Staphylococcus aureus (S. aureus) is an organism that can be problematic to eradicate, particularly for certain valves or components that have Ethylene Propylene Diene M-class (EPDM) membranes. Through iterative testing in four studies at room temperature (RT), we have developed an effective method for cleaning-in-place (CIP) for the ÄKTA process™ chromatography system.
We have previously shown the ability to clean the ÄKTA process™ chromatography system from two other bacterial strains recommended by the United States Pharmacopoeia (USP 38), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). For more information see Cleaning-in-place of ÄKTA process™ with sodium hydroxide.
Introduction
The efficiency of your cleaning method can be evaluated using microbial challenge tests. We will describe the cleaning procedure of ÄKTA process™ chromatography system after challenge with S. aureus, an organism that can be difficult to fully remove from all parts of the flow path. A series of four cleaning studies were performed to identify the most effective steps to include in a cleaning method using UNICORN™ control software.
ÄKTA process™ automated liquid chromatography system is built for process scale-up and large-scale biopharmaceutical manufacturing. The system is available in three flow rate ranges that extend up to 2000 L/h for large volume manufacturing and can be constructed using either electropolished stainless steel (SS) or polypropylene (PP), depending on process conditions and plant requirements.
A predefined cleaning method was used to evaluate surfaces and the hygienic design of an ÄKTA process™ chromatography system (a 1” PP system was used) using S. aureus as the challenge organism and 1 M NaOH as the cleaning agent. All surfaces in contact with the process flow were pre-cleaned, challenged, cleaned, and evaluated. Microbial sampling was performed at pre-determined sites and flow through samples were collected from the tested system during the run and after finishing the cleaning method.
Results and discussion
Study 1 and 2 for S. aureus
All results from viable count (VC) plates in study 1 and 2 showed that the challenge with the S. aureus organisms were correct (106-108 colony forming units [CFU]/mL) meeting the acceptance criteria. Control samples in both studies showed that experimental handling procedures and material used for evaluation of the studies met the acceptance criteria. The microbial air samples showed that bioburden level in the air in the lab were low in both studies. All liquid samples were also free from contamination.
Post-cleaning samples taken in study 1 and 2, did not contain any microorganisms meeting the acceptance criteria. Finally, endotoxin levels were < 0.5 endotoxin units [EU]/mL for both studies also meeting the acceptance criteria.
Study 1
Among the 29 swab samples taken, one of the samples (27) proved to be positive for the challenging organism S. aureus, which means that the acceptance criteria were not met. The challenging organism was found in outlet valve number 10, which shows that those valve types easily can hold on to this type of cocci shaped organisms. Contaminants other than the challenging organism were also found in one of the membrane valves positioned in the process control valve (PCV) component and in one of the valves related to filter unit two. Organisms found here were Ralstonia mannitolilytica and Stenotrophomonas maltophilia, respectively. Contaminants were also found at two other positions (8 and 24). The first position was the downward pointing pressure sensor and the second one the horizontal positioned conductivity cells closest to the outlet valves. Organisms found were Staphylococcus haemolyticus and Burkholderia cepacian, respectively, common among the human flora of bacteria. Those two contaminants were false positives and likely to be related to compromised aseptic handling. The same reasoning goes for the other two contaminants at positions 3 and 13, respectively.
It was also discovered that the filters were undersized, which consequently lowered the pump speed significantly below the set value of 75%. This was changed for ones with higher flow capacity for future studies. It was also decided to implement more mechanical cleaning of the valves during the period of static hold in the method by opening and closing all valves in the system in a loop-way fashion. By this, the movement of the cleaning agent in contact with the membrane would lead to a mechanical impact and increased contact time for the parts of the membrane that are hidden when valves are closed. It was further decided that all membranes would be inspected before the next study and that a new method for draining the system of liquid after the cleaning procedure was completed needed to be devised.
Study 2
One of the swab samples taken (6) representing the wetted surface of PCV C proved to be positive for the challenging organism S. aureus which means that the acceptance criteria was not met. This result along with the results from the other two swab samples (3 and 27) presenting other contaminants than the challenging one confirms again that the valves and specifically the PCVs are among the most difficult components to clean. Sample 3 and 27 tested positive for Brevibacterium casei (Gram positive rod), Rhodococcus coprophilus/erythropolis/globerulus (Gram positive rod), and Gram-positive cocci/rod (not identified), respectively.
