Cell Culture Bioprocesses Research Articles

Enhanced cell growth, production, and mAb quality produced in Chinese hamster ovary-K1 cells by supplementing polyamine in the media.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing.

Determining appropriate input excitation for model identification of a continuous bio-process

This manuscript addresses the problem of determining input excitation for data driven model identification appropriate for cell culture bio-processes in general, and for an industrial bioreactor used for the production of monoclonal antibodies, in particular. The design space is set up to give us the operating parameters for the key objective of demonstrating the feasibility of using far more perturbations than typically done in bio process identification, although significantly less than other applications, to yield data rich enough for the purpose of data driven modelling (and subsequently, control). A proprietary mechanistic model developed by Sartorius for their Cellca cell line is first introduced to serve as a test bed, based on AMBR 250® (Sartorius registered trademark for integrated high throughput bioreactor systems). Subsequently, this test bed is used to address the question of determining the frequency of input perturbation sufficient to identify a data driven dynamic model. To this end, the test bed is used to generate data at various frequencies and a linear time invariant model identified. The predictive capability of the identified model is used to ascertain the frequency of changes in data generation such that the changes are acceptable from a biological standpoint, and yet generate sufficiently rich data. In particular, a frequency of perturbations at once every three days is found to balance these tradeoffs for the monoclonal antibody process under consideration. The results from the manuscript are meaningful both from a specific results standpoint (as illustrated by subsequent adoption by Sartorius), but also by providing a mechanism to ascertain such information for other bio-processes.

Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli.

Deriving new value from waste streams through secondary processes is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be recycled and used as a feed in secondary microbial fermentation to produce new recombinant protein products. Our results show that CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich microbiological media (LB). The titre of recombinant protein produced following induction in a 4-hour expression screen was approximately equivalent in the CDSM fed cultures to that of baseline, and this was maintained in a 16-hr preparative fermentation. To understand the protein production achieved in CDSM fed culture we performed a quantitative analysis of proteome changes in the E. coli using mass spectrometry. This analysis revealed significant upregulation of protein synthesis machinery enzymes and significant downregulation of carbohydrate metabolism enzymes. We conclude that spent cell culture media, which represents 100s of millions of litres of waste generated by the bioprocessing industry annually, may be valorized as a feed resource for the production of recombinant proteins in secondary microbial fermentations. Data is available via ProteomeXchange with identifier PXD026884.

Low-molecular-weight impurity in Poloxamer 188 responsible for atypical cell culture performance for mAb production

During a recent manufacturing campaign for a monoclonal antibody using a fed-batch process, poor cell culture performance was observed across two manufacturing sites with similar scales and equipment. Root cause analysis indicated that the poor cell culture performance was linked to the production basal media. Genealogy of the precursor raw materials used in the media revealed that a particular lot of Poloxamer 188 (P188) was the common link to the poor-performing media lots. P188 serves a critical role in protecting cells against shear in cell culture bioprocesses. However, the small-scale studies suggested that the poor cell culture performance was cytostatic in nature rather than being caused due to lack of shear protection. Several P188 lots were tested analytically using SEC-MS and RP-LC-MS methods and a unique low molecular weight species was identified in the suspect lot of poloxamer. The impurity was identified to be polypropylene oxide (PPO), a reaction intermediate in P188 synthesis. Spiking studies with PPO further confirmed its cytostatic nature. This case study highlights yet another scenario where lot-to-lot variability continues to impact bioprocesses and re-emphasizes the need for robust analytical and cell-culture raw material screening methods.

Producción de antraquinonas por cultivo in vitro de suspensiones celulares y raíces transformadas de Rubia Tinctorum: un modelo para la enseñanza en el estudio de bioprocesos por cultivo de tejidos de plantas

Plant cell culture bioprocesses constitute an excellent tool for the formation of undergraduate, magister, and Ph.D. students. In fact, students can acquire experience in the field of plant biochemistry and molecular biology; plant primary and secondary metabolism; and biochemical engineering. In the present manuscript, the production of anthraquinones in different plant cell tissue cultures is showed as easy and versatile model to introduce magister students in the field of plant secondary metabolite production. AQs are extracted with ethanol 80% and spectrophotometrically quantified, which also makes it environmentally friendly and easy to manipulate in the laboratory. The students were able to analyse biomass and product formation kinetics in batch suspension and hairy root cultures and evaluate the effect of MeJa elicitation an extended used strategy to trigger secondary metabolite production in plant cell cultures. Moreover, the performance of in situ product removal, which has been extensively used in biocatalyst and microbial fermentations, was evaluated. In summary, an inexpensive, accessible, and robust laboratory work is presented that can be adapted to undergraduate, magister, and/or Ph.D. students' courses in different plant biology, chemistry, and biochemical engineering programs.

Model Transferability and Reduced Experimental Burden in Cell Culture Process Development Facilitated by Hybrid Modeling and Intensified Design of Experiments.

Reliable process development is accompanied by intense experimental effort. The utilization of an intensified design of experiments (iDoE) (intra-experimental critical process parameter (CPP) shifts combined) with hybrid modeling potentially reduces process development burden. The iDoE can provide more process response information in less overall process time, whereas hybrid modeling serves as a commodity to describe this behavior the best way. Therefore, a combination of both approaches appears beneficial for faster design screening and is especially of interest at larger scales where the costs per experiment rise significantly. Ideally, profound process knowledge is gathered at a small scale and only complemented with few validation experiments on a larger scale, saving valuable resources. In this work, the transferability of hybrid modeling for Chinese hamster ovary cell bioprocess development along process scales was investigated. A two-dimensional DoE was fully characterized in shake flask duplicates (300 ml), containing three different levels for the cultivation temperature and the glucose concentration in the feed. Based on these data, a hybrid model was developed, and its performance was assessed by estimating the viable cell concentration and product titer in 15 L bioprocesses with the same DoE settings. To challenge the modeling approach, 15 L bioprocesses also comprised iDoE runs with intra-experimental CPP shifts, impacting specific cell rates such as growth, consumption, and formation. Subsequently, the applicability of the iDoE cultivations to estimate static cultivations was also investigated. The shaker-scale hybrid model proved suitable for application to a 15 L scale (1:50), estimating the viable cell concentration and the product titer with an NRMSE of 10.92% and 17.79%, respectively. Additionally, the iDoE hybrid model performed comparably, displaying NRMSE values of 13.75% and 21.13%. The low errors when transferring the models from shaker to reactor and between the DoE and the iDoE approach highlight the suitability of hybrid modeling for mammalian cell culture bioprocess development and the potential of iDoE to accelerate process characterization and to improve process understanding.

Cyclodextrins: a tool in plant cell and organ culture bioprocesses for the production of secondary metabolites

Plant cell culture bioprocesses have become an alternative source for bioactive compound production to the traditional extraction technology from plants grown in nature or through agricultural techniques. Thus, environmental conditions could make the supply of these secondary metabolites intermittent and heterogeneous. Therefore, plant cell cultures assure a continuous product supply, homogeneous production, fulfilling GMP requirements. Elicitation has been an extensively used strategy to boost secondary metabolite production in plant cell cultures. Cyclodextrins, the naturally occurring cyclic oligosaccharides of glucose residues, has emerged as an elicitor that can trigger plant cell defense responses and secondary metabolite accumulation. Moreover, CDs can form complex with most of the plant secondary compounds, thus contribute to product removal, which results in the elimination of feedback inhibition of product biosynthesis and also prevents product degradation and toxicity against the plant cells. In the present manuscript, we collect and compare the effects of cyclodextrins on different secondary metabolite pathways in plant cell culture processes. We review different case studies, which includes the production of phenylpropanoids, terpenes, alkaloids, naphthoquinones and anthraquinones derivatives. In the present manuscript, we collect and compare the effects of cyclodextrins on different secondary metabolite pathways in plant cell culture, which include the production of phenylpropanoids, terpenes, alkaloids, naphthoquinones and anthraquinones derivatives. Cyclodextrins trigger secondary metabolite production in plant cell cultures and product release. Moreover, they form complexes with these bioactive compounds, reduce feedback inhibition biosynthesis and their degradation.

Technoeconomic analysis of semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures.

Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms in its ability to produce complex biologics with simple chemically defined media and reduced biosafety concerns. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous technoeconomic models or analyses have been published. Here we present technoeconomic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes.

Successful scale up expansion of Wharton's jelly mesenchymal stromal cells in different commercial xeno-free and serum-free media

Background & Aim Cell-based therapies are becoming widely accepted in regenerative medicine and their demand and potential is increasing. Human multipotent Mesenchymal Stromal Cells (MSC) derived from the Wharton's jelly (WJ) of the umbilical cords can be obtained from samples typically discarded from public cord blood banking programmes. However, current cell culture bioprocesses for the derivation, scale up expansion, and GMP-compliant product manufacturing for early phase clinical trials use serum and xenogeneic proteins in medium formulation. Therefore, the use of xeno-free and serum-free media (XSFM) formulations is required to ensure more homogenous cell populations. The aim of this study was to compare four commercially available XSFM as an alternative to human serum-containing media in WJ-MSC culture. Methods, Results & Conclusion Cryopreserved MSC derived from WJ in serum-conditions were cultured in four commercial XSFM, namely PRIME-XV MSC Expansion XSFM, MSC Nutristem® XF, MesenCult™-ACF Plus Medium, and MSC-Brew GMP Medium and were further compared with conventional expansion medium Dulbecco's modified Eagle's medium (DMEM) containing 2 mM glutamine and supplemented with 10% of human serum B (hSerB). Cells were successfully expanded in culture in all XSFM conditions. Cumulative population doublings and harvest cell densities were higher in early passages for 3 out of 4 XSFM than serum-containing expansion medium. WJ-MSC proliferation was higher than 1 × 105 cells/cm2 in early culture (passage 2) while it decreased under 1 × 105 cells/cm2 in late culture (passage 8) in XSFM conditions. Spindle shaped morphology was kept upon passages in XSFM conditions. In all cases, cells were highly positive for CD73, CD90 and CD105 and negative for CD31, CD45 and HLA-DR expression in passage 1 and 6 and multilineage capacity to osteogenic, adipogenic and chondrogenic lineages was confirmed after in vitro induction. These data demonstrate that WJ-MSC can be successfully cultured and expanded in commercially available xeno-free and serum-free media and retain all necessary characteristics attributed to MSC for potential therapeutic use.

Real-time characterization of mammalian cell culture bioprocesses by magnetic sector MS.

Mammalian cell culture processes were characterized upon the analysis of the exhaust-gas composition achieved through the on-line integration of a magnetic sector MS analyser with benchtop bioreactors. The non-invasive configuration of the magnetic sector MS provided continuous evaluation of the bioreactor's exhaust gas filter integrity and facilitated the accurate quantification of O2 and CO2 levels in the off-gas stream which ensured preserved bioreactor sterility prior to cell inoculation and provided evidence of the ongoing cellular respiratory activity throughout the cultures. Real-time determination of process parameters such as the Respiratory Quotient (RQ) allowed for precise pin-pointing of the occurrence of shifts in cellular metabolism which were correlated to depletion of key nutrients in the growth medium, demonstrating the suitability of this technology for tracking cell culture process performance.

Cell-culture growth conditions resulting in the oxidation of a recombinant antigen-binding fragment

Use of Quality-by-Design (QbD) tools is becoming an important part of the bioprocessing industry when developing a process for manufacturing operations to ensure the robustness and reproducibility of the biologic product. In the present study, a QbD tool, Design of Experiments (DOE), was utilized to optimize a bioprocess for the production of a CHO recombinant antigen-binding fragment (rFab) in small-scale bioreactors. DOE studies evaluated percent dissolved oxygen, temperature, and feeding strategy specific to this Chinese Hamster Ovary (CHO) clone. It was determined that these factors influenced cell viability, yield of the recombinant protein, and metabolic byproduct formation. To ensure the quality of the target molecule in the cell-culture process, small-scale purifications and analytical evaluation of the target molecule were completed prior to cell-culture scale-up to ensure that oxidation of the rFab, presence of free light chain, and truncation of thiol group were not observed. Analysis of the purified rFab by mass spectrometry indicated that rFab oxidation occurred under poor cell-culture conditions. PCR profile array results also revealed increased transcription of the oxidative genes Superoxide Dismutase 3, Myeloperoxidase, Dual Oxidase Like 2, Nuclear Receptor Coactivator 7, NADPH Oxidase Organizer 1, Mitochondria Uncouple Protein 3, Eosinophil Peroxidase, Lactoperoxidase Like, Serum Albumin Like, and Glutathione S-Transferase Pi 1 in this CHO strain. The present study suggests a mechanism and pathway for the oxidation of an rFab molecule during cell-culture bioprocess optimization. The present study also demonstrated the importance of utilizing the QbD tool of DOE to optimize the cell-culture bioprocess prior to scaling up into the large-scale production bioreactor.

Impact of mammalian cell culture conditions on monoclonal antibody charge heterogeneity: an accessory monitoring tool for process development

Recombinant monoclonal antibodies are predominantly produced in mammalian cell culture bioprocesses. Post-translational modifications affect the micro-heterogeneity of the product and thereby influence important quality attributes, such as stability, solubility, pharmacodynamics and pharmacokinetics. The analysis of the surface charge distribution of monoclonal antibodies provides aggregated information about these modifications. In this work, we established a direct injection pH gradient cation exchange chromatography method, which determines charge heterogeneity from cell culture supernatant without any purification steps. This tool was further applied to monitor processes that were performed under certain process conditions. Concretely, we were able to provide insights into charge variant formation during a fed-batch process of a Chinese hamster ovary cell culture, in turn producing a monoclonal antibody under varying temperatures and glucose feed strategies. Glucose concentration impacted the total emergence of acidic variants, whereas the variation of basic species was mainly dependent on process temperature. The formation rates of acidic species were described with a second-order reaction, where a temperature increase favored the conversion. This platform method will aid as a sophisticated optimization tool for mammalian cell culture processes. It provides a quality fingerprint for the produced mAb, which can be tested, compared to the desired target and confirmed early in the process chain.

Physiological alterations of GS-CHO cells in response to adenosine monophosphate treatment

Growth-arrested strategies (e.g. hypothermia and hyperosmolarity) have been widely employed to enhance cell-specific productivity (qP) in mammalian cell culture bioprocess. In addition to enhanced qP, alterations in cell physiology, such as cell size and cell cycle phase, have also attracted extensive attention under growth-arrested conditions. However, to date, very few reports on associations between physiological changes in growth-inhibiting approaches have been published. In this study, we explored associations between the physiological changes of GS-CHO cells in response to adenosine monophosphate (AMP) treatment. In dose response studies, AMP treatment resulted in suppressed proliferation, accumulated S-phase cells, increased cell size and enhanced qP. Subsequently, six GS-CHO clones exhibited the physiological alterations in varying degrees when treated with 7 mM AMP. But more importantly, a significant positive correlation between total intracellular protein content and mean electronic volume, an indicator of cell size (P < 0.01) was found, indicating that total intracellular protein was the determining factor in increasing cell size in this growth-arrested strategy. Besides, our results provide additional evidence that treatment with growth-arrested agents may increase cell size; the agent per se did not cause the increased productivity.

Parallel comparison of in situ Raman and NIR spectroscopies to simultaneously measure multiple variables toward real-time monitoring of CHO cell bioreactor cultures

For the first time, the ability of in situ Raman and Near Infrared (NIR) spectroscopies to predict in real-time the concentration of viable Chinese Hamster Ovary (CHO) cells, glucose, lactate, glutamine, ammonium and antibodies in bioreactors has been compared in parallel. PLS models were developed using Raman and NIR spectra recorded at same time points and in same cultures. Multivariate figures of merit were established to thoroughly compare the performances of models. Raman spectroscopy was found to better predict concentrations of glucose, lactate and antibody, while NIR spectroscopy predicted better glutamine and ammonium ion concentrations. By applying the models developed on spectra recorded continuously during a batch culture, both spectroscopies were able to monitor simultaneously the general trends of the concentration of different analytes, while Raman predictions were more accurate at the beginning of the culture. Moreover, other chemometric methods (partial least square regression of type 2 and spectral combination) were tested, but leading to only limited model performance improvements. Both spectroscopies are suitable tools for real-time monitoring of cell culture bioprocesses, while Raman spectroscopy provides several advantages concerning sensibility, selectivity and limit of detection. These results provide useful information for academic researchers and pharmaceutical industries to choose the most adequate spectroscopic technology for their cell culture bioprocesses.

Kinetic Modeling of Mammalian Cell Culture Bioprocessing: The Quest to Advance Biomanufacturing

Kinetic modeling is the most suitable framework to describe the dynamic behavior of mammalian cell culture although its industrial application is still in its infancy. Herein, the authors reviewed mammalian bioprocess relevant kinetic models, and found that the simple unstructured-unsegregated approach utilizing empirical Monod-type kinetics based on limiting substrates and inhibitory metabolites is commonly used due to its traceability and simple formalism. Notably, the available kinetic models are typically small to moderate in size, and the development of large-scale models is severely hampered by the scarcity of kinetic data and limitations in current parameter estimation methods. The recent availability of abundant high-throughput multi-omics datasets from mammalian cell cultures have now paved the way to improve parameterization of kinetic models, and integrate regulatory, signaling, and product quality related intracellular events, as well as cellular metabolism within the modeling framework. Ultimately, the authors foresee that multi-scale modeling is the way forward in building predictive kinetic models of mammalian cell culture to advance biomanufacturing.

Quality by control: Towards model predictive control of mammalian cell culture bioprocesses.

The industrial production of complex biopharmaceuticals using recombinant mammalian cell lines is still mainly built on a quality by testing approach, which is represented by fixed process conditions and extensive testing of the end-product. In 2004 the FDA launched the process analytical technology initiative, aiming to guide the industry towards advanced process monitoring and better understanding of how critical process parameters affect the critical quality attributes. Implementation of process analytical technology into the bio-production process enables moving from the quality by testing to a more flexible quality by design approach. The application of advanced sensor systems in combination with mathematical modelling techniques offers enhanced process understanding, allows on-line prediction of critical quality attributes and subsequently real-time product quality control. In this review opportunities and unsolved issues on the road to a successful quality by design and dynamic control implementation are discussed. A major focus is directed on the preconditions for the application of model predictive control for mammalian cell culture bioprocesses. Design of experiments providing information about the process dynamics upon parameter change, dynamic process models, on-line process state predictions and powerful software environments seem to be a prerequisite for quality by control realization.

Raman Spectroscopy as a Real-Time In Situ Analyzer for Cell Culture Bioprocesses

The development of fast and reliable methods for protein determination are of great relevance to a diversity of areas from industry to diagnostics. Molecular Imprinted Materials (MIM) has proved to be an interesting methodology for protein analysis however further studies of the effect of the experimental parameters and starting materials in the performance of the MIM are still required. Caffeic acid (CAF) is employed for the first time as a monomer to tailor a synthetic receptor for a protein target. This was done by bulk-electropolymerization, applying a constant potential of +2.0 V, for 30 s, on a carbon screen-printed electrode, immersed in a solution of protein and CAF prepared in phosphate buffer. Annexin A3 (ANXA3) was selected as protein target due to the fact that this is an emerging biomarker in prostate cancer.The assembly of the protein imprinted material (PIM) was followed by Electrochemical Impedance Spectroscopy (EIS) and Raman Spectroscopy. A non-imprinted material (NIM) was prepared in parallel as control.Square wave voltammetry (SWV) was used to monitor the electrochemical signal of the [Fe(CN)6]3−/[Fe(CN)6]4− redox for the quantification of ANXA3.The optimized PIM-based device showed average detection limits (LOD) of 0.095 ng/mL, a linear behavior against log (concentration) between 0.10, and 200 ng/mL and good selectivity. The NIM-based device showed random behavior against protein concentration. Finally, the PIM-sensor was successfully applied to the analysis of ANXA3 in spiked urine samples.

Bioreactor Process Parameter Screening Utilizing a Plackett-Burman Design for a Model Monoclonal Antibody

Consistent high-quality antibody yield is a key goal for cell culture bioprocessing. This endpoint is typically achieved in commercial settings through product and process engineering of bioreactor parameters during development. When the process is complex and not optimized, small changes in composition and control may yield a finished product of less desirable quality. Therefore, changes proposed to currently validated processes usually require justification and are reported to the US FDA for approval. Recently, design-of-experiments-based approaches have been explored to rapidly and efficiently achieve this goal of optimized yield with a better understanding of product and process variables that affect a product's critical quality attributes. Here, we present a laboratory-scale model culture where we apply a Plackett-Burman screening design to parallel cultures to study the main effects of 11 process variables. This exercise allowed us to determine the relative importance of these variables and identify the most important factors to be further optimized in order to control both desirable and undesirable glycan profiles. We found engineering changes relating to culture temperature and nonessential amino acid supplementation significantly impacted glycan profiles associated with fucosylation, β-galactosylation, and sialylation. All of these are important for monoclonal antibody product quality.

Automated disposable small-scale bioreactor for high-throughput process development: implementation of the 24 bioreactor array

Aim: A disposable reactor system (250 ml scale) has now been developed into a fully automated 24 reactor array which includes process parameter monitoring and control, advanced feed strategies, automated event triggering, robotic arm liquid handling and sample storage. Results: Process and analytical comparability was shown for a range of Pichia, Escherichia coli and CHO cell-culture bioprocesses up to pilot and commercial scale. This established the system as a scale-down model for process development and characterization of large-scale industrial processes. Conclusion: This work demonstrated the ability of the automated system to accelerate process development by executing a single statistical design of experiments, with a wider range of parameters, up to 3–5-times faster than conventional approaches.

Advances in process monitoring tools for cell culture bioprocesses

The productivity of cell culture manufacturing for biologics has increased momentously in the past decades. Increasingly, the process research efforts are devoted into improving product quality and consistency. Consistent process performance and successful implementation of quality by design practice requires well‐utilized process analytical technology. This review summarizes recent progress and current status of bioprocess monitoring. Many sensors for bioprocess monitoring have been available for decades while new ones, especially spectrometric sensors, are making their way into cell culture bioprocesses. On‐line sampling devices have grown mature in the past decade thus making many instruments traditionally used for off‐line analysis available for at‐line use. With a general trend of using better defined medium for cell cultivation and increasing emphasis of process analytical tools, the spectrometric methods are also making headway in cell culture process monitoring. The integration of those sensing technologies will be important to advance the real‐time monitoring of the state of cellular physiology for the control for process consistency and product quality.