Perfusion Process Research Articles

Perfusion Process Intensification for Lentivirus Production Using a Novel Scale-Down Model.

Process intensification has become an important strategy to lower production costs and increase manufacturing capacities for biopharmaceutical products. In particular for the production of viral vectors like lentiviruses (LVs), the transition from (fed-)batch to perfusion processes is a key strategy to meet the increasing demands for cell and gene therapy applications. However, perfusion processes are associated with higher medium consumption. Therefore, it is necessary to develop appropriate small-scale models to reduce development costs. In this work, we present the use of the acoustic wave separation technology in combination with the Ambr 250 high throughput bioreactor system for intensified perfusion process development using stable LV producer cells. The intensified perfusion process developed in the Ambr 250 model, performed at a harvest rate of 3 vessel volumes per day (VVD) and high cell densities, resulted in a 1.4-fold higher cell-specific functional virus yield and 2.8-fold higher volumetric virus yield compared to the control process at a harvest rate of 1 VVD. The findings were verified at bench scale after optimizing the bioreactor set-up, resulting in a 1.4-fold higher cell-specific functional virus yield and 3.1-fold higher volumetric virus yield.

Experimental determination of maximum shear stress in Mobius® Breez perfusion microbioreactors and comparative analysis with stirred tank bioreactors

Perfusion processes have experienced increased popularity in recent years due to their ability to sustain high cell densities and productivities in biopharmaceutical production, offering advantages over traditional batch and fed-batch cultivation methods. The Mobius® Breez microbioreactor significantly reduces experimental effort by downsizing the classical volume of perfusion bioreactors to the mL range and thus represents a valuable tool for process development. However, miniaturization has raised questions regarding comparability with traditional bioreactors in terms of the physical environment, such as hydrodynamic shear stress. Therefore, the maximum hydrodynamic shear stress, cultivation performance, and membrane-wall contact were evaluated to elucidate the system's behavior. Findings reveal two distinct operational conditions, distinguished by the presence or absence of membrane-wall contact, resulting in varying levels of hydrodynamic stress. Conditions lacking membrane contact demonstrate stress levels within safe operating thresholds for CHO cells, while those involving membrane contact exceed these thresholds, potentially leading to cell damage. Through the identification of critical frequencies of membrane motion, this study offers insights for optimizing microbioreactor operation and enhancing overall bioprocess efficiency.

New Horizons in Nuclear Cardiology: Imaging of Peripheral Arterial Disease.

Lower extremity peripheral artery disease (PAD) is characterized by impairment of blood flow associated with arterial stenosis and frequently coexisting microvascular disease and is associated with high rates of morbidity and mortality. Current diagnostic modalities have limited accuracy in early diagnosis, risk stratification, preprocedural assessment, and evaluation of therapy and are focused on the detection of obstructive atherosclerotic disease. Early diagnosis and assessment of both large vessels and microcirculation may improve risk stratification and guide therapeutic interventions. SPECT and PET imaging have been shown to be accurate to detect changes in perfusion in preclinical models and clinical disease, and have the potential to overcome limitations of existing diagnostic modalities, while offering novel information about perfusion, metabolic, and molecular processes. This review provides a comprehensive reassessment of radiotracer-based imaging of PAD in preclinical and clinical studies, emphasizing the challenges that arise due to the complex physiology in the peripheral vasculature. We will also highlight the latest advancements, underscoring emerging artificial intelligence and big data analysis, as well as clinically relevant areas where the field could advance in the next decade.

Inflammatory Gene Expression in Livers Undergoing Ex Situ Normothermic Perfusion Is Attenuated by Leukocyte Removal From the Perfusate.