The UNICORN™ method for cleaning was updated with loops that opened and closed valves in the inlet, column, and outlet blocks several times during the hold period. The aim was to introduce more mechanical cleaning and longer contact time for the entire wet membrane surface during the open state of the valve.
A plan was also developed for how to drain the system by opening the flow path at strategic points without the risk of contaminating the sampling positions. Liquids pouring out at those strategic points were collected in containers. It had also been decided before we started study 2 that the last step in the method would be to apply sterile filtered 20% ethanol (storage solution). In this way, we improved the aseptic handling and reduced risks with large spills of saline solution, which easily gives rise to salt deposits and corrosion if you fail to remove all the spillage.
The ULTA™ capsule 0.2 µm filters used in study one for the incoming liquids (0.9% NaCl, purified water (PW) and 20% ethanol) were replaced with new filters, Parker Murus PROPOR HC 0.2 µm, in study 2 with better flow capacity. Now the determined flow of 75% could be achieved in all steps of the method.
Study 3 and 4 for S. aureus
All results from VC plates in study 3 and 4 showed that the challenge with the S. aureus organisms met the acceptance criteria (106 - 108 CFU/mL). Control samples in both studies showed that experimental handling procedures and material used for evaluation of the studies also meet the acceptance criteria. The microbial air samples showed that bioburden level in the air in the lab were low in both studies compared to results from previous studies. All liquid samples (A through H) were free from contamination (Table 1). Post-cleaning samples taken in studies 3 and 4 did not contain any microorganisms and endotoxin levels were < 0.5 EU/mL in both studies.
Table 1. Sampling points, liquid samples, and control samples used in studies 3 and 4
| Sample name | Procedure | Description | Sampling method | Sample name | Procedure | Description | Sampling method |
| A | Pre-cleaning procedure | Pre-cleaning solution: 1 M NaOH, sample taken before application. Sampling from CIP inlet 2 | Test method 2 bioburden filtration test |
I | Cleaning procedure | Storage solution: 0.22 µm filtered 20% ethanol, sample taken before application Sampling from CIP inlet 1 |
Test method 5 endotoxin analysis |
| B | Pre-cleaning procedure | Storage solution: 0.22 µm filtered 20% ethanol, sample taken before application Sampling from CIP inlet 1 |
Test method 2 bioburden filtration test |
J | Post cleaning procedure | Storage solution: 0.22 µm filtered 20% ethanol, sample taken from the flowthrough |
Test method 2 bioburden filtration test |
| C | Challenging procedure | Priming solution: 0.22 µm filtered 0.9% NaCl, sample taken before application Sampling from CIP inlet 3 |
Test method 2 bioburden filtration test |
K | Post cleaning procedure | Storage solution: 0.22 µm filtered 20% ethanol, sample taken from flowthrough |
Test method 5 endotoxin analysis |
| D | Challenging procedure | Preparation of inoculum, suspension without the challenging organism, 0.9% NaCl | Test method 2 bioburden filtration test |
L | Challenging procedure | Inoculum (start concentration), sample taken before application | Test method 4 viable count test |
| E | Cleaning procedure | Wash solution: 0.22 µm filtered purified water, sample taken before application Sampling from CIP inlet 1 |
Test method 2 bioburden filtration test |
M | Challenging procedure | Post-application sample taken from the flowthrough directly after finished application | Test method 4 viable count test |
| F | Cleaning procedure | Cleaning solution: 1 M NaOH, sample taken before application. Sample from CIP inlet 2. | Test method 2 bioburden filtration test |
N | Challenging procedure | Pre-cleaning sample taken from the flowthrough after 16 to 18 h of incubation with the challenging suspension | Test method 4 viable count test |
| G | Cleaning procedure | Wash solution: Priming solution: 0.22 µm filtered 0.9% NaCl, sample taken before application Sampling from CIP inlet 3 |
Test method 2 bioburden filtration test |
||||
| H | Cleaning procedure | Storage solution: 0.22 µm filtered 20% ethanol, sample taken before application Sampling from CIP inlet 1 |
Test method 2 bioburden filtration test |
Table 2. Locations for collecting swab samples on ÄKTA process™ system (see Figs 1 through 8 for pictures of the swab points)
