High-performance countercurrent membrane purification for host cell protein removal from monoclonal antibody products

The transition to continuous biomanufacturing has led to renewed interest in alternative approaches for downstream processing of monoclonal antibody (mAb) products. In this study, we examined the potential of using high-performance countercurrent membrane purification (HPCMP) for the removal of host cell proteins (HCPs) derived from Chinese Hamster Ovary cells in the purification of a mAb. Initial studies used several model proteins to identify appropriate operating conditions for the hollow fiber membrane modules. HPCMP was then used for mAb purification, with mAb yield >95% and more than 100-fold reduction in HCP. Stable operation was maintained for 48 h for feeds that were first prefiltered through the 3M^TM Harvest RC chromatographic clarifier to remove DNA and other foulants. In addition, the Process Mass Intensity for HPCMP can be much less than that for alternative HCP separation processes. These results highlight the potential of using HPCMP as part of a fully continuous mAb production process.     

Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy

Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m² for X0HC and from 105 to 47 L/m² for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%–50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.     

Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography

Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.     

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

Evaluation of membranes for use in on-line cell separation during mammalian cell perfusion processes

In this study two microporous hollow fibre membranes were evaluated for their use as cell retention device in continuous perfusion systems. A chemically modified permanent hydrophillic PTFE membrane and a hydrophilized PP membrane were tested. To investigate the filtration characteristics under process conditions each membrane was tested during a long term perfusion cultivation of a hybridoma cell line. In both cultivations the conditions influencing membrane filtration (e.g. transmembrane flux) were kept constant. Filtration behaviour was investigated by monitoring transmembrane pressure and protein permeability. Transmembrane pressure was measured on-line with an autoclavable piezo-resistive pressure sensor. Protein permeability was determined by quantitative evaluation of unreduced, Coomassie stained SDS-PAGE. The membrane fouling process influences the filtration characteristics of both membranes in a different way. After fermentation the PP membrane was blocked by a thick gel layer located in the big outer pores of the asymmetric membrane structure. The hydraulic resistance was higher but the protein permeability was slightly better than of the PTFE membrane. For this reason the PP membrane should be preferred. On the other hand, transmembrane pressure decreases slower when the PTFE membrane is used, which favours this membrane for long term cultivations, especially when low molecular weight proteins (<30 KD) are produced.Abbreviations PP Polypropylene - PTFE Polytetrafluoroethylene     

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two‐dimensional difference gel electrophoresis (2D‐DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D‐DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D‐DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample. Biotechnol. Bioeng. 2010; 105: 306–316. © 2009 Wiley Periodicals, Inc.     

Comparison of host cell protein removal by depth filters with diatomaceous earth and synthetic silica filter aids using model proteins

A number of studies have demonstrated that depth filtration can provide significant adsorptive removal of host cell proteins (HCP), but there is still considerable uncertainty regarding the underlying factors controlling HCP binding. This study compared the binding characteristics of two fine grade depth filters, the X0SP (polyacrylic fiber with a synthetic silica filter aid) and X0HC (cellulose fibers with diatomaceous earth (DE) as a filter aid), using a series of model proteins with well-defined physical characteristics. Protein binding to the X0SP filter was dominated by electrostatic interactions with greatest capacity for positively-charged proteins. In contrast, the X0HC filter showed greater binding of more hydrophobic proteins although electrostatic interactions also played a role. In addition, ovotransferrin showed unusually high binding capacity to the X0HC, likely due to interactions with metals in the DE. Scanning Electron Microscopy with Energy Dispersive Spectroscopy was used to obtain additional understanding of the binding behavior. These results provide important insights into the physical phenomena governing HCP binding to both fully synthetic and natural (cellulose + DE) depth filters.     

Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process

An increasing number of non-mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log₁₀ reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.     

Host cell protein dynamics in the supernatant of a mAb producing CHO cell line

The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile.     

Efficient solubilization of inclusion bodies

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.     

Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions,LC-MS/MS,and multivariate analysis

Host cell protein (HCP) impurities are generated by the host organism during the production of therapeutic recombinant proteins, and are difficult to remove completely. Though commonly present in small quantities, if levels are not controlled, HCPs can potentially reduce drug efficacy and cause adverse patient reactions. A high resolution approach for thorough HCP characterization of therapeutic monoclonal antibodies is presented herein. In this method, antibody samples are first depleted via affinity enrichment (e.g., Protein A, Protein L) using milligram quantities of material. The HCP-containing flow-through is then enzymatically digested, analyzed using nano-UPLC-MS/MS, and proteins are identified through database searching. Nearly 700 HCPs were identified from samples with very low total HCP levels (< 1 ppm to ～10 ppm) using this method. Quantitation of individual HCPs was performed using normalized spectral counting as the number of peptide spectrum matches (PSMs) per protein is proportional to protein abundance. Multivariate analysis tools were utilized to assess similarities between HCP profiles by: 1) quantifying overlaps between HCP identities; and 2) comparing correlations between individual protein abundances as calculated by spectral counts. Clustering analysis using these measures of dissimilarity between HCP profiles enabled high resolution differentiation of commercial grade monoclonal antibody samples generated from different cell lines, cell culture, and purification processes.     

Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions,LC-MS/MS,and multivariate analysis

Host cell protein (HCP) impurities are generated by the host organism during the production of therapeutic recombinant proteins, and are difficult to remove completely. Though commonly present in small quantities, if levels are not controlled, HCPs can potentially reduce drug efficacy and cause adverse patient reactions. A high resolution approach for thorough HCP characterization of therapeutic monoclonal antibodies is presented herein. In this method, antibody samples are first depleted via affinity enrichment (e.g., Protein A, Protein L) using milligram quantities of material. The HCP-containing flow-through is then enzymatically digested, analyzed using nano-UPLC-MS/MS, and proteins are identified through database searching. Nearly 700 HCPs were identified from samples with very low total HCP levels (< 1 ppm to ∼10 ppm) using this method. Quantitation of individual HCPs was performed using normalized spectral counting as the number of peptide spectrum matches (PSMs) per protein is proportional to protein abundance. Multivariate analysis tools were utilized to assess similarities between HCP profiles by: 1) quantifying overlaps between HCP identities; and 2) comparing correlations between individual protein abundances as calculated by spectral counts. Clustering analysis using these measures of dissimilarity between HCP profiles enabled high resolution differentiation of commercial grade monoclonal antibody samples generated from different cell lines, cell culture, and purification processes.     

Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies

The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the β-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins.     

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington’s disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.     

Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product

Chinese hamster ovary (CHO) cells are often used to produce therapeutic monoclonal antibodies (mAbs). CHO cells express many host cell proteins (HCPs) required for their growth. Interactions of HCPs with mAbs can sometimes result in co‐purification of trace levels of ‘hitchhiker’ HCPs during the manufacturing process. Purified mAb‐1 product produced in early stages of process optimization had high HCP levels. In addition, these lots formed delayed‐onset particles containing mAb‐1 and its heavy chain C‐terminal fragments. Studies were performed to determine the cause of the observed particle formation and to optimize the purification for improved HCP clearance. Protease activity and inhibitor stability studies confirmed that an aspartyl protease was responsible for fragmentation of mAb‐1 resulting in particle formation. An affinity resin was used to selectively capture aspartyl proteases from the mAb‐1 product. Mass spectrometry identified the captured aspartyl protease as CHO cathepsin D. A wash step at high pH with salt and caprylate was implemented during the protein A affinity step to disrupt the HCP–mAb interactions and improve HCP clearance. The product at the end of purification using the optimized process had very low HCP levels, did not contain detectable protease activity, and did not form particles. Spiking of CHO cathepsin D back into mAb‐1 product from the optimized process confirmed that it was the cause of the particle formation. This work demonstrated that process optimization focused on removal of HCPs was successful in eliminating particle formation in the final mAb‐1 product. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1360–1369, 2015     

Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 microm ceramic membrane successfully recovered the granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by in-fermenter Benzonase digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and resuspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations.     

