Mammalian Cell Culture Broth Research Articles

Near-infrared spectroscopy based monitoring of all 20 amino acids in mammalian cell culture broth

The biopharmaceutical industry extensively employs Chinese hamster ovary (CHO) cell culture for monoclonal antibody production. Amino acids represent an essential source of nutrients in all CHO cell culture media, and their concentration is known to significantly impact cell viability, titre, and monoclonal antibody critical quality attributes. In this study, a robust Fourier transform near-infrared spectroscopy (FT-NIR) based quantification method has been developed for of all 20 amino acids (0–24 mM), as well as concentrations of glucose (0–6.7 mg mL−1), lactate (0–2.7 mg mL−1), and trastuzumab (0–2.5 mg mL−1) in the CHO cell culture. Near infra-red absorbance spectrum in the range of 4000–11,000 cm−1 were acquired, and spectra pre-processing through smoothening and derivatives were employed to enhance key characteristic signals. High-performance liquid chromatography with pre-column derivatization was used as the orthogonal analytical tool for quantification. Principal component analysis and partial least squares regression were employed for region selection and calibration model development, respectively. The results demonstrate that a good calibration statistic with the acceptable coefficient of determinations for both calibration (Rc2 = 0.94–0.99) and prediction (Rp2 = 0.83–0.98) could be achieved, along with high RPD values (>3) for all components except alanine (2.4). The external validation study also exhibited a satisfactory outcome (REV2 = 0.89–0.99, RMSE = 0.04–1.04), validating the model's ability to predict the concentrations of the respective species. The calibration models were successfully applied for at-line monitoring of two perfusion runs on a 10 L scale. To our knowledge, this is the first application where NIR spectroscopy-based measurement of all 20 amino acids in mammalian cell culture samples has been demonstrated. The proposed tool can play a critical role as biopharma manufacturers implement continuous processing as well as for facilitating process analytical technology-based control of mammalian cell culture processes.

Evaluation of recent Protein A stationary phase innovations for capture of biotherapeutics

We describe a comprehensive evaluation of 12 Protein A stationary phases for capture of biotherapeutics. We first examine the morphological properties of the stationary phases using a variety of orthogonal techniques including electron microscopy, particle sizing, pressure-flow behavior, and isocratic pulse response. A panel of nine proteins spanning a wide range of structures and biochemical properties was then used to assess equilibrium uptake, mass transport, dynamic binding capacity, and elution pH. Process performance and product quality were also examined under realistic bioprocess conditions using clarified mammalian cell culture broth. Equilibrium isotherms were found to be highly favorable, with equilibrium binding capacity for monoclonal and bispecific antibodies ranging from 47–100 mg/mL packed bed across all stationary phases tested. Effective pore diffusivities, De, were obtained by fitting the chromatography general rate model to breakthrough data. The fitted De values for monoclonal antibodies ranged from 1.1–5.7 × 10−8 cm2/s. The stationary phases had high dynamic binding capacities for the model proteins. The highest dynamic capacities for monoclonal and bispecific antibodies were seen with MabSelect SuRe pcc and MabSelect PrismA, which ranged from 58–74 mg/mL packed bed at 4 min residence times. Product capture using clarified cell culture broth as a feedstock showed high yields and elution pool volumes that ranged from 2–3 column volumes in most cases. Host cell protein, DNA, and aggregate levels in the elution pool were dependent on the specific nature of protein being purified, and levels were consistent between stationary phases. Lastly, we perform an analysis of bivariate correlations and discuss considerations for process design and optimization.

Application of CFD in Bioprocessing: Separation of mammalian cells using disc stack centrifuge during production of biotherapeutics

Centrifugation continues to be one of the most commonly used unit operations for achieving efficient harvest of the product from the mammalian cell culture broth during production of therapeutic monoclonal antibodies (mAbs). Since the mammalian cells are known to be shear sensitive, optimal performance of the centrifuge requires a balance between productivity and shear. In this study, Computational Fluid Dynamics (CFD) has been successfully used as a tool to facilitate efficient optimization. Multiphase Eulerian-Eulerian model coupled with Gidaspow drag model along with Eulerian-Eulerian k-ε mixture turbulence model have been used to quantify the complex hydrodynamics of the centrifuge and thus evaluate the turbulent stresses generated by the centrifugal forces. An empirical model has been developed by statistical analysis of experimentally observed cell lysis data as a function of turbulent stresses. An operating window that offers the optimal balance between high productivity, high separation efficiency, and low cell damage has been identified by use of CFD modeling.

An automated laboratory-scale methodology for the generation of sheared mammalian cell culture samples.