| Swab number | Component | Description | Sampling method | Swab number | Component | Description | Sampling method |
| 1 | Inlet A | Wetted part of EPDM membrane of GEMÜ valve, inlet position 2. | Test method 3 swab test | 16 | Filter units top valve | Horizontal valve channel, filter unit 1, pointing to the right. | Test method 3 swab test |
| 2 | Pressure control valve | Tip of pressure sensor, directed upward. | Test method 3 swab test | 17 | Filter units top valve | Horizontal valve channel, filter unit 2, pointing to the left. | Test method 3 swab test |
| 3 | Pressure control valve | Wetted part of EPDM membrane of valve, directed downward. | Test method 3 swab test | 18 | Air trap and filter valve block | Wetted part of EPDM membrane of GEMÜ valve position 4 | Test method 3 swab test |
| 4 | Pressure control valve | Wetted part of EPDM membrane of valve, directed upward. | Test method 3 swab test | 19 | Air trap and filter valve block | Wetted part of EPDM membrane of GEMÜ valve position 8 | Test method 3 swab test |
| 5 | Pressure control valve | Tip of pressure sensor, directed downward. | Test method 3 swab test | 20 | pH sensor (vertical) | Tip of pH dummy including O-ring, directed upward. | Test method 3 swab test |
| 6 | In-line dilution valve | Wetted part of EPDM membrane of valve, directed upward. | Test method 3 swab test | 21 | Pressure sensor (horizontal) | Tip of the pressure sensor, directed horizontally. | Test method 3 swab test |
| 7 | Pressure sensors, vertical | Tip of pressure sensor, directed upward. | Test method 3 swab test | 22 | Pressure sensor, vertical | Tip of the pressure sensor, directed upward. | Test method 3 swab test |
| 8 | Pressure sensors, vertical | Tip of pressure sensor, directed downward. | Test method 3 swab test | 23 | Conductivity sensor, horizontal | O-ring on conductivity sensor. | Test method 3 swab test |
| 9 | Conductivity sensor, horizontal | O-ring on conductivity sensor. | Test method 3 swab test | 24 | Conductivity sensor, horizontal | Insert with O-ring in conductivity cell supporting sensor. | Test method 3 swab test |
| 10 | Conductivity sensor, horizontal | Insert with O-ring in conductivity cell supporting sensor. | Test method 3 swab test | 25 | pH sensor | Tip of pH dummy including O-ring, directed upward. | Test method 3 swab test |
| 11 | Air trap and filter valve block | Wetted part of EPDM membrane of GEMÜ valve position 2 | Test method 3 swab test | 26 | Outlets | Wetted part of EPDM membrane of GEMÜ valve position 2. | Test method 3 swab test |
| 12 | Air trap and filter valve block | Wetted part of EPDM membrane of GEMÜ valve position 9. | Test method 3 swab test | 27 | Outlets | Wetted part of EPDM membrane of GEMÜ valve position 10. | Test method 3 swab test |
| 13 | Air trap and filter valve block | Wetted part of EPDM membrane of GEMÜ valve position 10. | Test method 3 swab test | 28 | ILD cart, pressure sensors | Tip of pressure sensor, directed upward. | Test method 3 swab test |
| 14 | Air trap and filter valve block | Wetted part of EPDM membrane of GEMÜ valve position 1 | Test method 3 swab test | 29 | ILD cart, pressure sensors | Tip of pressure sensor, directed downward. | Test method 3 swab test |
| 15 | Air trap top valve | Horizontal valve channel. | Test method 3 swab test |
Table 3. Control samples
| Sample number | Type | Description | Sampling method | |
| 30 | Microbial air control | Air samples taken in the room where the study was performed | Test method 1 microbial air sampling |
|
| 31 | Negative control | Sterile NaCl 0.9% solution | Test method 2 bioburden filtration test |
|
| 32 | Negative control | Swab directly transferred into the isotonic solution | Test method 3 swab test | |
| 33 | Negative control | Mini swab directly transferred into the isotonic solution | Test method 3 swab test | |
| 34 | Negative control | TSA-plate (pre-casted), directly bagged | N/A | |
| 35 | Positive control | TSA-plate (pre-casted) (Milliflex Oasis) | Test method 2 bioburden filtration test |
|
| 36 | Positive control | Swab dipped into inoculum | Test method 3 swab test | |
| 37 | Positive control | Mini swab dipped into inoculum | Test method 3 swab test | |
| 38 | Positive control | TSA-plate (pre-casted), the viable count plates of the inoculum will represent this plate | N/A | |
Study 3
The results of the serial dilution of the liquid samples (L, M and N) of the challenging suspension are shown in Table 4. The viable count plates confirm the S. aureus suspension was fully present prior to cleaning.
