Repurposing fed-batch media and feeds for highly productive CHO perfusion processes

Perfusion is a cell culture mode that is gaining popularity for the manufacture of monoclonal antibodies and their derivatives. The cell culture media supporting perfusion culture need to support higher cell densities than those used in fed-batch culture. Therefore, when switching from a fed-batch to a perfusion mode, a new medium need to be developed which supports high cell densities, high productivity, and favorable product quality. We have developed a method for deriving perfusion culture media based on existing fed-batch media and feeds. We show that we can obtain culture media that successfully support perfusion cultures in a single-use rocking bioreactor system at cell-specific perfusion rates below 25 pL⁻¹ cell⁻¹ day⁻¹. High productivities and favorable product quality are also achievable.     

Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality

During the development of a new drug product, it is a common strategy to develop a first-generation process with the aim to rapidly produce material for pre-clinical and early stage clinical trials. At a later stage of the development, a second-generation process is then introduced with the aim to supply late-stage clinical trials as well as market needs. This work was aimed at comparing the performance of two different CHO cell culture processes (perfusion and fed-batch) used for the production of a therapeutically active recombinant glycoprotein at industrial pilot-scale. The first-generation process was based on the Fibra-Cel packed-bed perfusion technology. It appeared during the development of the candidate drug that high therapeutic doses were required (>100mg per dose), and that future market demand would exceed 100 kg per year. This exceeded by far the production capacity of the first-generation process, and triggered a change of technology from a packed-bed perfusion process with limited scale-up capabilities to a fed-batch process with scale-up potential to typical bioreactor sizes of 15m(3) or more. The productivity per bioreactor unit volume (in product m(-3)year(-1)) of the fed-batch process was about 70% of the level reached with the first-generation perfusion process. However, since the packed-bed perfusion system was limited in scale (0.6m(3) maximum) compared to the volumes reached in suspension cultures (15m(3)), the fed-batch was selected as second-generation process. In fact, the overall process performance (in product year(-1)) was about 18-fold higher for the fed-batch compared to the perfusion mode. Data from perfusion and fed-batch harvests samples indicated that comparable product quality (relative abundance of monomers dimers and aggregates; N-glycan sialylation level; isoforms distribution) was obtained in both processes. To further confirm this observation, purification to homogeneity of the harvest material from both processes, followed by a complementary set of studies (e.g. full physico-chemical characterization, assessment of in vitro and in vivo bioactivity, comparative pharmacokinetics and pharmacodynamics studies in relevant species, etc.) would be required. Finally, this illustrates the need to fix the production process early during the development of a new drug product in order to minimize process conversion efforts and to shorten product development time lines.     

Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process

Controlled feeding of nutrient supplements to a cell culture to enhance monoclonal antibody productivity has been practiced widely in high-yield, fed-batch processes. In this study, a similar feeding concept has been applied to a perfused culture and evaluated for the effects on bioreactor productivity and product quality. Our experimental results show that, by using such a "controlled-fed perfusion" approach, the volumetric antibody productivity (antibody per liter per day) was significantly increased by nearly twofold over the perfusion process, and surpassed fed-batch and batch processes by almost tenfold. The substantial boost in the overall productivity is attributable primarily to the combined effects of increased cell density as well as reduced product dilution. Both were achieved through careful nutrient supplementation in conjunction with metabolite minimization. As the manufacturing process evolved from roller bottles to the controlled-fed perfusion bioreactor system, the immunoreactivity and the cDNA sequences of the antibody were well preserved. However, the product glycosylation distribution patterns did alter. The controlled-feed perfusion process demonstrated a unique encompassment of the advantages of fed-batch and perfusion methods; that is, high product concentration with high volume throughput. Therefore, it may be very suitable for large-scale production of monoclonal antibodies.     