Continuous disk-stack centrifugation is typically used for the removal of cells and cellular debris from mammalian cell culture broths at manufacturing-scale. The use of scale-down methods to characterise disk-stack centrifugation performance enables substantial reductions in material requirements and allows a much wider design space to be tested than is currently possible at pilot-scale. The process of scaling down centrifugation has historically been challenging due to the difficulties in mimicking the Energy Dissipation Rates (EDRs) in typical machines. This paper describes an alternative and easy-to-assemble automated capillary-based methodology to generate levels of EDRs consistent with those found in a continuous disk-stack centrifuge. Variations in EDR were achieved through changes in capillary internal diameter and the flow rate of operation through the capillary. The EDRs found to match the levels of shear in the feed zone of a pilot-scale centrifuge using the experimental method developed in this paper (2.4×105 W/Kg) are consistent with those obtained through previously published computational fluid dynamic (CFD) studies (2.0×105 W/Kg). Furthermore, this methodology can be incorporated into existing scale-down methods to model the process performance of continuous disk-stack centrifuges. This was demonstrated through the characterisation of culture hold time, culture temperature and EDRs on centrate quality.

Overcoming the clarification challenges of high cell density culture

Cell line engineering and process intensification efforts have led to high performing fed-batch cell culture processes where cell density can reach >30x106 cells/mL. These high performance processes also come with new challenges, in particular developing efficient and scalable primary recovery processes. Centrifugation coupled with depth filtration is a standard approach for clarification of mammalian cell culture broth. However, with high cell density cultures, it can become challenging to obtain low turbidity clarified cell culture fluid (CCCF), and the filtration surface area required for clarification may become limiting when scaling up.

Automated harvesting and 2-step purification of unclarified mammalian cell-culture broths containing antibodies

Therapeutic monoclonal antibodies represent one of the fastest growing segments in the pharmaceutical market. The growth of the segment has necessitated development of new efficient and cost saving platforms for the preparation and analysis of early candidates for faster and better antibody selection and characterization. We report on a new integrated platform for automated harvesting of whole unclarified cell-culture broths, followed by in-line tandem affinity-capture, pH neutralization and size-exclusion chromatography of recombinant antibodies expressed transiently in mammalian human embryonic kidney 293T-cells at the 1-L scale. The system consists of two bench-top chromatography instruments connected to a central unit with eight disposable filtration devices used for loading and filtering the cell cultures. The staggered parallel multi-step configuration of the system allows unattended processing of eight samples in less than 24h. The system was validated with a random panel of 45 whole-cell culture broths containing recombinant antibodies in the early profiling phase. The results showed that the overall performances of the preparative automated system were higher compared to the conventional downstream process including manual harvesting and purification. The mean recovery of purified material from the culture-broth was 66.7%, representing a 20% increase compared to that of the manual process. Moreover, the automated process reduced by 3-fold the amount of residual aggregates in the purified antibody fractions, indicating that the automated system allows the cost-efficient and timely preparation of antibodies in the 20–200mg range, and covers the requirements for early in vitro and in vivo profiling and formulation of these drug candidates.

Quantification of l-alanyl- l-glutamine in mammalian cell culture broth: Evaluation of different detectors

l-Alanyl- l-glutamine (also known as Ala-Gln or GlutaMAX) is widely used as a stable l-glutamine source in cell culture for the production of biopharmaceuticals. System approaches for the optimization of production processes require the analysis of all major substrates and products. We have compared four alternative detection systems for l-alanyl- l-glutamine in culture broth. Matrix effects prevented the use of ultraviolet or evaporative light scattering detection. Fluorescence detection used in routine amino acid protocols is compatible with culture broth and has a broad linear dynamic range. Mass spectrometry has superior sensitivity and can be integrated into quantitative metabolomic workflows.

Effects of solution environment on mammalian cell fermentation broth properties: Enhanced impurity removal and clarification performance

The processing of recombinant proteins from high cell density, high product titer cell cultures containing mammalian cells is commonly performed using tangential flow microfiltration (MF). However, the increased cellular debris present in these complex feed streams can prematurely foul the membrane, adversely impacting MF capacity and throughput. In addition, high cell density cell culture streams introduce elevated levels of process-related impurities, which increase the burden on subsequent purification operations to remove these complex media components and impurities. To address this challenge, an evaluation of mammalian cell culture broth buffer properties was examined to determine if enhanced impurity removal and clarification performance could be achieved. A framework is presented here for establishing optimized mammalian cell culture buffer conditions, involving trade-offs between product recovery and purification and improved clarification at manufacturing-scale production. A reduction in cell culture broth pH to 4.7-5.0 induced flocculation and impurity precipitation which increased the average feed particle-size. These conditions led to enhanced impurity removal and improved MF throughput and filter capacity for several mammalian systems. Feed conditions were further optimized by controlling ionic composition along with pH to improve product recovery from high cell density/high product titer cell cultures.