Among the swab samples taken, two samples (12 and 26) had contaminants other than the challenge organism. The contaminants, Kocuria varians and Clavibacter michiganensis, were found on the EPDM membranes valves in the filter/air block (valve position 9) and in the outlet block (valve position 2). The acceptance criteria stated that 10% of the swab samples (maximum 3 in this case) can have other organisms than the challenging one and since only two of the samples were positive in this case, this acceptance criteria was met. This meant that this cleaning method can efficiently clean the system.
Table 4. Viable count plates from study 3 for S. aureus of the liquid suspensions collected for samples L, M, and N (The average CFU was calculated from the × 105 values.)
| Sample L Inoculum (taken before application) |
CFU/mL (× 105) | CFU/mL (× 106) | ||
| S. aureus | A | 375 | 22 | |
| S. aureus | B | 233 | 31 | |
|
Sample M post infection |
CFU/mL (× 105) | CFU/mL (× 106) | ||
| S. aureus | A | 211 | 37 | |
| S. aureus | B | 321 | 32 | |
| Sample N pre-cleaning (taken from the flowthrough after 16-18 h of incubation) |
CFU/mL (× 105) | CFU/mL (× 106) | ||
| S. aureus | A | 200 | 26 | |
| S. aureus | B | 208 | 19 | |
The following changes from study 2 were implemented:
- All of the inlet and outlet manifolds were flushed, which ensured that the longest flow path of the inlet/outlet valve blocks are flushed with the correct solution before the actual cleaning starts. Flushing was done through valve 9 and 10 in inlet block A, valve 5 and 6 in inlet block B, and valve 1 and 2 in inlet block C (ILD cart) using 75% pump speed. Approximately 6 to 7 L of solution per valve were used. Outlet block where valves 9 and 10 are located, were used for flushing with approximately 14 L.
- Each inlet position was cleaned with flow speed 75% for 0.5 min in block A, B, and C. After this run, the system was run in ILD mode using all 3 pumps at the same time. This ensured proper cleaning of the PCV by extending the contact time through its valves. Pump speed was set to 75% pump speed. Flow speed through each PCV valve in ILD mode was 25% of pump speed. All valves of the inlet and outlet blocks were flushed during this mode in a pre-determined manner to expand the contact time of all wetted surfaces.
- The air trap and filter units were set in-line more frequently to extend contact time in these components and in the flow path in the connected valve block. This ensured that the whole flow path in this block was efficiently cleaned.
- During application of the cleaning agent, different combinations of flow paths in the inlet, outlet, and column valve blocks together with intermediate and associated components like pressure sensors, conductivity and pH cells, air trap, and filter units were put in-line in different flow distribution combinations to enhance the total contact time of the whole wetted surface of the flow path.
- During the 1 h of hold time with the cleaning agent, all valves in the system were opened at least 12 times for 0.5 min each time.
Study 4
The results of the serial dilution of the liquid samples (L, M, and N) of the challenging suspension are shown in Table 5. The viable count plates confirm the S. aureus suspension was fully present prior to cleaning. Among the swab samples taken, one sample (28), representing the pressure sensor pointing downward on the ILD cart tested positive for a contaminate but it was not identified as the challenging organism S. aureus but as a bacterium coming from the Moraxella group (Gram negative rod/cocci). Since only one of the samples were positive in this case, the acceptance criteria was met. This means that the cleaning method with an extra 1 min on the outlet used in this study can also efficiently clean the system.
