Two-stage depth filter perfusion culture for recombinant antibody production by recombinant Chinese hamster ovary cell

A rCHO cell line of DUKX origin 26*-320, producing recombinant antibody against the human platelet, was cultivated in a two-stage depth filter perfusion system (DFPS) for 20 days in order to attain high recombinant antibody concentration. The productivity of the first stage DFPS bioreactor reached 53 times that of the batch culture in a controlled stirred tank reactor and was showed 12.1 mg/L antibody concentration at a perfusion rate of 6.0 d⁻¹. Glucose concentration in the first DFPS was maintained at 1.5 g/L to avoid cell damage in the perfusion culture. A second stage DFPS system was attached to the first DFPS, which resulted in a low glucose concentration of 0.02 g/L and a high antibody concentration of 23.9 mg/L. The two-stage depth filter perfusion culture yielded 60% higher product concentration than the batch and 49-fold higher productivity of 69.3 mg/L/d in comparison with that (1.4 mg/L/d) in a batch system. Furthermore, antibody concentration of the second stage was 97% higher than that of the first stage, and the antibody productivities were comparable to that of the first stage. This two-stage DFPS system also showed potential for higher titer production of recombinant antibody and high volumetric productivity for long-term culture of bio-pharmaceutical substances.     

Fermentanomics: Relating quality attributes of a monoclonal antibody to cell culture process variables and raw materials using multivariate data analysis

Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real‐time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration‐related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1586–1599, 2015     

A systematic approach for scale-down model development and characterization of commercial cell culture processes

The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.     

A direct computer control concept for mammalian cell fermentation processes

In the last 10 years, new assignments and the special demands of mammalian cells to the culture conditions caused the develoepment of complex small scale fermentation setups. The use of continuous fermentation and cell retention devices requires appropriate process control systems. An arrangement for control and data-acquisition of complex laboratory-scale bioreactors is presented. The fundamental idea was the usage of a standard personal computer, which is connected to pumps, valves and sensors via ADA-transformation. The possibility of free programming allowed the development of user-oriented software, especially designed for the far-reaching requirements of a university laboratory in the field of animal cell culture. Control of aeration, pumps, data-acquisition and data-storage are combined within one program, which allows the automation of standard operations like measurement of k_La- or OTR-values. Pump control algorithms for all common fermentation strategies (batch, fed batch, chemostat, perfusion) are included and can be selected any time during cultivation. Oxygen partial pressure and pH are controlled via direct digital control (ddc), providing simple adaption of control parameters and set points to current fermentation conditions.     

A novel approach for the simultaneous quantification of a therapeutic monoclonal antibody in serum produced from two distinct host cell lines

Therapeutic monoclonal antibodies (mAbs) possess a high degree of heterogeneity associated with the cell expression system employed in manufacturing, most notably glycosylation. Traditional immunoassay formats used to quantify therapeutic mAbs are unable to discriminate between different glycosylation patterns that may exist on the same protein amino acid sequence. Mass spectrometry provides a technique to distinguish specific glycosylation patterns of the therapeutic antibody within the same sample, thereby allowing for simultaneous quantification of the same mAb with different glycosylation patterns. Here we demonstrate a two-step approach to successfully differentiate and quantify serum mixtures of a recombinant therapeutic mAb produced in two different host cell lines (CHO vs. Sp2/0) with distinct glycosylation profiles. Glycosylation analysis of the therapeutic mAb, CNTO 328 (siltuximab), was accomplished through sample pretreatment consisting of immunoaffinity purification (IAP) and enrichment, followed by liquid chromatography (LC) and mass spectrometry (MS). LC-MS analysis was used to determine the percentage of CNTO 328 in the sample derived from either cell line based on the N-linked G1F oligosaccharide on the mAb. The relative amount of G1F derived from each cell line was compared with ratios of CNTO 328 reference standards prepared in buffer. Glycoform ratios were converted to concentrations using an immunoassay measuring total CNTO 328 that does not distinguish between the different glycoforms. Validation of the IAP/LC-MS method included intra-run and inter-run variability, method sensitivity and freeze-thaw stability. The method was accurate (%bias range = -7.30–13.68%) and reproducible (%CV range = 1.49–10.81%) with a LOQ of 2.5 μg/mL.     