Ex situ normothermic perfusion (ESNP) is a method to evaluate and potentially recondition organs before transplantation. However, increased expression of inflammatory molecules, including by tissue-resident immune cells, may occur during the perfusion process, potentially negating the beneficial effects of perfusion. We used RNA sequencing to assess gene expression in 31 livers undergoing ESNP, including 23 donated after circulatory death (DCD) and 8 donated after brain death. In 7 DCD livers, a leucocyte filter was added to the circuit during perfusion. Biopsies were available for transcriptomic assessment in all cases at the start of perfusion and at varying time points postperfusion. During ESNP in DCD livers, we observed an increase in proinflammatory, profibrinolytic, and prorepair pathway genes. SERPINE1, encoding plasminogen activator inhibitor-1, was among the genes most significantly upregulated during perfusion in DCD livers, potentially promoting fibrin clot persistence in vasculature. We also found increased expression of monocyte and neutrophil recruiting chemokine and proinflammatory cytokine transcripts during ESNP, but several prorepair molecules, including thymic stromal lymphopoietin, were also upregulated. In both DCD and donation after brain death livers, interferon-gamma response genes were enriched, whereas oxidative phosphorylation genes decreased in organs with high perfusate alanine transaminase, a biomarker associated with adverse clinical outcomes. The inclusion of a leukocyte filter in the perfusion circuit mitigated the induction of inflammation/immune pathway genes during perfusion and was associated with enrichment in oxidative phosphorylation genes. Leukocyte removal during ESNP abrogates transcriptional changes that are associated with unfavorable clinical outcomes, potentially benefiting human livers undergoing ESNP.

A novel approach for perfusion process design based on a "Grey-Box" kinetic model.

Perfusion cell-culture mode has caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, perfusion-culture processes can support higher cell densities, higher productivities and longer process times. However, due to the inherent operational complexity and high running costs, the development and design of perfusion-culture processes remain challenging. Here, we present a model-based approach to design optimized perfusion cultures of Chinese Hamster Ovary cells. Initially, four batches of bench-top reactor continuous-perfusion-culture data were used to fit the model parameters. Then, we proposed the model-based process design approach, aiming to quickly find out the "theoretically optimal" operational parameters combinations (perfusion rate and the proportion of feed medium in perfusion medium) which could achieve the target steady-state VCD while minimizing both medium cost and perfusion rate during steady state. Meanwhile, we proposed a model-based dynamic operational parameters-adjustment strategy to address the issue of cell-growth inhibition due to the high osmolality of concentrated perfusion medium. In addition, we employed a dynamic feedback control method to aid this strategy in preventing potential nutrient depletion scenarios. Finally, we test the feasibility of the model-based process design approach in both shake flask semi-perfusion culture (targeted at 5 × 107 cells/ml) and bench-top reactor continuous perfusion culture (targeted at 1.1 × 108 cells/ml). This approach significantly reduces the number of experiments needed for process design and development, thereby accelerating the advancement of perfusion-mode cell-culture processes.

Enhancing protein productivities in CHO cells through adenosine uptake modulation – Novel insights into cellular growth and productivity regulation

Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch – a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.

High-density perfusion cultures of the marine bacterium Rhodovulum sulfidophilum for the biomanufacturing of oligonucleotides

Therapeutic oligonucleotides (ONs) are typically manufactured via solid-phase synthesis, characterized by limited scalability and huge environmental footprint, limiting their availability. Biomanufactured ONs have the potential to reduce the immunogenic side-effects, and to improve the sustainability of their chemical counterparts. Rhodovulum sulfidophilum was demonstrated a valuable host for the extracellular production of recombinant ONs. However, low viable cell densities and product titer were reported so far. In this work, perfusion cell cultures were established for the intensification of ON biomanufacturing. First, the perfusion conditions were simulated in 50 mL spin tubes, selected as a scale-down model of the process, with the aim of optimizing the medium composition and process parameters. This optimization stage led to an increase in the cell density by 44 % compared to the reference medium formulation. In addition, tests at increasing perfusion rates were conducted until achieving the maximum viable cell density (VCDmax), allowing the determination of the minimum cell-specific perfusion rate (CSPRmin) required to sustain the cell culture. Intriguingly, we discovered in this system also a maximum CSPR, above which growth inhibition starts. By leveraging this process optimization, we show for the first time the conduction of perfusion cultures of R. sulfidophilum in bench-scale bioreactors. This process development pipeline allowed stable cultures for more than 20 days and the continuous biomanufacturing of ONs, testifying the great potential of perfusion processes.

Biomass specific perfusion rate as a control lever for the continuous manufacturing of biosimilar monoclonal antibodies from CHO cell cultures.