Table 5. Viable count-plates from study 4 for S. aureus of the liquid suspensions collected for samples L, M, and N (The average CFU is calculated from the × 105 values.)
| Sample L Inoculum (taken before application) |
CFU/mL (× 105) | CFU/mL (× 106) | ||
| S. aureus | A | 163 | 27 | |
| S. aureus | B | 179 | 20 | |
|
Sample M post infection |
CFU/mL (× 105) | CFU/mL (× 106) | ||
| S. aureus | A | 175 | 35 | |
| S. aureus | B | 200 | 21 | |
| Sample N pre-cleaning (taken from the flowthrough after 16-18 h of incubation) |
CFU/mL (× 105) | CFU/mL (× 106) | ||
| S. aureus | A | 149 | 21 | |
| S. aureus | B | 180 | 14 | |
The following change was made to the cleaning method:
- An extra minute of flushing using 75% pump speed was added for each valve in the outlet block. The reason for this was that valve 2 (sampling point 26) had been free of contamination in all previously conducted studies on this system up to study 2.
Recommendations on how to set up a UNICORN™ method that efficiently cleans ÄKTA process™ systems for S. aureus
Results from studies 1 through 4 for S. aureus were compiled and analyzed with a focus on the ability of the 1 M NaOH cleaning agent to inactivate contaminating bacteria in relation to the parameters flow speed, contact time, and flow distributions. Our goal was to summarize the conclusions drawn from these studies into recommendations on how to set up a UNICORN™ method that efficiently cleans all configurations of the ÄKTA process™ system. A special focus has been placed on components that have been challenging to clean such as the (PCV), waste tubes, and outlet valves.
Preparation before system cleaning
Pre-sterilized manifolds and seals should be used on inlets and outlets for efficient cleaning of the complete wetted pathway. If the system is configured with a CIP block, a manifold should initially be used for this block as well. Attached inlet manifolds should be connected using a T-branched tube that in turn is connected to the outlet of the CIP block. The manifold on the outlet side should be positioned with the inlet of the manifold pointing upwards to create a liquid lock. This enhances the contact time with the cleaning agent of the wetted surface of each valve. pH probes should be replaced by dummies and cleaned according to manufactures recommendations.
Cleaning of the CIP block
Connect the tube that distributes the cleaning agent to the inlet of the manifold attached onto the CIP block. Put the system in bypass mode with inlet A1 and outlet 1 opened. Flush each inlet valve of the CIP block with 1 M NaOH for 20 s with pump speed at 50% and leave the system on hold for 1 h without any flow. Rinse away the cleaning agent in the CIP block with sterile filtered 20% ethanol. When it’s time to continue with the system cleaning, the manifold should be removed and tubes distributing the cleaning agent, the wash solution, and the storage solution should be connected to the CIP block.
Recommended parameters to be used in the UNICORN™ method during application of the 1 M NaOH cleaning agent
Phase 1: cleaning with flow
The section of the UNICORN™ method where the 1 M NaOH cleaning agent is applied should be designed so that the whole surface of the wetted flow path is in contact with the cleaning agent for as a long as possible during the total application time. This means that distribution of the cleaning agent should be distributed as equally as possible over the entire wetted surface with respect to time. The longer the contact time, the more effective the cleaning. By following the recommendations below the whole wetted surface including the surface in non-mentioned components will have enough contact time to be efficiently cleaned.
- Flush all inlet manifolds to ensure that the “longest” flow path of the inlet valve blocks are flushed with the correct solution before the actual cleaning starts. Flushing is done through valve 9 and 10 in inlet block A, valve 5 and 6 in inlet block B, valve 1 and 2 in inlet block C (ILD cart) using 75% pump speed. Approximately 6 to 7 L of solution per valve should be used. The same applies for the outlet block with valves 9 and 10 which should be flushed approximately 14 L of solution.
Inlet valve blocks
- Flush all manifolds through the last valves of each inlet block with approximately 7 L.
