Kinetic modeling: A tool for temperature shift and feeding optimization in cell culture process development.

Mammalian cells have dominated the biopharmaceutical industry for biotherapeutic protein production and tremendous efforts have been devoted to enhancing productivity during the cell culture process development. However, determining the optimal process conditions is still a huge challenge. Constrained by the limited resources and timeline, usually it is impossible to fully explore the optimal range of all process parameters (temperature, pH, dissolved oxygen, basal and feeding medium, additives, etc.). Kinetic modeling, which finds out the global optimum by systematically screening all potential conditions for cell culture process, provides a solution to this dilemma. However, studies on optimizing temperature shift and feeding strategies simultaneously using this approach have not been reported. In this study, we built up a kinetic model of fed-batch culture process for simultaneous optimization of temperature shift and feeding strategies. The fitting results showed high accuracy and demonstrated that the kinetic model can be used to describe the mammalian cell culture performance. In addition, five more fed-batch experiments were conducted to test this model's predicting power on different temperature shift and feeding strategies. It turned out that the predicted data matched well with experimental ones on viable cell density (VCD), metabolites, and titer for the entire culture duration and allowed selecting the same best condition with the experimental results. Therefore, adopting this approach can potentially reduce the number of experiments required for culture process optimization.

Effect of temperature on synthesis of clavulanic acid and impurity substance G during fermentation by Streptomyces clavuligerus.

Effect of temperature on synthesis of Clavulanic acid (CA) and impurity substance G during fermentation by Streptomyces clavuligerus were investigated. Results show that fermentation at 24 °C is the most favorable for CA synthesis though the fermentation duration was 20-30 hours longer than fermentation at 26 and 28 °C. Meanwhile, the impurity substance G was only 110 mg/L in the end broth of fermentation at 24 °C, which was significantly lower than 148 and 180 mg/L of fermentation at 26 and 28 °C, respectively. Correlation of specific growth rate and CA synthesis was statistically analyzed based on data of 10 batches of industrial fermentation. Two temperature-shift strategies were investigated in 50 L fermenter. Fermentation with 26-24 °C temperature strategy achieved 5097 mg/L CA titer, meanwhile the fermentation duration was shortened 24 hours comparing with fermentation at constant 24 °C. Fermentation with 26-24 °C control strategy was validated in a 60 m3 industrial fermenter, in which 4960 mg/L of CA was achieved while impurity G substance was decreased to titer 65 mg/L from 200 to 300 mg/L of normal production.

Subphysiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells.

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) splicing, has thus far, received little attention. In this study, we utilized RNASeq for transcriptomic analysis of a monoclonal antibody (mAb) producing CHO K1 cell line subjected to a temperature shift. More than 2,465 instances of differential splicing were observed 24 hr after the reduction of cell culture temperature. A total of 1,197 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using quantitative polymerase chain reaction in the mAb-producing CHO K1 cell line used for RNASeq and a further two CHO K1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of these changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilized to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

Expanding the Chinese hamster ovary cell long noncoding RNA transcriptome using RNASeq.

Our ability to study Chinese hamster ovary (CHO) cell biology has been revolutionised over the last decade following the development of next generation sequencing technology and publication of reference DNA sequences for CHO cells and the Chinese hamster. RNA sequencing has not only enabled the association of transcript expression with bioreactor conditions and desirable bioprocess phenotypes but played a key role in the characterisation of protein coding and small noncoding RNAs. The annotation of long noncoding RNAs, and therefore our understanding of their role in CHO cell biology, has been limited to date. In this manuscript, we use high-resolution RNASeq data to more than double the number of annotated lncRNA transcripts for the CHO K1 genome. In addition, the utilisation of strand-specific sequencing enabled the identification of more than 1,000 new antisense and divergent lncRNAs. The utility of monitoring lncRNA expression is demonstrated through an analysis of the transcriptomic response to a reduction of cell culture temperature and identification of simultaneous sense/antisense differential expression for the first time in CHO cells. To enable further studies of lncRNAs, the transcripts annotated in this study have been made available for the CHO cell biology community.