The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures

To investigate the effect of dextran sulfate (DS), a widely used anti‐aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM‐0.025 and CS13‐1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13‐1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13‐1.00 cells, and showed the highest maximum mAb concentration. For SM‐0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N‐glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13‐1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3–1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113–1122, 2016     

Progress toward forecasting product quality and quantity of mammalian cell culture processes by performance‐based modeling

The production of biopharmaceuticals requires highly sophisticated, complex cell based processes. Once a process has been developed, acceptable ranges for various control parameters are typically defined based on process characterization studies often comprising several dozens of small scale bioreactor cultivations. A lot of data is generated during these studies and usually only the information needed to define acceptable ranges is processed in more detail. Making use of the wealth of information contained in such data sets, we present here a methodology that uses performance data (such as metabolite profiles) to forecast the product quality and quantity of mammalian cell culture processes based on a toolbox of advanced statistical methods. With this performance based modeling (PBM) the final product concentration and 12 quality attributes (QAs) for two different biopharmaceutical products were predicted in daily intervals throughout the main stage process. The best forecast was achieved for product concentration in a very early phase of the process. Furthermore, some glycan isoforms were predicted with good accuracy several days before the bioreactor was harvested. Overall, PBM clearly demonstrated its capability of early process endpoint prediction by only using commonly available data, even though it was not possible to predict all QAs with the desired accuracy. Knowing the product quality prior to the harvest allows the manufacturer to take counter measures in case the forecasted quality or quantity deviates from what is expected. This would be a big step towards real‐time release, an important element of the FDA's PAT initiative. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1119–1127, 2015     

Control of galactosylated glycoforms distribution in cell culture system

Cell culture process conditions including media components and bioreactor operation conditions have a profound impact on recombinant protein quality attributes. Considerable changes in the distribution of galactosylated glycoforms (G0F, G1F, and G2F) were observed across multiple CHO derived recombinant proteins in development at Eli Lilly and Company when switching to a new chemically defined (CD) media platform condition. In the new CD platform, significantly lower G0F percentages and higher G1F and G2F were observed. These changes were of interest as glycosylation heterogeneity can impact the effectiveness of a protein. A systematic investigation was done to understand the root cause of the change and control strategy for galactosylated glycoforms distribution. It was found that changes in asparagine concentration could result in a corresponding change in G0F, G1F, and G2F distribution. A follow‐up study examined a wider range of asparagine concentration and it was found that G0F, G1F, and G2F percentage could be titrated by adjusting asparagine concentration. The observed changes in heterogeneity from changing asparagine concentration are due to resulting changes in ammonium metabolism. Further study ascertained that different integrated ammonium level during the cell culture process could control G0F, G1F, and G2F percentage distribution. A mechanism hypothesis is proposed that integrated ammonium level impacts intracellular pH, which further regulates β‐1, 4 galactosyltransferase activity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:547–553, 2014     

Simulation of an iterative learning control system for fed-batch cell culture processes

This paper describes an iterative learning control scheme for fed-batch operation where repetitive trajectory tracking tasks are required. The proposed learning strategy is model-independent, and it takes advantage of the repetitive feature of system operations with a certain degree of intelligence and requires only small size of dynamic database for the learning process. The convergence of the learning process is proven. An example of simultaneously tracking two predefined trajectories by iterative learning control with two control inputs is given to illustrate the methodology. Satisfactory performance of the learning system can be observed from the simulation results.     

A novel scale-up method for mammalian cell culture in packed-bed bioreactor

A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2×10⁷ cells ml^–1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.     