- Run 0.5 min at 75% pump speed per inlet valve starting with the last valve position in each block ascending to the first valve. This step is executed one block at a time.
- Run for at least 2.5 min at 25% pump speed per inlet valve starting with last valve position in each block ascending to the first valve. This step is executed with all PCV valves opened and all pumps running at the same time with the pump speed set to 75%.
Note: Run parameters in step 2 above needs to be adjusted depending on how the ÄKTA process™ chromatography system is configured since each PCV valve needs a total contact time with flow of at least 30 min. The time should be divided equally on each valve in each inlet block. Systems can be configured with a third PCV called inlet C where an ILD system with its own pump can be attached. Systems that have the ILD functionality can utilize its ability to run all three pumps simultaneously. The same parameters and method design mentioned above should be practiced and implemented for this extra unit in the cleaning method. Flow speed downstream of the PCV will be collectively 75% of maximum. One bar back pressure can be advantageously used in all steps throughout the whole cleaning procedure.
Air trap and Filter units
- Fill and drain the air trap four times consecutively with the pump speed set to 75%.
- Open the air trap top valve and run for 1.5 min with the pump speed set to 75%. This step cleans the top valve and the waste tube of the air trap.
- If the system is equipped with filter units, fill then drain those consecutively four times with the pump speed set to 75%.
- Open the filter top valves and run for 1.5 min with the pump speed at 75% through each valve. This step cleans the top valves and waste tubes connected to the filter units.
Note: The drain procedure for the air trap and filter units are conducted via the air trap/filter valve block. Effluent from either unit is collected into the common waste collection cup positioned below this block. Draining of the air trap or filter unit is only allowed when there are no pumps running unless you are running the out through drain function. This allows the pump effluent to flow through the air trap or filter unit and out to the common waste. The flow speed through this waste tube is solely based on gravitational speed if this function is not used. This valve and connected waste tube is flushed with four-unit volumes of the air trap and four-unit volumes from each existing filter unit by first filling and then draining the units. If the ÄKTA process™ chromatography system is configured with no or only one filter unit, all missing drain volumes should be compensated by running one or two times with the out through drain function. Run parameters per missing unit with this function should be set to 1 min in combination with pump speed at 75%. This will generate a flow speed of 37.5% through the valve and waste tube. All wetted pathways in the air trap/filter valve block including bypass flow paths will be efficiently cleaned if the recommended steps are followed.
Column valve blocks
Direct the flow in the following order and run for 4 min at 75% pump speed for each flow distribution: downflow column 1, bypass bottom, up flow column 1, bypass top, bypass both, downflow column 2, bypass bottom, up flow column 2, bypass top, then bypass both.
Outlet valve blocks
- Flush the manifold using the last valve of the outlet block with approximately 14 L of solution.
- Run for 4 min at 75% pump speed per outlet valve starting with the first valve position in the block ascending to the last valve.
Phase 2: hold time without flow
This phase has no flow and lasts for 1 h. All valves in the system are opened and closed in a loop during this time. This is comparable to a mechanical cleaning process. Valves that are included in this loop are inlets, column valves, and outlets. All the valves should be opened and closed twelve or more times during this phase.
Conclusions
In conclusion, we have shown:
- ÄKTA process™ chromatography systems can be efficiently cleaned from the challenging organism S. aureus using the cleaning methods tested in these studies.
- The most challenging components regarding bioburden in ÄKTA process™ chromatography systems are membrane valves since the bacteria S. aureus is strongly attracted to the EPDM membrane.
- PCV requires special focus in regard to bioburden and is a critical part for the design of cleaning methods.
- How the operator combines contact time, flow rate, and back pressure has a great impact on how to clean a surface. It is the interaction between these factors that you must focus on when designing a cleaning process.
- Recommendations on how to set up a UNICORN™ method that effectively cleans ÄKTA process™ chromatography system based on data analyzed from the last four completed studies has been developed to facilitate future optimizations of cleaning methods for similar systems.