Continuous manufacturing enables high volumetric productivities of biologics such as monoclonal antibodies. However, it is challenging to maintain both high viable cell densities and productivities at the same time for long culture durations. One of the key controls in a perfusion process is the perfusion rate which determines the nutrient availability and potentially controls the cell metabolism. Cell Specific Perfusion Rate (CSPR) is a feed rate proportional to the viable cell density while Biomass Specific Perfusion Rate (BSPR) is a feed rate proportional to the biomass (cell volume multiply bycell density). In this study, perfusion cultures were run at three BSPRs in the production phase. Low BSPR favored a growth arresting state that led to gradual increase in cell volume, which in turn led to an increase in net perfusion rate proportional to the increase in cell volume. Consequently, at low BSPR, while the cell viability and cell density decreased, high specific productivity of 55 pg per cell per day was achieved. In contrast, the specific productivity was lower in bioreactors operating at a high BSPR. The ability to modulate the cell metabolism by using BSPR was confirmed when the specific productivity increased after lowering the BSPR in one of the bioreactors that was initially operating at a high BSPR. This study demonstrated that BSPR significantly influenced cell growth, metabolism, and productivity in cultures with variable cell volumes.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to4.64×107TU mL-1in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38×107TU mL-1in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of>1×107TUmL-1day-1for up to 29 consecutive days in a non-optimized 5L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Improved sieving coefficient in perfusion cell culture with reduced effective filtration length of hollow fibers.

The hollow fiber filter is the primary cell-retention device used in high-density perfusion cell culture and often used in an alternating tangential flow (ATF) configuration. The limited commercially available diaphragm pumps for ATF prevent utilization of vertical space when scaling beyond 500 L. Stacking hollow fiber filters coupled with viscous cell culture imposes vacuum pressure exceeding facility capabilities. Additionally, the longer filter assembly increases the hold-up volume and exceeds the diaphragm pump's fluid exchange capacity. The conventional tangential flow filtration (TFF) configuration circumvents this issue by exchanging culture from the bioreactor and cell-retention device in a unidirectional recirculation loop; however, the increased filter length when scaled up exacerbates the TFF's inherent issue with product retention from Starling flow. Stacking commercially available 20 cm TFF filters to make up the similar single-module length TFF used for the platform 3 and 50 L perfusion process at 41.5 and 65 cm, respectively, attempts to reduce fouling caused by Starling flow. The permeate of a single-module filter is partitioned into short independent segments through serially stacked filters, each harvested separately. By partitioning the permeate, the sieving coefficient increased for both 3 and 50 L scales. Reduction of Starling flow was confirmed with lower total hydraulic membrane resistance throughout the culture. This work demonstrates a method for increasing sieving coefficient and filter capacity by stacking TFF filters with independent permeate streams.

Research on the Law of Coal Pillar Spontaneous Combustion and Fire Prevention and Control Technology.

In coal pillar fires, the fire source is hard to be detected and the adjacent goaf is extremely likely to be affected. Such fires would give rise to thermal and dynamic disasters in mines, further causing casualties and environmental disruption. In this study, with the coal pillar spontaneous combustion (CPSC) accident in Chahasu Coal Mine taken as the research object, the oxygen uptake and limit of oxygen concentration of CPSC were explored. Based on the research results, a similar model was constructed, where a control group was used to simulate the hazardous area of CPSC in a bid to investigate the law of CPSC. Moreover, the polymer colloid perfusion system was constructed and the drilling parameters and perfusion process parameters were determined, and practices concerning spontaneous fire control were carried out. Here are the conclusions: First, air leakage in the coal pillar may lead to coal spontaneous combustion due to the impact of rib spalling, threatening 2-6 m above the middle of the coal pillar at a shallow position. Second, coal pillar grouting, injecting polymer colloids for cooling, and coal pillar cement reinforcement prove to be effective ways to prevent CPSC fires and combat recombustion.

