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Development of a Hydrodynamic Cleaning Cycle for Ultrafiltration / Diafiltration Processes used for Monoclonal Antibody Formulation Youngbin Baek, Deyu Yang, and Andrew L. Zydney Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b02608 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Industrial & Engineering Chemistry Research

Development of a Hydrodynamic Cleaning Cycle for Ultrafiltration / Diafiltration Processes used for Monoclonal Antibody Formulation Youngbin Baek, Deyu Yang, and Andrew L. Zydney*

Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802

Submitted to Industrial & Engineering Chemistry Research Special Issue, Richard Noble Festschrift

*E-mail: [email protected]

ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Ultrafiltration / diafiltration (UFDF) processes are used for final formulation of high value biotherapeutics like monoclonal antibodies. Membrane fouling and cleaning are both critical factors governing the performance of these processes, but there is very limited data on either of these phenomena, particularly at the high monoclonal antibody concentrations used in current UFDF systems. The objective of this study was to compare the effectiveness of different cleaning regimens for composite regenerated cellulose membranes in Pellicon 3 tangential flow filtration cassettes after UFDF of a monoclonal antibody product. The water permeability after UFDF was 20 ± 5% smaller than the clean membrane permeability due to fouling. Membranes could be successfully cleaned with 0.5 N NaOH, but this caused an increase in permeability and a loss in solute rejection due to an opening of the membrane pores. Cleaning with DI water was not very effective under normal flow conditions but provided very good recovery of water permeability when the flow was into the retentate exit port and out through the feed port, without any damage of the membrane as seen with NaOH. These results demonstrate the potential of using a hydrodynamic cleaning cycle without any aggressive cleaning chemicals for antibody fouled membranes.

Keywords: Ultrafiltration; TFF; Clean-in-place; Monoclonal antibody; Fouling


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INTRODUCTION Monoclonal antibody (mAb) therapeutics have revolutionized the treatment of cancer and many autoimmune diseases.1,2 The global market for mAbs now exceeds $100 billion per year, with hundreds of new antibody-based therapeutics currently in clinical trials. mAbs continue to be the primary driver of growth in the biopharmaceutical industry. The production of mAb products typically follows a platform process involving initial capture by Protein A affinity chromatography followed by multiple polishing steps (e.g., ion exchange and hydrophobic interaction chromatography), with final formulation by ultrafiltration (UF) and diafiltration (DF) using tangential flow filtration (TFF).3 Diafiltration is used to remove low molecular weight impurities and achieve the desired buffer for the formulated drug product while UF provides the targeted mAb concentration for storage and delivery.4 High concentration formulations (well above 100 g/L) are typically required to deliver the desired therapeutic dosage in a single subcutaneous injection.5 Commercial UFDF systems for mAb processing can employ several hundred square meters of membrane area to process a single product batch.6 The cost-effective application of such large TFF systems typically requires the development of appropriate membrane cleaning protocols to reduce the cost associated with the membranes / modules. In addition, the use of clean-in-place reduces the labor required for installation of the TFF cassettes and minimizes the risk of failure due to improper installation. Despite the importance of membrane cleaning in mAb processing, there have been relatively few quantitative studies of membrane fouling and cleaning during UFDF of mAb products, particularly at the very high concentrations currently employed in industry. Rosenberg et al.7 examined UF of a series of mAbs using TFF cassettes with Sartocon Slice flat sheet


