedure.
Which recovery method provided the highest product recovery?
Both methods delivered nearly identical results. The Downstream Biocontainer method recovered 26.6 mL, while the Secondary Tube method recovered 26.5 mL, demonstrating comparable performance.
How effective are these recovery methods at reducing sterile filtration losses?
Both recovery methods reduced product loss from approximately 15% of batch volume to only 2%, significantly improving overall process yield.
Why is product recovery important in small-volume bioprocessing?
In small batch processes, even modest fluid losses can have a substantial impact on product yield. Recovering fluid downstream of sterilizing grade filters helps maximize valuable product recovery and improve manufacturing efficiency.
Can these recovery methods be used in closed single-use systems?
Yes. Both methods were specifically designed and evaluated for use in closed single-use systems (SUS), supporting aseptic processing without introducing additional contamination risk.
What volume remained nonrecoverable after applying the recovery methods?
After implementing either recovery method, only 4.2 to 4.3 mL of fluid remained nonrecoverable, representing approximately 2% of the sterile-filtered batch volume.
What contributes to the remaining nonrecoverable volume?
Most of the residual fluid volume remained within the sterilizing filter membrane and filter capsule. This hold-up volume is primarily influenced by filter design and filtration area rather than the recovery method used.
How can manufacturers improve sterile filtration yield in single-use systems?
Manufacturers can improve yield by incorporating recovery strategies such as the Downstream Biocontainer or Secondary Tube methods, optimizing flow path design, minimizing hold-up volumes, and installing systems in a free-draining orientation whenever possible.
Do these recovery methods require system inversion during production?
No. System inversion was performed in this study for gravimetric measurement purposes only. In commercial applications, the assembly can be installed in a free-draining orientation from the outset, eliminating the need for inversion.
CY59864-11Sep26-AR