Ultra scale‐down approaches for clarification of mammalian cell culture broths in disc‐stack centrifuges

Ultra-scale down (USD) methodology developed by University College London for cell broth clarification with industrial centrifuges was applied to two common cell lines (NS0 and GS-CHO) expressing various therapeutic monoclonal antibodies. A number of centrifuges at various scales were used with shear devices operating either by high speed rotation or flow-through narrow channels. The USD methodology was found effective in accounting for both gravitational and shear effects on clarification performance with three continuous centrifuges at pilot and manufacturing scales. Different shear responses were observed with the two different cell lines and even with the same cell line expressing different products. Separate particle size analysis of the treated broths seems consistent with the shear results. Filterability of the centrifuged solutions was also evaluated to assess the utility of the USD approach for this part of the clarification operation.

Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugation and its verification

This article describes the use of ultra scale-down studies requiring milliliter quantities of process material to study the clarification of mammalian cell culture broths using industrial-scale continuous centrifuges during the manufacture of a monoclonal antibody for therapeutic use. Samples were pretreated in a small high-speed rotating-disc device in order to mimic the effect on the cells of shear stresses in the feed zone of the industrial scale centrifuges. The use of this feed mimic was shown to predict a reduction of the clarification efficiency by significantly reducing the particle size distribution of the mammalian cells. The combined use of the rotating-disc device and a laboratory-scale test tube centrifuge successfully predicted the separation characteristics of industrial-scale, disc stack centrifuges operating with different feed zones. A 70% reduction in flow rate in the industrial-scale centrifuge was shown to arise from shear effects. A predicted 2.5-fold increase in throughput for the same clarification performance, achieved by the change to a centrifuge using a feed zone designed to give gentler acceleration of the bioprocess fluid, was also verified at large-scale.

Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection

The direct separation detection of amino acids by anion exchange chromatography with integrated pulsed amperometric detection was optimized for the analysis of typical mammalian cell culture broth samples. Existing gradient elution conditions were adapted, considering the additions of peptone (2g/L) and 10vol% fetal calf serum to the medium as well as changing concentrations of glucose from 5.5g/L up to complete consumption. Samples had to be analyzed in two dilutions with water (1:33.3 and 1:200) due to the strongly varying amino acid concentrations in the samples as a result of the medium composition and cell metabolism. The method was validated in a linear working range for the most common amino acids (2.5–7.5 and 1.25–3.75μM for cystine/cysteine with 15μl injection volume). The relative standard deviation of the method for all amino acids was less than 5%, with detection limits of less than 0.6μM and quantitation limits of less than 1.6μM. As an example, data for the amino acid composition of different media used for the production of inactivated influenza vaccines in cell culture are shown.

Filter Life After Cell Death: Impact of Viable, Non-Viable Apoptotic and Necrotic Mammalian Cells on Depth Filter Blinding

Intermittent and hitherto inexplicable blinding of depth filters have been experienced during the large-scale primary downstream processing of mammalian cell culture broths. Distinct populations of either healthy viable mammalian cells or those that had suffered cell death via apoptosis or necrosis were assessed for their ability to impact on the resistance of a depth filter of a type that is commonly used to remove cells in bioprocesses. Apoptotic and necrotic cells were found to induce a pressure drop 1.7 and 15.0 times greater than the corresponding pressure drop induced by viable cultures, respectively. There were no apparent explanations for this in terms of the size distribution of debris and cells. However, DNA removed from the cell culture supernatant was separated electrophoretically and the necrotic culture was clearly identified as containing chiefiy high molecular weight DNA, whereas the supernatant of viable and apoptotic cultures contained low molecular weight DNA. Further treatment of necrotic cultures with DNase 1 or benzonase resulted in these cultures having a reduced impact on the resistance of the depth filter, as compared with untreated necrotic cultures. The significance of high molecular weight DNA in the filtration of biological materials was clearly indicated.

Interaction of mammalian cell culture broth with adsorbents in expanded bed adsorption of monoclonal antibodies

The interaction of a mammalian cell culture broth with two commercially available adsorbents for the use in expanded bed adsorption (EBA) has been studied. A cation exchange resin (Streamline SP) and an affinity adsorbent (Streamline rProtein A) were compared with regard to adsorption of hybridoma cells during sample application as well as potential cell damage. The results showed that hybridoma cells interact significantly with an expanded bed of cation exchange adsorbents but not with the Protein A adsorbent. After application of 17–20 sedimented bed volumes a saturation of the Streamline SP resin with cells was noted. With both adsorbents no measurable cell damage was found and IgG1 was recovered in approximately 95% yield. The capacity for IgG1 adsorption at 3% breakthrough was 2.7 mg IgG1/ml Streamline rProtein A at a constant fluid velocity of 380 cm/h and 1.0 mg IgGl/ml Streamline SP at 215–240 cm/h fluid velocity.

Fluorometric determination of DNA in mammalian cell cultures by PicoGreen

An intercalating fluorochrome, PicoGreen, was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 91.8% of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples.