Sustainable cleaning of ÄKTA pilot™ 600 chromatography system

The ÄKTA pilot™ 600 chromatography system is designed for use in process development, scale-up and scale-down applications, in both GMP and non-GMP environments. Its front-facing design and snap connectors provide easy access to all components that require cleaning.

Precleaning of the ÄKTA pilot™ 600 chromatography system

We autoclaved the system tubing prior to use and sprayed interfaces between the system’s connection points and the tubing with 70% ethanol. CIP was then carried out using the recommended sustainable method. During the storage solution application phase, we used purified water (PW) instead of the recommended 10 mM NaOH. This substitution was made to prepare the system for the challenge procedure and to ensure that the cleaning agent, 1 M NaOH, was thoroughly flushed out.

Outlet tubes for ports 1 to 3 were fitted with sections of tubing covered in aluminum foil. This precaution was taken to reduce the risk of false positives during post-cleaning sample collection, which involves cutting the tubing with sterilized scissors.

Between the completion of the cleaning step and the start of the combined wash/storage step, we replaced all inlet tubes with freshly autoclaved tubes to prevent any residual 1 M NaOH from compromising the purity of the PW. We collected samples from both the cleaning solution and the PW prior to their application.

Preparation and application of the challenge organism suspension

We primed the precleaned system with 0.9% NaCl prior to the application of the challenge organism suspension (see experimental procedures in Table 1). Tryptic soy agar (TSA) plates streaked with the challenge organism were incubated at 37°C overnight. Fresh colonies from these plates were transferred to 200 mL of autoclaved tryptic soy broth (TSB) and incubated with shaking at 37°C overnight.

Calculations were performed to determine the volume of preculture required to be added to approximately 2700 mL of filtered 0.9% NaCl solution in order to achieve final concentrations of 10⁶ to 10⁸ CFU/mL in the challenge suspension. These calculations were based on the measured optical density (OD) of the preculture and the assumption that one OD corresponds to approximately 1.5 × 10⁸ colony-forming units (CFU)/mL for S. aureus.

We placed the inlet tubes of the system in a container filled with approximately 2700 mL of the challenge organism suspension, while the outlet tubes were placed in a separate container (see experimental procedures in Table 1). Upon completion of the procedure, a post-application sample was collected from the applied challenge suspension via Outlet Tube 1. The system was then left in contact with the challenge suspension for 16 to18 h.

Cleaning procedure

Prior to initiating the cleaning procedure, we collected a precleaning sample from the applied challenge suspension via Outlet Tube 1. Cleaning was then carried out using the recommended sustainable method. We collected samples of all liquids before their application to the system.

Upon completion of the cleaning method, we also collected three post-cleaning samples to evaluate the effectiveness of the procedure.

Microbial sampling

We took microbial samples at predetermined sites after the system was drained of fluids (see the swab sampling points in Table 4 and shown visually in Figure 2). Applied solutions on the system were also sampled (Table 1).

We then performed microbial sampling using one of the following methods:

1. Test method 1: sampling of air for airborne microorganisms with a microbial air sampler (MAS). We positioned an MAS loaded with an agar plate at a suitable measuring point. When the measuring started, a predefined volume of surrounding air passed through the machine. Microorganisms were collected on the agar surface by impaction.
2. Test method 2: bioburden filtration test for S. aureus. We collected sample solutions (minimum 50 mL) in sterile tubes and then filtered through 0.45 µm cellulose nitrate membrane filters. We then incubated filters on agar plates at 30°C to 35°C for 5 d after which the plates were inspected for CFUs.
3. Test method 3: swab test for S. aureus. Surface samples were taken with swabs. We inserted the swab into the tube containing the 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. The 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.
4. Test method 4: viable count test for S. aureus. We diluted samples of challenging organism suspensions 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 the challenge organism was determined in the sampled suspensions.

Criteria for acceptance

• Concentration of viable challenging organisms should be 10⁶ to 10⁸ CFU/mL in inoculum (K), post-application (L) and precleaning sample (M).
• Post-cleaning samples H, I and J should contain zero CFU/mL of the challenge organism.
• Sampling points 1 through 57 should contain zero 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 57 and H through J can contain contamination other than the challenge organism (six samples in total).

Tables 1 to 3 present the results from the cleaning studies, including data on challenge organism suspensions, sampling points, liquid samples, and control samples. Identified contaminations are listed by name in the tables. Sampling points related to swabs are visually illustrated in Figure 1, and additional information about each sampling point is provided in Tables 1 and 2.

The results from viable count plates of the liquid samples (K, L, and M), which contained the challenge suspension, are shown in Table 3. These plates confirmed that the S. aureus suspension was fully present prior to cleaning, with concentrations ranging from 10⁶ to 10⁸ CFU/mL, thereby meeting the predefined acceptance criteria. Microbial air samples indicated that the bioburden level in the laboratory air was within the normal range. All liquid samples A through G were free from contamination, indicating a low risk of false-positive results originating from these liquids (Table 4). Control samples collected during the study demonstrated that the experimental procedures and materials used for evaluation functioned as intended, and all acceptance criteria were fulfilled (Table 5).