CY44034-xxXXX2024-AN
TR- 31126210
Related Content
Materials and methods
The cleaning studies consisted of five parts:
- Pre-cleaning of parts and ÄKTA processTM chromatography system (Liquid samples)
- Challenging of ÄKTA process™ chromatography system (Liquid samples)
- Cleaning with pre-defined method (Liquid samples)
- Swab sampling at predetermined positions
- Evaluation of swab samples and liquid samples after 5 d
Pre-cleaning of parts
The ÄKTA process™ chromatography system was first cleaned externally with purified water (PW) and wiped with lint free towels wetted with 70% ethanol.
Tubes, valves, manifolds, seals, TC-clamps, and blinds (TC-fitting end caps) upstream of the system were autoclaved (121°C for 20 min) before attachment. Filters (0.2 µm 10", Parker Murus PROPOR HC) were attached on the pipes of the incoming NaCl and PW/ethanol liquids. All liquids, except for NaOH were filtered (0.2 µm). Inlet blocks A, B, and C were equipped with manifolds and connected to each other and the CIP-block outlet using ordinary and T-branched tubes. Also, the outlet block was equipped with a manifold together with sampling equipment (autoclaved at 121°C for 20 min). The manifold on the outlet side was positioned in such a way that the tube that collects all outgoing liquids pointed upwards to create a liquid lock.
Pre-cleaning of ÄKTA process™ chromatography system
An autoclaved manifold was attached onto the four CIP inlets. All seals and interaction points were treated with Klercide (70% ethanol, Ecolab USA, Inc.). The tube distributing the 1 M NaOH cleaning agent was attached to the manifold inlet. The system was run manually during cleaning of the CIP block. Each inlet was opened and flushed with 1 M NaOH for approximately 20 s at a pump speed of 50%. The system was set in bypass mode with Inlet A1 and outlet 1 valves open during those flushes of the CIP valves. After the flushes, the system was left for 1 h of static hold. A rinse of the wetted flow path was followed with sterile filtered 20% ethanol. All valves on the CIP block were opened and closed a couple of times during run with pump speed at 75%. The whole wetted flow path was filled with 20% ethanol and the system stored in this state until the next procedure was started.
The manifold on the CIP inlet was removed and the tubes distributing the liquids PW/20% ethanol, 1 M NaOH, and 0.9% NaCl, were attached to positions 1 through 3 in that order. Before attaching the tubes to the inlet positions on the CIP inlet, autoclaved sampling equipment for testing incoming solutions was attached. Position 4 was capped.
A UNICORN™ method was created to automate the pre-cleaning procedure. The procedure included a rinse with filtered water, application of 1 M NaOH cleaning agent including a static hold for 1 h, and a wash out step using sterile filtered 20% ethanol. Liquids were applied with 25% to75% of maximum pump speed.
Preparation and application of challenging organism suspension
The pre-cleaned system was primed with 0.9% NaCl prior to application of the challenging organism suspension.
The challenging step (step 2 above) consisted of three parts:
- Inoculum (Sample L), which is the liquid sample taken of the prepared suspension with S. aureus before application on the ÄKTA process™ chromatography system.
- Post-infection (Sample M), which is the liquid sample taken directly after application of the suspension on the ÄKTA process™ chromatography system. This sample is taken to ensure that nothing has happened to the organisms as they flowed through the flow path.
- Pre-cleaning (Sample N), which is the liquid sample taken after the challenging suspension has been incubated in the flow path of the ÄKTA process™ chromatography system for 16 to 18 h and just before the start of the predetermined cleaning method.
TSA-plate streaked with challenging organism were incubated at 37°C overnight. Fresh colonies from this plate were transferred to 200 mL of autoclaved TSB-media and left shaking in 37°C overnight. Based on measured optical density (OD) of the pre-culture and the assumption that 1 OD is approximately 1.5 × 108 CFU/mL for S. aureus, calculations were made on the volumes needed of the pre-culture to be added in ~70 L (study 3) and ~100 L (study 4) of filtered 0.9% NaCl solution to obtain the final concentrations of ~107 CFU/mL for the challenging organism in the suspension. Results of the viable count plates for the liquid samples are shown in Table 4 for study 3 and Table 5 for study 4.