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regenerated cellulose membranes. The modules were cleaned with 1 N NaOH between cycles to restore the water permeability to within 10% of the initial (clean) value. However, the focus of this work was on minimizing mAb aggregation, with no details provided on either the extent of fouling or any long-term changes in the membrane. Arunkumar et al.8 used 0.5 N NaOH to clean a series of TFF cassettes used for UFDF of several mAbs and an Fc-fusion protein, but again no details were provided on either the extent of fouling or the effectiveness of the cleaning. Binabaji et al.9 compared the performance of hollow fiber modules and TFF cassettes during UF of a mAb up to concentrations of more than 200 g/L. The flat sheet composite regenerated cellulose membranes were successfully cleaned with 0.3 N NaOH while the hollow fiber polyethersulfone membranes used 0.5 N NaOH, but no quantitative information was provided on the permeability before / after cleaning. The most detailed study of membrane fouling / cleaning during mAb ultrafiltration was performed by Hung et al.10 using an IgG1 mAb in different buffer formulations. Data were obtained up to mAb concentrations of 280 g/L using polyethersulfone MidiKros hollow fiber modules. The water permeability after the UF process decreased between 29 and 79% depending upon the buffer composition, which the authors attributed to differences in the degree of concentration polarization in the different buffers arising primarily from differences in mAb viscosity. The polyethersulfone hollow fibers were cleaned using 0.5 N NaOH for 30 min, although no details were provided on the effectiveness of the cleaning cycle. The objective of this study was to obtain quantitative data on membrane fouling and cleaning after UFDF of a highly concentrated mAb with the goal of developing a hydrodynamic (flow-based) cleaning process that would effectively restore the membrane permeability while minimizing the use of harsh cleaning chemicals. The results clearly demonstrate the potential of


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using appropriate hydrodynamic conditions to clean composite regenerated cellulose UF membranes in TFF cassettes fouled during mAb processes.

MATERIALS AND METHODS Materials. A highly purified monoclonal antibody (mAb) was provided by Bristol-Myers Squibb (Devens, MA). The mAb was initially in a 20 mM phosphate buffer with 58 mM NaCl at pH 7 at a concentration of 10 g/L. 20 mM histidine buffer with 250 mM sucrose at pH 6.5 was chosen as the diafiltration buffer. Histidine is a commonly used buffer for mAb formulation,11 and sucrose is effective at reducing mAb aggregation during storage.12 L-histidine, L-histidine HCl, and NaOH were purchased from Sigma Aldrich (St. Louis, MO). Sucrose was obtained from MP Biomedicals LLC (Santa Ana, CA). Pellicon 3 tangential flow filtration (TFF) cassettes with an internal D-Screen and 30 kDa UltracelTM composite regenerated cellulose UF membranes (MilliporeSigma Corp., Bedford, MA) were used in all UFDF experiments. The effective membrane area (A) was 88 cm2. Data were also obtained in a stirred ultrafiltration cell (Amicon 8200, Millipore Corp.) with 25 mm diameter disks cut from a large flat sheet of a 30 kDa UltracelTM membrane.

Ultrafiltration / Diafiltration. The Pellicon 3 cassettes were initially flushed with at least 500 mL of deionized (DI) water (NANOpure Diamond water purification system, Barnstead Themolyne Corp., Dubuque, IA) to remove any storage solution and thoroughly wet the membrane pores. The membrane water permeability was evaluated from the slope of data for


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the filtrate flux (Jv) as a function of the transmembrane pressure (TMP) using deionized (DI) water as the feed solution:

Lp =

Jv TMP

(1)

Ultrafiltration was performed in a batch concentration mode with the retentate recycled back to the feed reservoir while the permeate was continuously removed. The feed flow rate was fixed at 45 mL/min, which corresponds to a feed flux of 310 L/h/m2 where the feed flow rate has been normalized by the membrane area. The average transmembrane pressure (TMP) was kept constant at 100 kPa (15 psi) by adjusting a pressure regulator on the retentate exit. At very high protein concentrations, the pressure drop through the module became quite high due to the large viscosity of the mAb solution. Under these conditions, the pressure regulator on the retentate was fully opened, leading to a somewhat higher average TMP.13 Diafiltration was performed using the same flow rates / pressures as during UF but with DF buffer added at a flow rate equal to the permeate flow. The feed (retentate) volume was constant at 50 mL. 20 mM histidine buffer with 250 mM sucrose at pH 6.5 was used as the DF buffer. The UFDF process was performed in 3 steps: an initial UF to concentrate the mAb solution to 60 g/L, a 10-diavolume DF into the histidine buffer, and a final UF to achieve a mAb concentration of 180 g/L. Feed samples were taken periodically throughout the process for offline analysis of the mAb concentration based on the UV absorbance at 280 nm as measured using a DU530 UV-vis spectrophotometer (Beckman Instruments, Fullerton, CA). Filtrate flow rates were evaluated by timed collection at appropriate points throughout the UFDF experiment.