Post-cleaning samples (H–J), collected after the cleaning procedure, did not contain the challenge organism in either study 1 or study 2, thereby meeting the acceptance criteria. However, other contaminants were detected in samples H and J in study 1. All swab samples from both studies were free from the challenge organism, confirming successful cleaning. Nonetheless, other contaminants were found in four swab samples in study 1 and three in study 2. In study 1, contamination was detected in pump B1 and in the restrictor, while in study 2, it was found only in pump B1.

Of the 60 samples evaluated (57 swab samples and three post-cleaning samples H–J), a total of four swab samples and two post-cleaning samples in study 1, and three swab samples in study 2, contained contaminants other than the challenge organism. As the maximum allowable number of samples containing non-target contaminants is six, the acceptance criteria were met in both studies.

Calculation of CO₂-equivalent emissions (kg CO₂-eq) for the recommended and the sustainable methods

Table 1. Standard cleaning method

Table 2. Alternative sustainable cleaning method

There is a reduction in 1.591 kg CO₂-eq going from the standard cleaning method to the alternative sustainable cleaning method. This equates to a 76.67% reduction. The method evaluated in this study demonstrates that the challenge organism S. aureus can be effectively inactivated, while also achieving approximately 77% reduction in carbon dioxide emissions compared to the previously recommended method.

When using 10 mM NaOH as a storage solution, it is essential to clearly label the system’s rinsing vessels, as the chemical is corrosive and must be handled with appropriate safety precautions.

Conductivity measurements indicate that applying 10 mM NaOH after completing the cleaning block can effectively replace the use of 1 M NaOH as cleaning agent. This enables a seamless transition of the system into storage mode in a single step.

The alternative sustainable cleaning method we recommend has been updated with consideration for carbon footprint, flow distribution, and dynamic contact time within the wetted flow path.

By replacing the previously recommended 0.9% NaCl for rinsing and 20% ethanol for storage with a single-step process using 10 mM NaOH for both rinsing and transitioning the system into storage mode, the carbon footprint has been significantly reduced, which can help you meet your facility’s sustainability goals.

Three step sustainable cleaning process

• Step 1 – Rinsing: Rinse with water (approximately 9 L)
• Step 2 – Cleaning: Clean with 1 M NaOH (approximately 11 L)
• Step 3 – Transition to storage: Rinse and switch to 10 mM NaOH (approximately 11 L)

Preparations before starting the cleaning method

The pH probe should be replaced with a dummy. Prepare a tube to connect the manual valve of the air trap to the IP port on the column valve.

Step 1: Rinsing

Place all inlet tubes in the vessel containing water and all outlet tubes in the waste container. Manually rinse the pH cell with 10 mL of water using a syringe. Replace the liquid in both rinsing system vessels connected to the system with approximately 250 mL of water.

Method programming for the rinsing step in UNICORN™ control software

• Block 1: Draining of Air trap — Completely drain the Air trap. Remove any tubing from the manual valve on the Air trap and open the valve.
• Block 2: Set up the Air trap for automated cleaning — Connect a tube between the manual Air trap valve and the IP port on the Column valve. Open the manual Air trap valve.
• Block 3: Inlet wash — Each Inlet is flushed with 250 mL at 600 mL/min, alternating pumps (e.g., Inlet B6, A6, B5, A5,…).
• Block 4: Mixer wash — Flush the Mixer valve with 100 mL at 400 mL/min in dual-flow, both in-line and bypass.
• Block 5: Air trap wash — Flush “In-line” 40 mL at 500 mL/min; overfill 100 mL at 500 mL/min (Column: “Pack to Waste 2”; Air trap: “Fill”); then “In-line” 1300 mL at 900 mL/min; overfill 300 mL at 900 mL/min (same positions). Close the Air trap valve after Block 5.
• Block 6: Column valve wash — At 900 mL/min dual-flow: quick 100 mL (“Column 1 down to Column 2 downflow”), 100 mL (“Bypass both”); wash Column 1 and 2 with 100 mL each in “Downflow” and “Up flow”, repeat twice; flush all bypass positions (100 mL each), finish with “Bypass both”; flush “Waste 2” (200 mL).
• Block 7: Outlet wash — Flush each Outlet valve with 200 mL at 900 mL/min dual-flow, sequentially from Outlet 1 onward.

Step 2: Cleaning

Place all inlet tubes in 1 M NaOH and all outlet tubes in waste. Manually rinse the pH cell with 10 mL of 1 M NaOH. Replace both rinsing vessels with ~ 250 mL of 1 M NaOH. Before Block 6, place outlet and waste tubes into the same container as the inlet tubes.