Different fermentation processes produced variants of an anti-CD52 monoclonal antibody that have divergent in vitro and in vivo characteristics

The anti-CD52 antibody has already been approved for the treatment of patients with resistant chronic lymphocytic leukemia, relapsing-remitting multiple sclerosis, and has demonstrable efficacy against stem cell transplantation rejection. A CHO cell line expressing a humanized anti-CD52 monoclonal antibody (mAb-TH) was cultivated in both fed-batch and perfusion modes, and then purified. The critical quality attributes of these mAb variants were characterized and the pharmacokinetics (PK) properties were investigated. Results showed that the perfusion culture achieved higher productivity, whereas the fed-batch culture produced more aggregates and acid components. Additionally, the perfusion culture produced similar fucose, more galactose and a higher proportion of sialic acid on the anti-CD52 mAb compared to the fed-batch culture. Furthermore, the perfusion process produced anti-CD52 mAb had higher complement-dependent cytotoxicity (CDC) efficacy than that produced by the fed-batch culture, a result probably linked to its higher galactose content. However, antibody produced by fed-batch and perfusion cultures showed similar PK profiles in vivo. In conclusion, perfusion is a more efficient method than fed-batch process in the production of functional anti-CD52 monoclonal antibody. Product quality variants of anti-CD52 mAb were found in different cell culture processes, which demonstrated different physiochemical and biological activities, but comparable PK properties. Whether these observations apply to all mAbs await further investigation.     

Effect of process change from perfusion to fed-batch on product comparability for biosimilar monoclonal antibody

A fed-batch process for the production of biosimilar monoclonal antibody was developed. Since the brand product is produced by perfusion process, the impact of process change from perfusion to fed-batch on product quality and cell performance was evaluated. Perfusion culture was performed at 0.47–1.00 (v/v/d) perfusion rate by spin-filter method with 15–17 μm mesh. Culture parameters such as pH (6.8–7.2), dissolved oxygen (40–70% air saturation), temperature (37 °C) and agitation speed (250 rpm) were applied in both culture modes. In terms of cell performance, volumetric productivity increased 3.7 times while process performance increased 7.5 times in fed-batch culture due to 10 times higher scalability. Considering the glycosylation pattern and charge variants, no significant changes in product quality were observed upon process change, although intact IgG level slightly decreased in fed-batch mode. The change of production media showed more effect on glycosylation patterns than the operation in different culture modes. Furthermore, there were no differences in biological activity, including TNFα, FcγRIIIa, and C1q-binding affinity. Through a scale-up study from 3 L to 12,500 L, it was confirmed that cell performance and product quality could be maintained. In conclusion, product quality of the fed-batch process was comparable to that of the reference product.     

Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention: characterization of separation efficiency and impact of perfusion on product quality

Several small-scale Chinese hamster ovary (CHO) suspension cultures were grown in perfusion mode using a new acoustic filtration system. The separation performance was evaluated at different cell concentrations and perfusion rates for two different CHO cell lines. It was found that the separation performance depends inversely on the cell concentration and perfusion rate. High media flow rates as well as high cell concentrations resulted in a significant drop in the separation performance, which limited the maximal cell concentration achievable. However, packed cell volumes of 10% to 16% (corresponding to 3 to 6. 10(7) cells/mL) could be reached and were maintained without additional bleeding after shifting the temperature to 33 degrees C. Perfusion, up to 50 days, did not harm the cells and did not result in a loss of performance of the acoustic filter as often seen with other perfusion systems. Volumetric productivities in perfusion mode were 2- to 12-fold higher for two cell lines producing two different glycoproteins when compared to fed-batch or batch processes using the same cell lines. Product concentrations were in the range of 20% to 80% of batch or fed-batch culture, respectively. In addition, using the protease-sensitive product rhesus thrombopoietin, we could show that cultivation in perfusion mode drastically reduced proteolysis when compared to a batch culture without addition of protease inhibitors such as leupeptin.     

Fed‐batch and perfusion culture processes: Economic,environmental, and operational feasibility under uncertainty

This article evaluates the current and future potential of batch and continuous cell culture technologies via a case study based on the commercial manufacture of monoclonal antibodies. The case study compares fed‐batch culture to two perfusion technologies: spin‐filter perfusion and an emerging perfusion technology utilizing alternating tangential flow (ATF) perfusion. The operational, economic, and environmental feasibility of whole bioprocesses based on these systems was evaluated using a prototype dynamic decision‐support tool built at UCL encompassing process economics, discrete‐event simulation and uncertainty analysis, and combined with a multi‐attribute decision‐making technique so as to enable a holistic assessment. The strategies were compared across a range of scales and titres so as to visualize how their ranking changes in different industry scenarios. The deterministic analysis indicated that the ATF perfusion strategy has the potential to offer cost of goods savings of 20% when compared to conventional fed‐batch manufacturing processes when a fivefold increase in maximum viable cell densities was assumed. Savings were also seen when the ATF cell density dropped to a threefold increase over the fed‐batch strategy for most combinations of titres and production scales. In contrast, the fed‐batch strategy performed better in terms of environmental sustainability with a lower water and consumable usage profile. The impact of uncertainty and failure rates on the feasibility of the strategies was explored using Monte Carlo simulation. The risk analysis results demonstrated the enhanced robustness of the fed‐batch process but also highlighted that the ATF process was still the most cost‐effective option even under uncertainty. The multi‐attribute decision‐making analysis provided insight into the limited use of spin‐filter perfusion strategies in industry. The resulting sensitivity spider plots enabled identification of the critical ratio of weightings of economic and operational benefits that affect the choice between ATF perfusion and fed‐batch strategies. Biotechnol. Bioeng. 2013; 110: 206–219. © 2012 Wiley Periodicals, Inc.     