Investigation of fouling in perfusion cell culture processes using alternating tangential flow filtration

Microfiltration membranes are increasingly used to retain the cells inside bioreactors while continuous harvest of the desired biopharmaceutical occurs in perfusion processes. One method of microfiltration is Alternating Tangential Flow (ATF) filtration, which involves moving the cultivation broth tangentially back and forth across the membrane, which results in a backwash effect that reduces fouling. In this study, fouling was investigated with asymmetric polysulfone hollow fibers operated in ATF mode attached to a bioreactor producing a recombinant protein in Chinese Hamster Ovary (CHO) cells. Fouling was assessed through different approaches, including determination of critical flux using an improved flux-step method. Fouling was studied through measurements of Transmembrane Pressure (TMP), protein transmission, membrane pore size and staining of the membrane after operation to visualize the distribution of biological fouling inside the membrane. For critical flux determination, fluxes of up to 69 LMH were used without exceeding the critical flux. No sign of fouling was observed for the short-term (<3.5 h) critical flux experiment. However, during prolonged operation at 8.3 LMH the TMP jumped to 0.9–0.95 bar, indicating fouling. At this state, the protein transmission remains at the same high level (>88 %).

Initial Experience of NIR-II Fluorescence Imaging-Guided Surgery in Foot and Ankle Surgery

Optical imaging in the second near-infrared (NIR-II; 900–1880 nm) window is currently a popular research topic in the field of biomedical imaging. This study aimed to explore the application value of NIR-II fluorescence imaging in footandankle surgeries. A lab-established NIR-II fluorescence surgical navigation system was developed and used to navigate foot and ankle surgeries which enabled obtaining more high-spatial-frequency information and a higher signal-to-background ratio (SBR) in NIR-II fluorescence images compared to NIR-I fluorescence images; our result demonstrates that NIR-II imaging could provide higher-contrast and larger-depth images to surgeons. Three types of clinical applicationscenarios (diabetic foot, calcaneal fracture, and lower extremity trauma) were included in this study. Using the NIR-II fluorescence imaging technique, we observed the ischemic region in the diabetic foot before morphologicalalterations, accurately determined the boundary of the ischemic region in the surgical incision, and fully assessed the blood supply condition of the flap. NIR-II fluorescence imaging can help surgeons precisely judge surgical margins, detect ischemic lesions early, and dynamically trace the perfusion process. We believe that portable and reliable NIR-II fluorescence imaging equipment and additional functional fluorescent probes can play crucial roles in precision surgery.

GMP implementation of a hybrid continuous manufacturing process for a recombinant non-mAb protein-A case study.

Advances in manufacturing technology coupled with the increased potency of new biotherapeutic modalities have created an external environment where continuous manufacturing (CM) can address a growing need. Amgen has successfully implemented a hybrid CM process for a commercial lifecycle program. In this process, the bioreactor, harvest, capture column, and viral inactivation/depth filtration unit operations were integrated together in an automated, continuous module, while the remaining downstream unit operations took place in stand-alone batch mode. CM operations are particularly suited for so-called "high mix, low volume" manufacturing plants, where a variety of molecules are manufactured in relatively low volumes. The selected molecule fit this mold and was manufactured in a low-capital micro-footprint suite attached to an existing therapeutic production facility. Use of a hybrid process within an already operating facility required less capital and minimized complexity. To enable this hybrid CM process, an established fed-batch process was converted to a perfusion process with continuous harvest. Development efforts included both process changes and the generation of a novel cell line adapted to long-term perfusion. Chromatography resins were updated, and purification processes adapted to handle variable inputs due to the fluctuations in harvest titer from the lengthy production process. A novel automated single-use (SU) viral inactivation (VI) skid was introduced, which entailed the development of a robust pH verification and alarm system, along with procedures for product isolation to allow discard of specific cycles. The CM process demonstrated consistent performance, meaning it met predefined performance criteria (including product quality attributes, or PQAs) when operated within established process parameters and manufactured according to applicable procedures. Using a 75% reduction in scale, it resulted in a five-fold reduction in process media and buffer usage, a fifteen-fold increase in mass per thaw, and an overall process productivity increase of 45-fold (as measured by grams drug substance per liter per day.) The hybrid CM process also enabled increased material demand to be met with no change in cost of goods manufactured or plant capacity, due to the repurposing of existing facility space and the flexible duration of the hybrid CM harvest. Overall, the success of the hybrid CM platform represents an exciting opportunity to reduce costs and increase process efficiency in industry.

Application of Multivariate Data Analysis on Historical Recombinant Adenovirus Zoster Vaccine Production Data for Upstream Process Improvements

In recent years, multivariate data analysis (MVDA) has been widely used for process characterization and fault diagnosis in the biopharmaceutical industry. This study aims to investigate the feasibility of using MVDA for the development and scale-up of a perfusion process for HEK293 cell-based recombinant adenovirus zoster vaccine (Ad-HER) production. The Principal Component Analysis (PCA) results suggested comparable performance among the ATF, PATFP, and BFP perfusion systems in benchtop-scale stirred-tank bioreactor (STR). Then a Batch Evolution Model (BEM) was built using representative data from 10 L STR with a BFP system to assess the Ad-HER perfusion process performance at pilot-scale bioreactor (50 L STR and 50 L wave bioreactor). Furthermore, another BEM model and Batch Level Model (BLM) were built to monitor process parameters over time and predict the final adenovirus titer in 50 L wave bioreactor. The loading plot revealed that lactate dehydrogenase activity, viable cell diameter, and base-added during the virus production phase could be used as preliminary indicators of adenovirus yield. Finally, an adenovirus titer of 2.0±0.3×1010 IFU/mL was achieved in the 50 L wave bioreactor with BFP system, highlighting the robustness of the Ad-HER perfusion process at pilot-scale. Overall, this study emphasizes the effectiveness of MVDA as a tool for advancing the understanding of recombinant adenovirus vaccine perfusion production process development and scale-up.

Platform development for high-throughput optimization of perfusion processes-Part II: Variation of perfusion rate strategies in microwell plates.

The biopharmaceutical industry is replacing fed-batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high cell densities (HCD) and improved productivities. HCDs are achieved by cell retention and continuous medium exchange, which is often based on the cell-specific perfusion rate (CSPR). To obtain a cost-productive process the perfusion rate must be determined for each process individually. However, determining optimal operating conditions remain labor-intensive and time-consuming experiments, as investigations are performed in lab-scale perfusion bioreactors. Small-scale models such as microwell plates (MWPs) provide an option for screening multiple perfusion rates in parallel in a semi-perfusion mimic. This study investigated two perfusion rate strategies applied to the MWP platform operated in semi-perfusion. The CSPR-based perfusion rate strategy aimed to maintain multiple CSPR values throughout the cultivation and was compared to a cultivation with a perfusion rate of 1 RV d-1. The cellular performance was investigated with the dual aim (i) to achieve HCD, when inoculating at conventional and HCDs, and (ii) to maintain HCDs, when applying an additional manual cell bleed. With both perfusion rate strategies viable cell concentrations up to 50 × 106 cells mL-1 were achieved and comparable results for key metabolites and antibody product titers were obtained. Furthermore, the combined application of cell bleed and CSPR-based medium exchange was successfully shown with similar results for growth, metabolites, and productivities, respectively, while reducing the medium consumption by up to 50% for HCD cultivations.

Variability in perfusion conditions and set-up parameters used in ex vivo human placenta models: A literature review

The ex vivo human placenta perfusion model has proven to be clinically relevant to study transfer- and fetal exposure of various drugs. Although the method has existed for a long period, the setup of the perfusion model has not been generalized yet. This review aims to summarize the setups of ex vivo placental perfusion models used to examine drug transfer across the placenta to identify generalized properties and differences across setups. A literature search was carried out in PubMed September 26, 2022. Studies were labeled as relevant when information was reported, between 2000 and 2022, on the setups of ex vivo placental perfusion models used to study drug transfer across the placenta. The placenta perfusion process, and the data extraction, was divided into phases of preparation, control, drug, and experimental reflecting the chronological timeline of the different phases during the entire placental perfusion process. 135 studies describing an ex vivo human placental perfusion experiment were included. Among included studies, the majority (78.5%) analyzed drug perfusion in maternal to fetal direction, 18% evaluated bi-directional drug perfusion, 3% under equilibrium conditions, and one study investigated drug perfusion in fetal to maternal direction. This literature review facilitates the comparison of studies that employ similar placenta perfusion protocols for drug transfer studies and reveals significant disparities in the setup of these ex vivo placental perfusion models. Due to interlaboratory variability, perfusion studies are not readily comparable or interchangeable. Therefore, a stepwise protocol with multiple checkpoints for validating placental perfusion is needed.

An intelligent continuous bladder irrigation monitoring and control system: Development and preliminary assessment

Abstract Objectives: To design an intelligent continuous bladder irrigation monitoring and control system and preliminarily assess its sensitivity of the early warning of flushing anomalies. Methods: According to the principle of bladder irrigation, an intelligent continuous bladder irrigation monitoring and control system was designed. The Amesim fluid simulation software was used to establish a parameterized simulation model for the bladder perfusion process and simulate the dynamic changes of bladder pressure and volume under different perfusion speeds to assess the sensitivity of the early warning of flushing anomalies. Results: The intelligent system consisted of 3 parts: the monitoring part (a flushing fluid inlet flow monitoring device, a flushing fluid outlet flow monitoring device, and a flushing fluid outlet color recognition sensor), the transition part (a control motherboard), and the regulation part (an alarm device, an electric control device for flushing fluid input speed, and a squeezing device for flushing waste fluid line). The data of the parameterized simulation model for the bladder perfusion process suggested that the sensitivity of the system was acceptable. Conclusion: The intelligent bladder irrigation system can automatically detect, report, and resolve signal abnormalities, thereby achieving the closed-loop management of bladder irrigation. The intelligent system might be used in clinical settings to reduce the burdens of nurses and promote patients’ recovery.

Robust platform for inline Raman monitoring and control of perfusion cell culture.

Perfusion cell culture has been gaining increasing popularity for biologics manufacturing due to benefits such as smaller footprint, increased productivity, consistent product quality and manufacturing flexibility, cost savings, and so forth. Process Analytics Technologies tools are highly desirable for effective monitoring and control of long-running perfusion processes. Raman has been widely investigated for monitoring and control of traditional fed batch cell culture process. However, implementation of Raman for perfusion cell culture has been very limited mainly due to challenges with high-cell density and long running times during perfusion which cause extremely high fluorescence interference to Raman spectra and consequently it is exceedingly difficult to develop robust chemometrics models. In this work, a platform based on Raman measurement of permeate has been proposed for effective analysis of perfusion process. It has been demonstrated that this platform can effectively circumvent the fluorescence interference issue while providing rich and timely information about perfusion dynamics to enable efficient process monitoring and robust bioreactor feed control. With the highly consistent spectral data from cell-free sample matrix, development of chemometrics models can be greatly facilitated. Based on this platform, Raman models have been developed for good measurement of several analytes including glucose, lactate, glutamine, glutamate, and permeate titer. Performance of Raman models developed this way has been systematically evaluated and the models have shown good robustness against changes in perfusion scale and variations in permeate flowrate; thus models developed from small lab scale can be directly transferred for implementation in much larger scale of perfusion. With demonstrated robustness, this platform provides a reliable approach for automated glucose feed control in perfusion bioreactors. Glucose model developed from small lab scale has been successfully implemented for automated continuous glucose feed control of perfusion cell culture at much larger scale.

Platform development for high-throughput optimization of perfusion processes:Part I: Implementation of cell bleeds in microwell plates.

The promise of continuous processing to increase yields and improve product quality of biopharmaceuticals while decreasing the manufacturing footprint is transformative. Developingand optimizing perfusion operations requires screening various parameters, which is expensive and time-consuming when using benchtop bioreactors. Scale-down models (SDMs) are the most feasible option for high-throughput data generation and condition screening. However, new SDMs mimicking perfusion are required, enabling experiments to be run in parallel. In this study, a method using microwell plates (MWP) operating in semi-perfusion mode with an implemented cell bleed step is presented. A CHO cell line was cultivated in a 24-well MWP (Vw = 1.2 mL) and grown at four high cell density (HCD) setpoints. Quasi steady-state condition was obtained by manually performing cell bleeds followed by a total medium exchange after centrifugation. Further, two HCD setpoints were scaled up (VW = 30 mL), comparing a squared six-well deepwell plate (DWP) to shake flasks (SF). This evaluation showed comparable results between systems (DWP vs.SF) and scales (MWP vs.DWP + SF). The results show that the well-plate-based methods are suitable to perform HCD and quasi steady-state cultivations providing a robust solution to industrially relevant challenges such as cell clone and media selection.