Method programming for the cleaning step in UNICORN™ control software

• Block 1: Inlet wash — 250 mL per Inlet at 600 mL/min, alternating pumps (B6, A6, B5, A5,…).
• Block 2: Mixer wash — 100 mL at 400 mL/min in dual-flow, in-line and bypass.
• Block 3: Air trap wash — Prepare Air trap (connect tube, open valve). Then: “In-line” 40 mL at 500 mL/min; overfill 100 mL at 500 mL/min (Column: “Pack to Waste 2”; Air trap: “Fill”); “In-line” 1300 mL at 900 mL/min; overfill 300 mL at 900 mL/min (same positions). Close Air trap valve after Block 3.
• Block 4: Column valve wash — Same sequence as in rinsing step (see above).
• Block 5: Outlet wash — 200 mL per Outlet at 900 mL/min dual-flow.
• Block 6: Recirculation 1 h — Dual pumps at 900 mL/min for 1 h; switch all valves every 30 s (loop):
  • Inlet valves: open each for 30 s sequentially.
  • Air trap valve: alternate In-line, Bypass, and maintenance “Fill” (with Column “Pack to Waste 2”).
  • Mixer valve: alternate In-line 30 s/Bypass 30 s.
  • Column valve: alternate 30 s each among: “Column 1 down → Column 2 downflow”, “Column 2 down → Column 1 downflow”, “Bypass both”, and “Pack to Waste 2” (combined with Air trap “Fill”).
  • Outlet valves: open each for 30 s sequentially.

Step 3: Transition to storage

Replace inlet tubes with autoclaved ones. Place all inlet tubes in 10 mM NaOH; outlets to waste. Manually rinse the pH cell with 10 mL of 10 mM NaOH. Replace both rinsing vessels with ~ 250 mL of 10 mM NaOH.

Note: 10 mM NaOH is corrosive; bottles must be clearly labeled.

Method programming in UNICORN™ control software to prepare the system for storage

• Block 1: Initial wash of inlets
  Each inlet is flushed with 100 mL of liquid at a flow rate of 600 mL/min. Begin with the highest Inlet for each Pump and alternate between Pumps during the procedure, for example, B6, A6, B5, A5, and so on. Repeat Block 1 twice.
• Block 2: Washing of inlets
  Each Inlet is flushed with 250 mL of liquid at a flow rate of 600 mL/min. Begin with the highest Inlet for each Pump and alternate between the Pumps during the procedure, for example, B6, A6, B5, A5, and so on.
• Block 3: Mixer wash
  Flush the Mixer valve with 100 mL at a flow rate of 400 mL/min in dual-flow mode, both in the in-line position and in the bypass position.
• Block 4: Air trap wash
  In this step, the Air trap is prepared for automatic cleaning by connecting a tube between the manual Air trap valve and the IP port on the Column valve. The manual Air trap valve should then be opened. Begin by flushing the Air trap valve in the “In-line” position with 40 mL at a flow rate of 500 mL/min in dual-flow mode. Next, overfill the Air trap with 100 mL at 500 mL/min in dual-flow mode, using the Column valve in the maintenance position “Pack to Waste 2” and the Air trap valve in the maintenance position “Fill.” Then, set the Air trap to “In-line” and flush with 1300 mL at 900 mL/min in dual-flow mode. Finally, overfill the Air trap again with 300 mL at 900 mL/min in dual-flow mode, using the same valve positions as before: Column valve in “Pack to Waste 2” and Air trap valve in “Fill.” The Air trap valve should be closed after Block 4 has been completed.
• Block 5: Column wash
  Flush the Column valve at a flow rate of 900 mL/min in dual-flow mode. Begin with a quick initial rinse using 100 mL in the position “Column 1 down to Column 2 downflow,” followed by 100 mL in “Bypass both.” Continue by washing Column positions 1 and 2 with 100 mL each in both “Downflow” and “Up flow” positions, repeating the sequence twice in the order downflow, up flow, downflow, up flow. After this, flush all bypass positions with 100 mL each, ending with “Bypass both.” Conclude the procedure by flushing the “Waste 2” position with 200 mL.
• Block 6: Outlet wash
  Flush each Outlet valve with 200 mL at a flow rate of 900 mL/min in dual-flow mode. Begin with Outlet position 1 and continue sequentially through the remaining Outlet positions, opening and flushing each in the same manner.

Fig 2. Location of swab points from this study. 2A: Inlet valves for pump A and pump B. 2B: Pump A inlet and outlet of pump head. 2C: Pump B screws. 2D. Pump B ceramic cylinder. 2E. Restrictor inlets. 2F. Restrictor internal components. 2G. Air trap valve. 2H. Mixer. 2I. Air trap outlets. 2J. Air trap screw top. 2K. Air trap internal components. 2L. Column valve. 2M. Outlet valve modules 1 and 2. See Table 4 for the descriptions of each swab location.

Table 3. Results from air and liquid samples collected in study 1 and 2

^* The results from the air sampling with MAS100EX microbial air sampler have been converted to a more statistical relevant value by using the formula Pr = N (1/N + 1/(N-1) + 1/(N-2) + … 1/(N-r+1)) (Feller, 1950). Pr equals probable statistical total, and r equals the number of CFU counted on a 90 mm Petri dish. The conversion is based upon the principle that as the number of viable particles being impinged on an individual plate increases, the probability of the next particle going into an "empty hole" decreases.

Table 4. Locations for collecting swab samples 1 through 57

Table 5. Control samples

^* Too numerous to count (TNTC).

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