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Membrane Cleaning. At the end of the experiment, the TFF system and module were cleaned using 0.5 N NaOH or DI water, with the flow directed either in through the feed port (and out through the retentate exit) or in through the retentate exit port (and out through what is normally the feed port), in both cases with the permeate exit port kept closed. Details are provided in the text. Cleaned membrane cassettes were stored in 0.1 N NaOH at 4°C between experiments. The effectiveness of the membrane cleaning was evaluated from changes in the membrane permeability before / after cleaning. In addition, the rejection characteristics of the membrane were evaluated by filtration of a 40 kDa dextran (Sigma Aldrich, St. Louis, MO) at a constant filtrate flux of 3 µm/s (11 L/m2/h). The dextran was prepared as a 1 g/L solution in 50 mM phosphate buffer at pH 7. The dextran rejection coefficient was evaluated as:

R = 1−

Cp

(2)

CF

Dextran concentrations in the permeate (Cp) and feed (CF) were evaluated by size exclusion chromatography with a Superdex 200 column using a refractive index detector (GE Healthcare Life Sciences).

RESULTS AND DISCUSSION Ultrafiltration Behavior. Figure 1 shows typical data for the filtrate flux during a UFDF process beginning with a 10 g/L mAb solution in the phosphate / NaCl buffer and ending with a 180 g/L mAb solution in the histidine / sucrose buffer. Data are shown for 3 separate modules: one used for the initial UF, one used for the UF and DF, and one used for the full UF-DF-UF


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cycle. The filtrate flux decreased from 26 µm/s (94 L/m2/h) to 17 µm/s (61 L/m2/h) over the initial UF due to the increase in the mAb concentration (from 10 to 60 g/L). The filtrate flux continued to decrease slightly during the DF into the histidine / sucrose buffer, and then decreased dramatically to only 2 µm/s (7 L/m2/h) in the final UF as the mAb concentration increased to 180 g/L.

Figure 1. Filtrate flux behavior during UFDF. Feed: 10 g/L mAb in 20 mM phosphate buffer with 58 mM NaCl at pH 7. DF buffer was 20 mM histidine with 250 mM sucrose at pH 6.5. Process involved an initial UF that concentrated the mAb to 60 g/L (UF-1), followed by a 10-diavolume DF, and then a final UF to 180 g/L (UF-2).

Most of the flux decline seen in Figure 1 is due to the increase in mAb concentration and the corresponding increase in the osmotic pressure and viscosity of the mAb solution as discussed elsewhere.9,13,14 The slight decline in flux during the DF was due to the increase in solution viscosity and the reduction in mAb diffusivity associated with the sucrose. In order to


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determine the contribution from membrane fouling, the water permeabilities before and after cleaning were evaluated after the initial UF, after the DF, and after the final UF, with results summarized in Figure 2 as the ratio of the permeability after the particular step in the UFDF process divided by the permeability of the fresh (clean) membrane. In each case, the membrane was first rinsed thoroughly with DI water to remove any labile protein from the TFF cassette. Membrane cleaning was performed with 0.5 N NaOH for 30 min. The permeability of the membrane declined by only 4% after UF-1, with a much greater loss in permeability seen after the DF and UF-2 due to the higher mAb concentrations in these steps, possibly in combination with the effects of the histidine / sucrose buffer. The final loss of permeability was 20 ± 5% based on results for several repeat experiments, which is considerably less fouling than was seen by Hung et al.10 after ultrafiltration of a mAb through polyethersulfone hollow fiber modules. This difference is likely related to differences in both the mAb and the filtration conditions in these studies. For example, Hung et al.10 found only a 29% decline in water permeability when using a buffer containing a mixture of histidine, trehalose, and citric acid compared to more than a 70% decline in permeability with the same mAb when using a buffer containing 10 mM histidine, 4% mannitol, and 0.1% Tween-80. Cleaning of the Pellicon 3 modules with 0.5 N NaOH for 30 min provided almost complete recovery of the initial water permeability (100 g/L) currently of interest in bioprocessing. This paper presents data examining the effects of NaOH and hydrodynamics on the cleaning of composite regenerated cellulose (Ultracel) TFF cassettes fouled during a typical UFDF process that would be used to formulate a highly concentrated mAb solution in a histidine / sucrose buffer. The water permeability after the UFDF process was approximately 20% less than that of the clean membrane, which is significantly less fouling than seen by Hung et al.10 in their UF experiments using hollow fiber polyethersulfone membranes. This reduced fouling is likely due to the greater hydrophilicity of the Ultracel membranes3. Exposure of the membranes to NaOH caused a significant increase in the membrane permeability and a corresponding reduction in the rejection of a 40 kDa dextran, both consistent with a significant increase in the effective membrane pore size. NaOH is well known to cause hydrolysis of cellulose acetate, leading to a reduction in the number of acetate groups on the cellulose backbone.17 NaOH can also catalyze the conversion of the hydroxyl groups on the glucose monomer to carboxylic acids, which would be expected to cause an increase in the negative charge and possible swelling of the polymer.18 Additional studies will be required to identify the underlying chemical modification of the regenerated cellulose that results in the initial decrease in the membrane permeability seen in Figure 3 followed by the significant increase in pore size upon longer term exposure to NaOH.