Effect of different cell culture conditions on the polypeptide integrity and N-glycosylation of a recombinant model glycoprotein

The effect of different short-term controlled cell culture conditions on the product quality of a genetically engineered human interleukin-2 N-glycosylation variant protein expressed from a baby hamster kidney cell line (BHK-21) has been investigated. A perfused 2-L stirred tank reactor was used. Products purified from the culture supernatant of cells grown under experimentally initiated nutrient limitations (glucose, amino acids, pO(2)) were characterized by their HPLC-elution profile, SDS-PAGE and western blotting, amino acid sequencing as well as for their N-linked carbohydrates, using "HPAEC-PAD fingerprinting" and methylation analysis. The glycoprotein products secreted from cells under the different culture conditions (kept for 24 h, after an adaption time period of 48 h) showed an almost identical oligosaccharide pattern. By contrast, short-term changes of the culture condition led to considerable differences in the ratio of glycosylated to unglycosylated protein forms. Significant amounts of NH(2)-terminally truncated polypeptide forms were observed. They lacked proponderantly the first two amino acids; however, under certain culture conditions forms lacking up to eight NH(2)-terminal amino acids were detected. (c) 1995 John Wiley & Sons, Inc.     

The effect of the dilution rate on CHO cell physiology and recombinant interferon-gamma production in glucose-limited chemostat culture

The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (mu) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (mu(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 muM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma. (c) 1993 John Wiley & Sons, Inc.     

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Perfusion medium was successfully developed based on our fed‐batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed‐batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2× to increase medium depth. The medium osmolality was also optimized where we found ～360 mOsm/kg was desirable resulting in a residual culture osmolality of ～300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N, and D were rebalanced to reduce lactate and ammonium levels, and increase the cell‐specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell‐specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell‐day yielding 1.2 g/L‐day at steady state. This perfusion process was then successfully scaled up to a 100 L single‐use bioreactor with an ATF6 demonstrating similar performance as a 2 L bioreactor with an ATF2. Large volume handling challenges in our fed‐batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug‐and‐play use with the bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:891–901, 2017     

Enhanced process understanding and multivariate prediction of the relationship between cell culture process and monoclonal antibody quality

This work investigates the insights and understanding which can be deduced from predictive process models for the product quality of a monoclonal antibody based on designed high‐throughput cell culture experiments performed at milliliter (ambr‐15^®) scale. The investigated process conditions include various media supplements as well as pH and temperature shifts applied during the process. First, principal component analysis (PCA) is used to show the strong correlation characteristics among the product quality attributes including aggregates, fragments, charge variants, and glycans. Then, partial least square regression (PLS1 and PLS2) is applied to predict the product quality variables based on process information (one by one or simultaneously). The comparison of those two modeling techniques shows that a single (PLS2) model is capable of revealing the interrelationship of the process characteristics to the large set product quality variables. In order to show the dynamic evolution of the process predictability separate models are defined at different time points showing that several product quality attributes are mainly driven by the media composition and, hence, can be decently predicted from early on in the process, while others are strongly affected by process parameter changes during the process. Finally, by coupling the PLS2 models with a genetic algorithm first the model performance can be further improved and, most importantly, the interpretation of the large‐dimensioned process–product‐interrelationship can be significantly simplified. The generally applicable toolset presented in this case study provides a solid basis for decision making and process optimization throughout process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1368–1380, 2017     

A fully predictive model for one-dimensional light attenuation by Chlamydomonas reinhardtii in a torus photobioreactor

The light attenuation in a photobioreactor is determined using a fully predictive model. The optical properties were first calculated, using a data bank of the literature, from only the knowledge of pigments content, shape, and size distributions of cultivated cells which are a function of the physiology of the current species. The radiative properties of the biological turbid medium were then deduced using the exact Lorenz-Mie theory. This method is experimentally validated using a large-size integrating sphere photometer. The radiative properties are then used in a rectangular, one-dimensional two-flux model to predict radiant light attenuation in a photobioreactor, considering a quasi-collimated field of irradiance. Combination of this radiative model with the predictive determination of optical properties is finally validated by in situ measurement of attenuation profiles in a torus photobioreactor cultivating the microalgae Chlamydomonas reinhardtii, after a complete and proper characterization of the incident light flux provided by the experimental set-up.     

