Cleaning Polyethersulfone Membranes After Ultrafiltration-Diafiltration in Monoclonal Antibody Production

April 1, 2006
Amal Ahmed

Amal Ahmed is bioprocess associate III at HGSI purification sciences department.

,
Ruta Waghmare

Ruta Waghmare is group leader, process development sciences at Millipore Corporation, Billerica, MA 01821.

,
David Kahn

David kahn is director of purification sciences department of Human Genome Sciences, Inc. (HGSI).

,
Yaling Wu

Yaling Wu is senior scientist in purification sciences department at Human Genome Sciences, Inc.

Volume 19, Issue 4

In the pharmaceutical industry, ultrafiltration (UF) membranes are used extensively in the downstream purification of recombinant proteins or monoclonal antibodies. However, the fouling of membranes after a unit operation?especially when recombinant proteins or monoclonal antibodies are highly concentrated?is a common problem. Typically, normalized water permeability (NWP) of a membrane can be reduced to about 20 percent of its original permeability at the end of an ultrafiltration-diafiltration (UF-DF) operation.

In the pharmaceutical industry, ultrafiltration (UF) membranes are used extensively in the downstream purification of recombinant proteins or monoclonal antibodies. However, the fouling of membranes after a unit operation—especially when recombinant proteins or monoclonal antibodies are highly concentrated—is a common problem. Typically, normalized water permeability (NWP) of a membrane can be reduced to about 20 percent of its original permeability at the end of an ultrafiltration-diafiltration (UF-DF) operation.

Yaling Wu

Several reports describe membrane cleaning procedures using acid, alkali, high temperature, enzymes, and hypochlorite.1-6 These methods, which often involve extensive cleaning with a series of chemical reagents, are time-consuming, unsafe, or expensive. For example, using NaOH combined with high temperature is effective for cleaning the membranes,2,6 but poses safety concerns in the laboratory and in manufacturing operations. Enzymatic cleaning is relatively safe, but can be expensive.5,6 More recently, an abstract by Davis and Abraham described the use of NaOH and hypochlorite for UF membrane cleaning, but it contained limited detailed information.7 Consequently, there is a great need to develop an effective, safe, economical, and easily scalable cleaning method for the UF-DF operation.

Manufacturers often choose polyethersulfone (PES) membranes for applications requiring high-flux, low-to-moderate protein binding, and high resistance to chemical cleaning and sanitization solutions. This article describes an effective, safe, and economic approach for cleaning a PES membrane at ambient temperature, using a scale-down simulation of the antibody UF-DF process with a cleaning solution of 250 ppm sodium hypochlorite in 0.5 M NaOH. The solution is fairly generic and can be used on most PES membranes. We show the results after 10 cycles of cleaning the membrane post UF-DF, in which the water permeability of the membrane was maintained. We also present total organic carbon (TOC), residual chlorine content, and process flux test data to demonstrate the effectiveness of cleaning and the ability to wash out the cleaning reagent.

MATERIALS AND METHODS

Ultrafiltration Modules

This study was carried out by using two PES Biomax 30kD cassettes with C-screen (0.01 m2 total area) in a Pellicon XL Labscale system (Millipore Corporation, Billerica, MA) with an external peristaltic pump. The Pellicon XL devices were chosen to conserve the amount of protein and buffers needed for the trials. The experimental set-up is shown in Figure 1. The cleaning method and operation conditions for the study are shown in Table 1 and a UF-DF cleaning process flow diagram, together with controls for cleaning effectiveness, is shown in Figure 2.

Figure 1. Experimental set-up

RESULTS AND DISCUSSION

The parameters used to measure the effectiveness of cleaning were NWP, process flux, TOC, and residual chlorine content.

Figure 2. Ultrafiltration-diafiltration cleaning process. Cleaning solution: 250 ppm NaOCl in 0.5 M NaOH. Storage solution: 0.1 N NaOH

Normalized Water Permeability

NWP is an established method for determining the cleanliness of a membrane cassette after cleaning. The method involves measuring the passage of clean water through the membrane under standard pressure and temperature conditions (Table 1). The rate of clean water flux through the membrane is measured as liters per meter squared per hour (LMH). Water flux divided by the transmembrane pressure (TMP) is the NWP (LMH/psi).

Table 1. UF-DF cleaning and operation condition parameters

As shown in Table 2, after the membrane was used in the UF-DF process, it was reduced to about 20 percent of the pre-process NWP. Previous experiments showed that cleaning the membrane with 0.5N NaOH was not sufficient to recover the initial water permeability of the membrane (data not shown). Therefore, the authors used a solution of 0.5N NaOH with 250 ppm hypochlorite solution in an attempt to recover the permeability of the membrane.