Improving product quality and productivity of bispecific molecules through the application of continuous perfusion principles

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12-day fed-batch process and an approximately 40-day high-density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15-fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed-batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity-capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity-determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds.     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Economics of fed-batch operation: a computer-aided approach

The widely anticipated economic potential of fed-batch operation was quantified for a therapeutic product by flowsheet simulation. A process for production of t-PA from CHO cells based on fed-batch operation was compared to a base process that operates in batch mode. Two cases of fed-batch operation were considered, case A, where the product concentration was assumed to be four times the concentration obtained with base process and case B, where the concentration was eight times. The simulator, Bioprocess Simulator (BPS) from Aspen Technology, reported the final bioreactor volume and the total amount of the continuous feeds added during the fed-batch operation. BPS was also used to simulate the downstream processes. Comparison of the economic performances of the processes revealed that return on investment (ROI) of the base process would increase by 112% by switching to case A fed-batch operation from batch mode. Case B, on the other hand, would result in an increase of 288%. The importance of downstream processing for recovery of high-value products became apparent from this study. A breakdown of equipment purchase cost showed that the contribution from the product recovery section increased from 62% for base case to 77% for case A as the product concentration increased by fed-batch operation. For a fixed recovery of 40%, contribution from the downstream section was found to decrease to 70% for case B compared to case A. It was concluded from the results that higher product concentration would not result in proportionate increase in ROI because of limitations in the recovery section. A sensitivity analysis was carried out on several uncertainties of the simulated fed-batch process.     

Analyzing the dynamics of cell growth and protein production in mammalian cell fed-batch systems using logistic equations

The logistic modeling approach was used to describe experimental viable cell density (X) and product concentration (P) data from two industrial fed-batch mammalian cell culture processes with maximum product concentrations in the 3.0-9.4 g/l range. In both cases, experimental data were well described by the logistic equations and the resulting specific growth rate and protein productivity profiles provided useful insights into the process kinetics. Subsequently, sensitivity equations for both the X and P models were analyzed which helped characterize the influence of model parameters on X and P time courses. This was augmented by conventional sensitivity analyses where five values of each model parameter, 25% apart, were used to generate X and P time courses. Finally, results from sensitivity analysis were used to simulate X and P time courses that were reflective of typical early- and late-stage fed-batch cell culture processes. Different combinations of the logistic model parameters were used to arrive at the same final product concentration demonstrating the ability of the logistic approach to describe the multitude of process paths that result in the same final product concentration. Overall, the capability of the logistic equations to well describe X and P data from fed-batch cultures, coupled with their ability to simulate the multitude of paths leading up to the desired cell density and product concentration profiles, make them a useful tool during mammalian cell fed-batch process development.     

13C metabolic flux analysis identifies limitations to increasing specific productivity in fed-batch and perfusion

Industrial cell culture requires substantial energy to generate protein. The protein generated is not only the product of interest (IgG in this case), but also the protein associated with biomass. Here, ¹³C-Metabolic Flux Analysis (¹³C-MFA) was utilized to compare the stationary phase of a fed-batch process to a perfusion process producing the same product by the same clone. The fed-batch process achieved significantly higher specific productivity, approximately 60% greater than the perfusion process. In spite of this, a general lack of difference was observed when globally comparing glycolysis, pentose phosphate pathway, and TCA cycle fluxes. In contrast, gross growth rate was significantly different, approximately 80% greater in the perfusion process. This difference was concealed by a significantly greater death rate in the perfusion process, such that net growth rates were both similar and near-zero. When considering gross growth rate and IgG specific productivity, total protein specific productivity (Biomass+ IgG) differed little, offering rationale for the observed central carbon pathway similarities. Significant differences were identified in anaplerotic branched-chain amino acid (BCAA) contributions by ¹³C-MFA. The perfusion process exhibited markedly higher (up to three times) BCAA catabolism, an observation often associated with increased death.     