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In order to avoid the use of NaOH, a series of cleaning studies were performed using just DI water as the cleaning solution. The cleaning efficiency was significantly increased when using flow in the reverse direction (into the module from retentate exit and out through the feed inlet) at high flow rates. The reverse flow likely provided better removal of deposited protein from the membrane and from regions in the spacer that were more difficult to access with flow entering from the feed port. A 60 min cleaning cycle using DI water at a flow rate of 400 L/h/m2 was able to restore the membrane permeability to 99% of the clean (fresh) permeability after a single UFDF and maintained the permeability to within 10% of the clean membrane permeability throughout a cycle of 5 UFDF processes, without the use of any harsh cleaning chemicals. In addition to cleaning, NaOH also provides sanitization, which would be lost when cleaning with DI water. Boschetti et al.19 have shown that 0.1 N NaOH provides effective inactivation of MVM after only 1 min of exposure, indicating that it may well be possible to use very short time NaOH exposure for sanitization. Alternatively, one could use a completely different sanitization strategy. For example, Rogers et al.20 showed that a solution of phosphoric acid, acetic acid, and benzyl alcohol provided very effective sanitization of Protein A affinity resins. Further study will be required to investigate the effectiveness of this “hydrodynamic” cleaning method using larger scale commercial membrane cassettes as well as available options to achieve the required bioburden reduction / sanitization.

ACKKNOWLEDGMENTS The authors would like to acknowledge Bristol-Myers Squibb for their donation of the monoclonal antibody used in this work and for their financial support of this project.


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FIGURE CAPTIONS Figure 1. Filtrate flux behavior during UFDF. Feed: 10 g/L mAb in 20 mM phosphate buffer with 58 mM NaCl at pH 7. DF buffer was 20 mM histidine with 250 mM sucrose at pH 6.5. Process involved an initial UF that concentrated the mAb to 60 g/L (UF-1), followed by a 10-diavolume DF, and final UF to 180 g/L (UF-2). Figure 2. Normalized water permeability both before and after cleaning with 0.5 N NaOH for a Pellicon 3 cassette after the initial UF, the DF, and final UF for the process examined in Figure 1. Figure 3. Effect of NaOH on the membrane permeability (top) and rejection coefficient of a 40 kDa dextran (bottom) as a function of exposure time to 0.5 and 1 N NaOH. Figure 4. Efficiency of different cleaning protocols for membranes fouled during the UFDF process. Cleaning was done with DI water or 0.5 N NaOH for 30 min using a feed flux of 300 L/h/m2 directed either from feed inlet to retentate exit (standard) or from retentate exit back through the feed port (reverse). Figure 5. Cleaning efficiency using DI water at a feed flux of 300 L/h/m2 as a function of the cleaning time with flow in both the standard and reverse (retentate exit to feed inlet) flow configurations. Figure 6. Cleaning efficiency with different feed flux using DI water for 30 min in both the standard and reverse (retentate exit to feed inlet) flow configurations. Figure 7. Normalized water permeabilities for five repeat cycles involving UFDF of the mAb solution followed by cleaning with DI water using a cleaning time of 60 min, feed flux of 400 L/h/m2, and reverse flow configuration.


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Development of a Hydrodynamic Cleaning Cycle for

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