Differential Phosphoproteomic Analysis of Recombinant Chinese Hamster Ovary Cells Following Temperature Shift.

Phosphorylation is one of the most important post-translational modifications, playing a crucial role in regulating many cellular processes, including transcription, cytoskeletal rearrangement, cell proliferation, differentiation, apoptosis, and signal transduction. However, to date, little work has been carried out on the phosphoproteome in CHO cells. In this study we have carried out a large scale differential phosphoproteomic analysis of recombinant CHO cells following a reduction of culture temperature (temperature shift). The reduction of culture temperature during the exponential phase of growth is commonly employed by the biopharmaceutical industry to increase product yield; however, the molecular mechanisms of temperature shift in CHO cells remain poorly understood. We have identified 700 differentially expressed phosphopeptides using quantitative label-free LC-MS/MS phosphoproteomic analysis in conjunction with IMAC and TiO2 phosphopeptide enrichment strategies, following a reduction in temperature from 37 to 31 °C. Functional assessment of the phosphoproteomic data using gene ontology analysis showed a significant enrichment of biological processes related to growth (e.g., cell cycle, cell division), ribosomal biogenesis, and cytoskeleton organization, and molecular functions related to RNA binding, transcription factor activity, and protein serine/threonine kinase activity. Differential phosphorylation of two proteins, ATF2 and NDRG1, was confirmed by Western blotting. This data suggests the importance of including the post-translational layer of regulation, such as phosphorylation, in CHO "omics" studies. This study also has the potential to identify phosphoprotein targets that could be modified using cell line engineering approaches to improve the efficiency of recombinant protein production.

NMR in drug design.

The use of NMR as a tool to determine 3 dimensional protein solution structures, once a darling of the pharmaceutical industry, has largely given way to study of the interaction of prospective drugs with macromolecular targets. Many of these approaches involve ligand-centered studies, which have the advantage of speed and efficiency, but there are also many approaches that take directly from our learnings in macromolecular NMR and provide greater structural detail yet are still optimized for rapid turn-around of information. In the evolution of NMR in the pharmaceutical industry, the unique strengths of NMR to provide dynamic and atomic level information continue to be exploited to discover and design new drugs. Numerous methods have been developed over the past two decades that fall into the categories of fragment-based pre-lead discovery, ligand binding studies and qualitative structural screening.

Induced gene expression in industrial Saccharomyces pastorianus var. carlsbergensis TUM 34/70: evaluation of temperature and ethanol inducible native promoters.

Induced gene expression is an important trait in yeast metabolic engineering, but current regulations prevent the use of conventional expression systems, such as galactose and copper, in food and beverage fermentations. This article examines the suitability of temperature-inducible native promoters for use in the industrial yeast strain Saccharomyces pastorianus var. carlsbergensis TUM 34/70 under brewing conditions. Ten different promoters were cloned and characterized under varying temperature shifts and ethanol concentrations using a green fluorescent protein reporter. The activities of these promoters varied depending upon the stress conditions applied. A temperature shift to 4°C led to the highest fold changes of PSSA3, PUBI4 and PHSP104 by 5.4, 4.5 and 5.0, respectively. Ethanol shock at 24°C showed marked, concentration-dependent induction of the promoters. Here, PHSP104 showed its highest induction at ethanol concentrations between 4% (v/v) and 6% (v/v). The highest fold changes of PSSA3 and PUBI4 were found at 10% (v/v) ethanol. In comparison, the ethanol shock at a typical fermentation temperature (12°C) leads to lower induction patterns of these promoters. Taken together, the data show that three promoters (PHSP104, PUBI4 and PSSA3) have high potential for targeted gene expression in self-cloning brewing yeast using temperature shifts.

Production of α-ketobutyrate using engineered Escherichia coli via temperature shift.

Alpha-ketobutyrate has been widely used in medicine and food additive industry. Because chemical and enzymatic methods are associated with many deficiencies, the recent focus shifted to fermentation for the production of α-ketobutyrate. In this study, a genetically engineered strain THRDΔrhtAΔilvIH/pWSK29-ilvA was constructed, starting from an L-threonine-producing strain, by overexpressing threonine dehydratase (TD), reducing α-ketobutyrate catabolism and L-threonine export. The shake flask cultivation of THRDΔrhtAΔilvIH/pWSK29-ilvA allowed the production of 16.2 g/L α-ketobutyrate. Accumulation of α-ketobutyrate severely inhibited the cell growth. To develop a better TD expression system and avoid the usage of the expensive inducer IPTG, a temperature-induced plasmid pBV220-ilvA was selected to transform the strain THRDΔrhtAΔilvIH for α-ketobutyrate production. The initial temperature was maintained at 35°C to guarantee normal cell growth, and then elevated to 40°C to induce the expression of TD. Under optimized conditions, the α-ketobutyrate titer reached 40.8 g/L after 28 h of fermentation, with a productivity of 1.46 g/L/h and a yield of 0.19 g/g glucose, suggesting large-scale production potential. Biotechnol. Bioeng. 2016;113: 2054-2059. © 2016 Wiley Periodicals, Inc.

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV-3) and possible latency of CyHV-3 by temperature shift in the cells.

A new cell line named CCF-K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV-3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV-3-infected CCF-K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV-3 was produced stably in CCF-K104 cells over 30 viral passages. Our findings showed that CCF-K104 is a useful cell line for isolation and productive replication of CyHV-3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real-time PCR showed that CyHV-3 was present with low viral DNA loads, suggesting that CyHV-3 may establish latent infection in CCF-K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV-3 genome arranged in a head-to-tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV-3.

Novel Methodology for Identifying the Occurrence of Ovulation by Estimating Core Body Temperature During Sleeping: Validity and Effectiveness Study.

BACKGROUND: Body temperature is the most-used noninvasive biomarker to determine menstrual cycle and ovulation. However, issues related to its low accuracy are still under discussion. OBJECTIVE: This study aimed to improve the accuracy of identifying the presence or absence of ovulation within a menstrual cycle. We investigated whether core body temperature (CBT) estimation can improve the accuracy of temperature biphasic shift discrimination in the menstrual cycle. The study consisted of 2 parts: experiment 1 assessed the validity of the CBT estimation method, while experiment 2 focused on the effectiveness of the method in discriminating biphasic temperature shifts. METHODS: In experiment 1, healthy women aged between 18 and 40 years had their true CBT measured using an ingestible thermometer and their CBT estimated from skin temperature and ambient temperature measured during sleep in both the follicular and luteal phases of their menstrual cycles. This study analyzed the differences between these 2 measurements, the variations in temperature between the 2 phases, and the repeated measures correlation between the true and estimated CBT. Experiment 2 followed a similar methodology, but focused on evaluating the diagnostic accuracy of these 2 temperature measurement approaches (estimated CBT and traditional oral basal body temperature [BBT]) for identifying ovulatory cycles. This was performed using urine luteinizing hormone (LH) as the reference standard. Menstrual cycles were categorized based on the results of the LH tests, and a temperature shift was identified using a specific criterion called the "three-over-six rule." This rule and the nested design of the study facilitated the assessment of diagnostic measures, such as sensitivity and specificity. RESULTS: The main findings showed that CBT estimated from skin temperature and ambient temperature during sleep was consistently lower than directly measured CBT in both the follicular and luteal phases of the menstrual cycle. Despite this, the pattern of temperature variation between these phases was comparable for both the estimated and true CBT measurements, suggesting that the estimated CBT accurately reflected the cyclical variations in the true CBT. Significantly, the CBT estimation method showed higher sensitivity and specificity for detecting the occurrence of ovulation than traditional oral BBT measurements, highlighting its potential as an effective tool for reproductive health monitoring. The current method for estimating the CBT provides a practical and noninvasive method for monitoring CBT, which is essential for identifying biphasic shifts in the BBT throughout the menstrual cycle. CONCLUSIONS: This study demonstrated that the estimated CBT derived from skin temperature and ambient temperature during sleep accurately captures variations in true CBT and is more accurate in determining the presence or absence of ovulation than traditional oral BBT measurements. This method holds promise for improving reproductive health monitoring and understanding of menstrual cycle dynamics.

Temperature stress improved exopolysaccharide yield from Tetragenococcus halophilus: Structural differences and underlying mechanisms revealed by transcriptomic analysis.

This study aimed to enhance exopolysaccharide production by Tetragenococcus halophilus, and results showed that low temperature (20 °C) significantly improved exopolysaccharide production. Based on the analysis of batch fermentation kinetic parameters, a temperature-shift strategy was proposed, and the exopolysaccharide yield was increased by 28 %. Analysis of the structure of exopolysaccharide suggested that low temperature changed the molecular weight and monosaccharide composition. Transcriptomic analysis was performed to reveal mechanisms of low temperature improving exopolysaccharide production. Results suggested that T. halophilus regulated utilization of carbon sources through phosphotransferase system and increased the expression of key genes in exopolysaccharide biosynthesis to improve exopolysaccharide production. Meanwhile, metabolic pathways involved in glycolysis, amino acids synthesis, two-component system and ATP-binding cassette transporters were affected at low temperature. Results presented in this paper provided a theoretical basis for biosynthetic pathway of exopolysaccharide in T. halophilus and aided to strengthen its production and application in many areas.

Response and resilience of karst subterranean estuary communities to precipitation impacts.

The impact of meteorological phenomena on ecosystem communities of karst subterranean estuaries (KSEs) remains unknown. KSEs are characterized by vertically stratified groundwater separated by a halocline and host endemic aquatic cave-adapted fauna (stygobionts). In October 2015, 8 days of heavy precipitation caused the first recorded mortality event in the KSE. This event was marked by a halocline shift 5 m deeper. The present study aimed to provide insights into resilience of KSEs faunal communities to temporal shifts in temperature and precipitation. Cave water temperature decreased on average 0.0068°C per mm of accumulated precipitation over 4 days, which can add up to, and surpass, the interannual temperature variation in cases of heavy precipitations. Biological surveys (2012-2021) conducted within cave systems El Aerolito and La Quebrada, in Cozumel, indicated that change in community structure was not detected and stygobionts were resilient; however, marine species inhabiting the caves were impacted. Overall, the faunal community at KSEs remains resilient within short-term meteorological phenomena despite shifts of non-stygobionts.

El impacto de fenómenos meteorológicos en las comunidades de los ecosistemas de estuarios subterráneos kársticos (KSE, por sus siglas en inglés) sigue siendo desconocido. Los KSE se caracterizan por aguas subterráneas estratificadas verticalmente separadas por una haloclina, y albergan fauna acuática endémica adaptada a la vida en cuevas (estigobiontes). En octubre de 2015, ocho días de fuertes precipitaciones causaron el primer evento de mortandad registrado en un KSE. Este evento estuvo marcado por un desplazamiento de la haloclina, resultando en una haloclina 5 m más profunda. El presente estudio tuvo como objetivo proveer información sobre la resiliencia de las comunidades faunísticas de los KSE a cambios temporales en la temperatura y la precipitación. La temperatura del agua de la cueva disminuyó en promedio 0.0068°C por mm de precipitación acumulado durante cuatro días, lo cual puede alcanzar, e incluso superar, la variación interanual de la temperatura debido a fuertes precipitaciones. Los estudios biológicos (2012­2021) realizados dentro de los sistemas de cuevas El Aerolito y La Quebrada, en Cozumel, indicaron que no se detectó un cambio en la estructura de la comunidad y que los estigobiontes fueron resilientes, sin embargo, las especies marinas que habitan las cuevas se vieron afectadas. En general, la comunidad de fauna de KSEs se mantiene resiliente a los fenómenos meteorológicos a corto plazo a pesar de los cambios en la fauna no estigobia.

Metabolomic rearrangement controls the intrinsic microbial response to temperature changes.

Temperature is one of the key determinants of microbial behavior and survival, whose impact is typically studied under heat- or cold-shock conditions that elicit specific regulation to combat lethal stress. At intermediate temperatures, cellular growth rate varies according to the Arrhenius law of thermodynamics without stress responses, a behavior whose origins have not yet been elucidated. Using single-cell microscopy during temperature perturbations, we show that bacteria exhibit a highly conserved, gradual response to temperature upshifts with a time scale of ~1.5 doublings at the higher temperature, regardless of initial/final temperature or nutrient source. We find that this behavior is coupled to a temperature memory, which we rule out as being neither transcriptional, translational, nor membrane dependent. Instead, we demonstrate that an autocatalytic enzyme network incorporating temperature-sensitive Michaelis-Menten kinetics recapitulates all temperature-shift dynamics through metabolome rearrangement, which encodes a temperature memory and successfully predicts alterations in the upshift response observed under simple-sugar, low-nutrient conditions, and in fungi. This model also provides a mechanistic framework for both Arrhenius-dependent growth and the classical Monod Equation through temperature-dependent metabolite flux.

Thermosensitive period for sex determination of the tropical freshwater turtle Malayemys macrocephala.

Many egg-laying reptiles possess temperature-dependent sex determination (TSD) in which outcome of gonadogenesis is determined by incubation temperature during a temperature-sensitive period of development. Prior studies on Malayemys macrocephala showed that incubation temperatures influence gonadal development and suggested that M. macrocephala exhibits TSD. However, information on the temperature-sensitivity period in this species was unknown until the current study. Turtle eggs were collected from rice fields in central Thailand from December 2016 to February 2017. In the laboratory, eggs were incubated at male-biased temperature (26 °C) and shifted to female-biased temperature (32 °C), or vice versa. Single shift experiments were performed systematically during embryonic stages 13-21. After hatching, sex of individual turtles was determined by histological analysis. We found that the sex determination of M. macrocephala is affected by temperature up to stage 16 of embryonic development.

Effective temperature shift strategy development and scale confirmation for simultaneous optimization of protein productivity and quality in Chinese hamster ovary cells.

Temperature shifts to lower culture temperatures are frequently employed in the manufacturing of protein therapeutics in mammalian cells to improve productivity, viability, or quality attributes. The direction and extent to which a temperature shift affects productivity and quality may vary depending on the expression host and characteristics of the expressed protein. We demonstrated here that two Chinese hamster ovary (CHO) clones expressing different human monoclonal antibodies responded differently to a temperature shift despite sharing a common parental CHO cell line. Within a single CHO line, we observed a nonlinear response to temperature shift. A moderate shift to 35°C significantly decreased final titer relative to the unshifted control while a larger shift to 32°C significantly increased final titer by 25%. Therefore, we proposed a systematic empirical approach to assess the utility of a temperature shift for faster implementation during process development. By testing multiple shift parameters, we identified optimum shift conditions in shake flasks and successfully translated findings to benchtop bioreactors and 1,000-L bioreactor scale. Significant differences in final antibody titer and charge variants were observed with temperature shift increments as small as Δ1.5°C. Acidic charge variants decreased monotonically with decreasing shift temperature in both cell lines; however, final antibody titer required simultaneous optimization of shift day and temperature. Overall, we were able to show that a systematic approach to identify temperature shift parameters at small scales is useful to optimize protein production and quality for efficient and confident translation to large-scale production.

Fermentation and Metabolic Pathway Optimization to De Novo Synthesize (2S)-Naringenin in Escherichia coli.

Flavonoids have diverse biological functions in human health. All flavonoids contain a common 2-phenyl chromone structure (C6-C3-C6) as a scaffold. Hence, in using such a scaffold, plenty of highvalue-added flavonoids can be synthesized by chemical or biological catalyzation approaches. (2S)-Naringenin is one of the most commonly used flavonoid scaffolds. However, biosynthesizing (2S)-naringenin has been restricted not only by low production but also by the expensive precursors and inducers that are used. Herein, we established an induction-free system to de novo biosynthesize (2S)-naringenin in Escherichia coli. The tyrosine synthesis pathway was enhanced by overexpressing feedback inhibition-resistant genes (aroGfbr and tyrAfbr) and knocking out a repressor gene (tyrR). After optimizing the fermentation medium and conditions, we found that glycerol, glucose, fatty acids, potassium acetate, temperature, and initial pH are important for producing (2S)-naringenin. Using the optimum fermentation medium and conditions, our best strain, Nar-17LM1, could produce 588 mg/l (2S)-naringenin from glucose in a 5-L bioreactor, the highest titer reported to date in E. coli.

Structural insights into human Arginase-1 pH dependence and its inhibition by the small molecule inhibitor CB-1158.

Arginase-1 is a manganese-dependent metalloenzyme that catalyzes the hydrolysis of L-arginine into L-ornithine and urea. Arginase-1 is abundantly expressed by tumor-infiltrating myeloid cells that promote tumor immunosuppression, which is relieved by inhibition of Arginase-1. We have characterized the potencies of the Arginase-1 reference inhibitors (2S)-2-amino-6-boronohexanoic acid (ABH) and N ω-hydroxy-nor-L-arginine (nor-NOHA), and studied their pH-dependence and binding kinetics. To gain a better understanding of the structural changes underlying the high pH optimum of Arginase-1 and its pH-dependent inhibition, we determined the crystal structure of the human Arginase-1/ABH complex at pH 7.0 and 9.0. These structures revealed that at increased pH, the manganese cluster assumes a more symmetrical coordination structure, which presumably contributes to its increase in catalytic activity. Furthermore, we show that binding of ABH involves the presence of a sodium ion close to the manganese cluster. We also studied the investigational new drug CB-1158 (INCB001158). This inhibitor has a low-nanomolar potency at pH 7.4 and increases the thermal stability of Arginase-1 more than ABH and nor-NOHA. Moreover, CB-1158 displays slow association and dissociation kinetics at both pH 9.5 and 7.4, as indicated by surface plasmon resonance. The potent character of CB-1158 is presumably due to its increased rigidity compared to ABH as well as the formation of an additional hydrogen-bond network as observed by resolution of the Arginase-1/CB-1158 crystal structure.

Model-based optimization of temperature and pH shift to increase volumetric productivity of a Chinese hamster ovary fed-batch process.

Industrial production processes, which utilize mammalian cells for the production of therapeutic proteins are routinely designed to include temperature and pH shifts. The process conditions are shifted away from growth promoting conditions towards a state of decreased metabolic activity. This results in the extension of the cultivation duration and therefore in a higher final product concentration. Although the correct timing of these shifts is essential for peak process performance, not many studies have been investigating this topic. In this work temperature and pH shift were optimized with a mechanistic model to increase the final product concentration in comparison to an established industrial fed-batch process. The major advantage of the mechanistic in comparison to a data-driven approach lies in the reduced number of experiments, which is needed. Therefore process development is faster, which decreases the time of the product to the market. Based on the optimization, an increased final product concentration of 14% was achieved in comparison to an already established industrial fed-batch process with the same cell line. Furthermore, the space-time-yield of the process did increase in comparison, resulting in a 20% increase of the final volumetric product concentration.

Systematic development of temperature shift strategies for Chinese hamster ovary cells based on short duration cultures and kinetic modeling.

Temperature shift (TS) to a hypothermic condition has been widely used during protein production processes that use Chinese hamster ovary (CHO) cells. The effect of temperature on cell growth, metabolites, protein titer and quality depends on cell line, product, and other bioreactor conditions. Due to the large numbers of experiments, which typically last 2-3 weeks each, limited systematic TS studies have been reported with multiple shift temperatures and steps at different times. Here, we systematically studied the effect of temperature on cell culture performance for the production of two monoclonal antibodies by industrial GS and DG44 CHO cell lines. Three 2-8 day short-duration methods were developed and validated for researching the effect of many different temperatures on CHO cell culture and quality attributes. We found that minor temperature differences (1-1.5 °C) affected cell culture performance. The kinetic parameters extracted from the short duration data were subsequently used to compute and predict cell culture performance in extended duration of 10-14 days with multiple TS conditions for both CHO cell lines. These short-duration culture methods with kinetic modeling tools may be used for effective TS optimization to achieve the best profiles for cell growth, metabolites, titer and quality attributes. Although only three short-duration methods were developed with two CHO cell lines, similar short-duration methods with kinetic modeling may be applied for different hosts, including both microbial and other mammalian cells.