Optimization of mass transfer for toxin removal and immunoprotection of hepatocytes in a bioartificial liver

This study was designed to determine optimal operating conditions of a bioartificial liver (BAL) based on mass transfer of representative hepatotoxins and mediators of immune damage. A microprocessor‐controlled BAL was used to study mass transfer between patient and cell compartments separated by a hollow fiber membrane. Membrane permeability (70, 150, or 400 kDa molecular weight cut‐off—MWCO), membrane convection (high: 50 mL/min; medium: 25 mL/min; low: 10 mL/min; diffusion: 0 mL/min), and albumin concentration in the cell compartment (0.5 or 5 g%) were considered for a total of 24 test conditions. Initially, the patient compartment contained pig plasma supplemented with ammonia (0.017 kDa), unconjugated bilirubin (0.585 kDa), conjugated bilirubin (0.760 kDa), TNF‐α (17 kDa), pig albumin (67 kDa), pig IgG (147 kDa), and pig IgM (900 kDa). Mass transfer of each substance was determined by its rate of appearance in the cell compartment. Membrane fouling was assessed by dextran polymer technique. Of the three tested variables (membrane pore size, convection, and albumin concentration), membrane permeability had the greatest impact on mass transfer (P < 0.001). Mass transfer of all toxins was greatest under high convection with a 400 kDa membrane. Transfer of IgG and IgM was insignificant under all conditions. Bilirubin transfer was increased under high albumin conditions (P = 0.055). Fouling of membranes ranged from 7% (400 kDa), 24% (150 kDa) to 62% (70 kDa) during a 2‐h test interval. In conclusion, optimal toxin removal was achieved under high convection with a 400‐kDa membrane, a condition which should provide adequate immunoprotection of hepatocytes in the BAL. Biotechnol. Bioeng. 2009; 104: 995–1003. © 2009 Wiley Periodicals, Inc.     

Host cell protein adsorption characteristics during protein a chromatography

Protein A chromatography is a critical and ‘gold‐standard’ step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI‐TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back‐bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D‐PAGE was then used to determine individual components associated with resin back‐bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1037–1044, 2012     

Anxa2- and Ctsd-knockout CHO cell lines to diminish the risk of contamination with host cell proteins

Chinese hamster ovary (CHO) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. While the majority of HCP impurities are effectively removed in typical downstream purification processes, clearance of a small population of HCP remains challenging. In this study, we knocked out the Anxa2 and Ctsd genes to assess the feasibility of knockout approaches for diminishing the risk of contamination with HCP. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were successfully established, and we confirmed the complete elimination of the corresponding HCP in cell lysates. Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type control during 8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the production of recombinant therapeutic proteins.     

Anaerobic submerged membrane bioreactor (AnSMBR) for municipal wastewater treatment under mesophilic and psychrophilic temperature conditions

A pilot scale anaerobic submerged membrane bioreactor (AnSMBR) with an external filtration unit for municipal wastewater treatment was operated for 100 days. Besides gas sparging, additional shear was created by circulating sludge to control membrane fouling. During the first 69 days, the reactor was operated under mesophilic temperature conditions. Afterwards, the temperature was gradually reduced to 20 °C. A slow and linear increase in the filtration resistance was observed under critical flux conditions (7 L/(m2 h)) at 35 °C. However, an increase in the fouling rate probably linked to an accumulation of solids, a higher viscosity and soluble COD concentrations in the reactor was observed at 20 °C. The COD removal efficiency was close to 90% under both temperature ranges. Effluent COD and BOD5 concentrations were lower than 80 and 25 mg/L, respectively. Pathogen indicator microorganisms (fecal coliforms bacteria) were reduced by log(10)5. Hence, the effluent could be used for irrigation purposes in agriculture.