High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: comparison to a batch Sf-9-BEV system

The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.     

Perfusion bioreactors for the production of recombinant proteins in insect cells

Conclusion High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates.For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.     

A protocol to transfer a fed-batch platform process into semi-perfusion mode: The benefit of automated small-scale bioreactors compared to shake flasks as scale-down model

Continuous processes such as perfusion processes can offer advantages compared to fed-batch or batch processes in bio-processing: improved product quality (e.g. for labile products), increased product yield, and cost savings. In this work, a semi-perfusion process was established in shake flasks and transferred to an automated small-scale bioreactor by daily media exchange via centrifugation based on an existing fed-batch process platform. At first the development of a suitable medium and feed composition, the glucose concentration required by the cells and the cell-specific perfusion rate were investigated in shake flasks as the conventional scale-down system. This lead to an optimized process with a threefold higher titer of 10 g/L monoclonal antibody compared to the standard fed-batch. To proof the suitability and benefit as a small-scale model, the established semi-perfusion process was transferred to an automated small-scale bioreactor with improved pH and dissolved oxygen control. The average specific productivity improved from 24.16 pg/(c*d) in the fed-batch process and 36.04 pg/c*d in the semi-perfusion shake flask to 38.88 pg/(c*d) in the semi-perfusion process performed in the controlled small-scale bioreactor, thus illustrating the benefits resulting from the applied semi-perfusion approach, especially in combination with controlled DO and pH settings. © 2019 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 35: e2757, 2019.     

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

ABSTRACT

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.     

Multi-stage high cell continuous fermentation for high productivity and titer

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.     

Mechanistic modeling of the loss of protein sieving due to internal and external fouling of microfilters

Fed‐batch and perfusion cell culture processes used to produce therapeutic proteins can use microfilters for product harvest. In this study, new explicit mathematical models of sieving loss due to internal membrane fouling, external membrane fouling, or a combination of the two were generated. The models accounted for membrane and cake structures and hindered solute transport. Internal membrane fouling was assumed to occur due to the accumulation of foulant on either membrane pore walls (pore‐retention model) or membrane fibers (fiber‐retention model). External cake fouling was assumed to occur either by the growth of a single incompressible cake layer (cake‐growth) or by the accumulation of a number of independent cake layers (cake‐series). The pore‐retention model was combined with either the cake‐series or cake‐growth models to obtain models that describe internal and external fouling occurring either simultaneously or sequentially. The models were tested using well‐documented sieving decline data available in the literature. The sequential pore‐retention followed by cake‐growth model provided a good fit of sieving decline data during beer microfiltration. The cake‐series and cake‐growth models provided good fits of sieving decline data during the microfiltration of a perfusion cell culture. The new models provide insights into the mechanisms of fouling that result in the loss of product sieving. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1323–1333, 2017     

Application of "oxygen uptake rate-amino acids" associated mode in controlled-fed perfusion culture

Controlled-fed perfusion, a new operation mode, which combines the advantages of fed-batch and perfusion, has been reported to enhance monoclonal antibody productivity. The aim of the present study was to further enrich this mode by an "oxygen uptake rate-amino acids (OUR-AA)" strategy in which the feeding of amino acids was controlled according to the variation of OUR during perfusion. And the effects of this strategy on bioreactor productivity and product quality were evaluated. Experimental results indicated that by using this "OUR-AA" approach in controlled-fed perfusion mode a high viable cell density of more than 1.9 x 10(7)cells/ml was achieved and the productivity of mAb reached 325 mg/l/d, which was significantly increased by nearly twofold over those of the perfusion and fed-batch process. The residual concentrations of selected amino acids were controlled at a relative steady level by OUR during the culture. The immunoreactivity and the purity of the antibody were well preserved as the culture process was evolving from flask to the controlled-fed perfusion mode. The primary application of "OUR-AA" approach in controlled-fed perfusion mode may present a novel control strategy to enhance the culture performance and to display the potential of this approach in automatic control field.