A predictive model to locate ancient forests in the Cross Timbers of Osage County, Oklahoma

The Cross Timbers are a mosaic of upland deciduous forest, savanna, and glade that typifies the broad ecotone between the eastern deciduous forest and the grasslands of the southern Great Plains. The pre-settlement Cross Timbers may have covered some 7,909,700 ha from central Texas, across Oklahama into eastern Kansas, and today may represent the least disturbed forest ecosystem of comparable size still left in the eastern United States. Extensive tree-ring research indicates that ancient forests dominated by 200 to 400 year old post-oaks ( Quercus stellata Wang) survive throughout the Cross Timbers, particularly in Oklahoma. These ancient forests persist largely because the Cross Timbers formation is non-commercial for timber production, and has not experienced large-scale industrial logging. Because ancient forest relics are often found on stressful non-commercial sites in the Cross Timbers and elsewhere, it is possible to design predictive models to locate the specific terrain where undisturbed forests are likely to survive. A predictive model for southern Osage County, Oklahoma, was developed based on the steep, infertile soils of the Niotaze-Darnell complex. We tested the model with field inspection and tree-ring analysis of fifty randomly selected belt transects, and 74% of the sampled terrain is still old-growth Cross Timbers woodland. This translates into 8200 ha of ancient Cross Timbers on this single site type in southern Osage County. The abundance of ancient forest in the Cross Timbers is not widely appreciated. However, large contiguous tracts of ancient Cross Timbers up to 700 ha were identified with this predictive model, strongly supporting inferences concerning the relatively undisturbed nature of this ecosystem.     

A comparison of different culture methods for hybridoma propagation and monoclonal antibody production

A major variable to consider in the production of biologicals from mammalian cell cultures is the mode of operation, be it a batch, continuous, perfusion, fed-batch or other production method. The final choice must consider a number of fundamental and economic issues. Here we present some antibody production data from different cell lines using different modes of production and discuss the important factors for consideration in choosing a production strategy. It was found that the productivity of batch cultures was lower than that obtained in continuous and perfused cultures, but that productivity could be improved by implementing suitable feeding strategies. The antibody productivity of one cell line, MCL1, during exponential phase was not affected by media type or glucose level. The maximum productivity of two cell lines in continuous culture was found to occur at dilution rates below the maximum, from 0.019 to 0.030 hr^–1.     

A comparison of orbitally‐shaken and stirred‐tank bioreactors: pH modulation and bioreactor type affect CHO cell growth and protein glycosylation

Orbitally shaken bioreactors (OSRs) support the suspension cultivation of animal cells at volumetric scales up to 200 L and are a potential alternative to stirred‐tank bioreactors (STRs) due to their rapid and homogeneous mixing and high oxygen transfer rate. In this study, a Chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5 L OSR and a 3 L STR, both operated with or without pH control. Effects of bioreactor type and pH control on cell growth and metabolism and on recombinant protein production and glycosylation were determined. In pH‐controlled bioreactors, the glucose consumption and lactate production rates were higher relative to cultures grown in bioreactors without pH control. The cell density and viability were higher in the OSRs than in the STRs, either with or without pH control. Volumetric recombinant antibody yields were not affected by the process conditions, and a glycan analysis of the antibody by mass spectrometry did not reveal major process‐dependent differences in the galactosylation index. The results demonstrated that OSRs are suitable for recombinant protein production from suspension‐adapted animal cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174–1180, 2016