The suspension was applied on the system with 10% of maximum pump speed bypassing the CIP valves. The procedure was automated with a UNICORN™ method. The infected system was left for 16 to 20 h at RT before starting the cleaning procedure. Overview of the challenging organism loading is described below for the inlets (A, B, and C), air trap, filter, all column positions, and all outlets.
Cleaning procedure
Liquids were filtered through 0.2 µm filters except for the 1 M NaOH cleaning agent. The procedure was the same as when pre-cleaning the system. In study 4, however, the contact time of the cleaning agent with focus on the outlet block valves was elongated to 1 min per valve. Endotoxin samples were collected.
Microbial sampling
Microbial samples were taken at predetermined sites shown in Figures 1 through 8. Applied solutions on the system were also sampled.
Fig 1. Swab samples were collected from inlet A (1), the pressure control valve (PCV) (2 through 5), and inline dilution inlet (6).
Fig 2. Swab samples were collected from the pressure sensors vertical (7 and 8), and conductivity sensor horizontal (9 and 10) positions.
Fig 3. Swab samples were collected from wetted membrane directing flow to the air trap, filter, and sample valves (11 through 14). Contamination was detected from sample 12 in study 3.
Fig 4. Swab samples were collected from the air trap horizontal valve channel (15).
Fig 5. Swab samples were collected from filter units horizontal valve channel (16 and 17), swabbing of outlet channel on valve block waste outlet channel (18), and lower valve inlet channel (19).
Fig 6. Swab samples were collected from the pH (vertical) and pressure sensor (horizontal) (20, 21), pressure sensor (vertical) (22), conductivity sensor (horizontal) (23 and 24), and pH sensor (25).
Fig 7. Swab samples were collected from the outlets (26 and 27). Contamination was detected from sample 26 in study 3.
Fig 8. Swab samples were collected from the inline dilution cart and pressure sensors (28 and 29). Contamination was detected from sample 28 in study 4.
Microbial sampling was performed by one of the following methods:
Test method 1, microbial air sampling
Sampling of air for airborne microorganisms was conducted with a Microbial Air Sampler (MAS). A MAS loaded with an agar plate is positioned at a suitable measuring point. When the measuring starts, a pre-defined volume of surrounding air is passed through the machine. Microorganisms will be collected on the agar surface by impaction.
Test method 2, bioburden filtration test for S. aureus
Sample solutions (minimum 50 mL) were collected in sterile tubes and then filtered through a 0.45 µm cellulose nitrate membrane filters. Filters were incubated on agar plates at 30°C to 35°C for 5 d after which the plates were inspected for CFUs.
Test method 3, swab test for S. aureus
Surface samples were taken with swabs. The swab was inserted into the tube containing isotonic swab rinse solution and vortexed for a minimum of 20 s. The solutions including the swabs were poured into Petri dishes and mixed with 30 mL of temperature controlled molten agar. Maximum temperature of the molten agar was 45°C. After solidification, plates were incubated at 30°C to 35°C for 5 d after which the plates were inspected for CFUs.
Test method 4, viable count test for S. aureus
Samples of challenging organism suspensions were diluted in series in 0.9% NaCl. Samples from the diluted suspensions were plated on agar plates and incubated at 30°C to 35°C for 1 to 2 d after which the plates were inspected for CFUs. The concentration of challenging organism was determined in the sampled suspensions.
Test method 5, endotoxin analysis
The method used for analysis of endotoxin content was based on the Limulus Amoebocyte lysate kinetic chromogenic assay.
Criteria for acceptance
The following acceptance criteria were used to determine if the cleaning method was successful:
- Concentration of each viable challenging organism used in the common load media should be > 106 CFU/mL in Inoculum (L), Post-infection sample (M), and Pre-cleaning sample (N).
- Post-cleaning sample (J) should contain 0 CFU/mL of the challenge organism.
- Endotoxin sample (K) should contain ≤ 0.5 EU/mL.
- Sample points 1 through 29 should contain 0 CFU/unit of the challenge organism.
- Control samples should confirm that methods, materials, and handling procedures are functional.
- A maximum of 10% of the sampling points 1 through 29 can contain other contaminants than the challenge organism (3 samples in total).