Table 2. Normalized water permeability of the membrane before and after ultrafiltration-diafiltration

Figure 3 shows NWP levels of a membrane following results from 10 cycles of a UF-DF step in a monoclonal antibody process, with cleaning after each use with 250 ppm hypochlorite in 0.5M NaOH at room temperature. The results in Table 2 show that after the 10 runs, the NWP was recovered to an average of 91 percent of the initial, pre-use value.

Figure 3. Results of normalized water permeability after cleaning the membrane post ultrafiltration-diafiltration

Process Flux (measure of process reproducibility)

The goal of a cleaning cycle is to ensure consistent process performance. Process flux is another measure of how well the membrane has been cleaned. The UF-DF process throughout the 10 cycles was carried out by maintaining the membrane load at 250 g monoclonal antibody/m2; the TMP at 17–20 psi, and the concentration at diafiltration, at 40 g/L. The final, target protein concentration and product recovery method were kept constant. The authors expected that if the cleaning method was effective, the process flux would remain relatively constant. The process flux was calculated by the following equation:

Figure 4. Results of process flux for ten cycles

{total permeate volume (L)}/ (total time (h))(filter area).

Figure 5 shows that the process flux for the 10 cycles remained fairly constant. The average process flux was approximately 45 LMH, although run four had a lower flux, which may have resulted from the load sample's lower temperature at the start of the experiment (data not shown).

Figure 5. Total organic carbon content after cleaning

Total Organic Carbon

The membrane cassette and the UF-DF system should be flushed with water after cleaning to displace cleaning and storage solutions and to remove residual process material. TOC measurements of the permeate and the retentate flush streams provide a reliable and highly sensitive means of detecting organic contamination in the system that may arise from product carryover resulting from poor cleaning procedures. In addition, a mock UF-DF run, containing no protein in the load, was performed after the normal cleaning procedure had been implemented following the tenth UF-DF cycle. A mock pool was recovered and analyzed for TOC. The results, shown in Figure 4, indicate that the TOC was less than 1,000 ppb (1.0 ppm) for all the runs except for runs two and four. The higher TOC content in runs two and four was likely because of the holding of the flush at room temperature for two weeks until the test could be performed.

The TOC content of the mock run flushes of the retentate–permeate flush was 255 ppb, less than the 500 ppb USP limit for water for injection (WFI). This result indicates that the cleaning protocol was very effective in removing any residual monoclonal antibody contamination.

Residual Total Chlorine Content

Table 3 summarizes the residual total chlorine content measured in the pre-use water flush of the cassettes for the 10 cycles. These results demonstrate that total chlorine is reduced adequately by the pre-use flush procedures that were implemented in the studies.

Table 3. Chlorine content after cleaning the ultrafiltration-diafiltration system

CONCLUSIONS

This study evaluated a cleaning procedure for polyethersulfone membranes using a Millipore Biomax 30kD membrane. The results showed that the cleaning solution of 0.5N NaOH with 250 ppm NaOCl at room temperature was effective in cleaning the membrane, following execution of an ultrafiltration–diafiltration step in a monoclonal antibody process. One 30-min cycle of cleaning the membrane with 3.0 L of cleaning solution per square-meter membrane area was sufficient to recover the normalized water permeability to 90% of its initial value. This cleaning methodology also ensures reproducibility of process flux and results in low total organic carbon (< the USP limit for WFI), and low residual chlorine content in the subsequent UF-DF process step. These studies demonstrate the adequacy of the cleaning procedure for a PES membrane in a specific device. Although the authors anticipate that the procedure is broadly applicable, additional testing on other devices is needed to ensure compatibility with the non-membrane components of the specific device.

Terminology

ACKNOWLEDGEMENTS

The authors would like to acknowledge Sylvia Isaacson and Mani Krishnan from Millipore Corporation for their valuable technical discussions, and the QA/QC group at Human Genome Sciences, Inc., for their assistance with the TOC analysis.

Yaling Wu is senior scientist I in purification sciences department at Human Genome Sciences,Inc., 14200 Shady Grove Road, Rockville, MD 20850; 301.610.5790; yaling_wu@Hgsi.comAmal Ahmed is bioprocess associate III at HGSI purification sciences department. David Kahn is director of purification sciences department of HGSI. Ruta Waghmare is group leader, process development sciences at Millipore Corporation, Billerica MA 01821.

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