Characterization and improvement of cell line performance via flow cytometry and cell sorting

The improvement of specific productivity is a continuous challenge for bioprocesses involving mammalian cells, and hence, high‐throughput methods and low‐cost strategies are needed for the selection of high producers. The aim of this study was the productivity improvement of the hybridoma cell line IV F 19.23. For this purpose, a cell surface affinity matrix assay was established to identify and select high producers. This assay is based on the binding of secreted monoclonal antibodies to an affinity matrix assembled on the outer cell membrane. A protein microarray approach was used to investigate and optimize the functionality of the affinity matrix. The protein microarray was particularly useful to identify critical steps of the staining method, such as unspecific binding, before it was applied to the hybridoma cell line. Secreting hybridomas were treated with the affinity matrix and then selected via flow‐cytometric cell sorting in four consecutive bulk sort rounds. The applied bulk strategy, allowing low screening costs, resulted in a 125% increase in specific productivity of the cell line in comparison to the initial population.     

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

A Chinese Hamster Ovary cell line, CHO1-15₅₀₀, producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.065 pg cell⁻¹ h⁻¹) and early decline (0.040 pg cell⁻¹ h⁻¹) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase. The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity of 0.080 pg c-h⁻¹. Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization of the animal cell production system. This information facilitates rational process design, including operating mode, modelling and control, and medium formulation.     

Computer programs for modeling mammalian cell batch and fed-batch cultures using logistic equations

A MATLAB^® toolbox was developed for applying the logistic modeling approach to mammalian cell batch and fed-batch cultures. The programs in the toolbox encompass sensitivity analyses and simulations of the logistic equations in addition to cell specific rate estimation. The toolbox was first used to generate time courses of the sensitivity equations for characterizing the relationship between the logistic variable and the model parameters. Subsequently, the toolbox was used to describe CHO cell data from batch and fed-batch mammalian cell cultures. Cell density, product, glucose, lactate, glutamine, and ammonia data were analyzed for the batch culture while fed-batch analysis included cell density and product concentration. In all instances, experimental data were well described by the logistic equations and the resulting specific rate profiles were representative of the underlying cell physiology. The 6-variable batch culture data set was also used to compare the logistic specific rates with those from polynomial fitting and discrete derivative methods. The polynomial specific rates grossly misrepresented cell behavior in the initial and final stages of culture while those based on discrete derivatives had high variability due to computational artifacts. The utility of logistic specific rates to guide process development activities was demonstrated using specific protein productivity versus growth rate trajectories for the 3 cultures examined in this study. Overall, the computer programs developed in this study enable rapid and robust analysis of data from mammalian cell batch and fed-batch cultures which can help process development and metabolic flux estimation.     

Hybridoma growth and productivity: effects of conditioned medium and of inoculum size

Apart from gas concentrations, temperature, and pH, generally only the initial conditions can be manipulated in batch culture. Inoculum size and initial conditioned medium concentration represent two important considerations for optimal batch production. Two hybridoma cell lines were used to assess the impact of these initial conditions on population growth and monoclonal antibody productivity in suspension batch culture. Varying initial cell concentration over the range of 1.0 × 105 cells mL-1 to 3.0 × 105 cells mL-1 did not affect maximum product titre or maximum volumetric cell-hours attained. Initial percent of conditioned medium up to 40 percent strongly impacted on population growth and productivity, with initial levels of 30 to 40% conditioned medium reducing or eliminating lag phase and increasing average viable cell density. However, specific productivity and product titre declined with increasing initial percent conditioned medium, even on a per volume of fresh medium basis. Glutamine and glucose depletion or ammonia toxicity could cause depressed product titres when conditioned medium is used. Glutamine and glucose levels can easily be replenished in conditioned medium at minimal cost, and ammonia can be removed. Specific productivity was higher during cyclic batch operating mode than during batch operating mode. This may be because cyclic batch operating mode results in an incidental volume of conditioned medium at the beginning of each cycle. A two stage, cyclic-batch/batch operating mode can be employed to fully utilize medium and maximize product titre.     

Use of the weighted jackknife method to calculate the variance in cellular-specific protein secretion rate: application to monoclonal antibody secretion rate kinetics in response to osmotic stress

The specific secretion rate (q, mug protein secreted/viable cell-h) and its variance are very useful to compare the capability of cell lines for protein secretion. An assessment of specific secretion rate variability is also beneficial and important when the specific secretion rate is to be used as an on-line process parameter to monitor culture production behavior or for in-process decisionmaking. Experimental errors in mammalian cell culture (e.g., protein concentration measurement and cell counting) and estimation error in the method of calculating q contribute to the total variance of the specific secretion rate. Although the variance of q is essential for comparing the differences between cell lines and the response of the same cell line to different nutrient or environmental conditions, few methods for calculating the variance of the specific secretion rate have been reported. As a model system, we have used the weighted jackknife method and the delta method to calculate the variance in the specific secretion rate of a murine monoclonal antibody (q(mAb)) determined by a differential method. These methods were applied to calculate q(mAb) and its standard deviation to determine the change in q(mAb) kinetics during batch culture of the 9.2.27 hybridoma in response to growth in hyperosmotic media or osmotic stress. Without osmotic stress, during exponential growth in DMEM + 5% FBS spinner culture, the estimate of q(mAb) decreases at least threefold. Results indicate that the 9.2.27 hybridoma responds to hyperosmotic media (400 mOsm, 470 mOsm) by significantly reducing the degree of q(mAb) decrease in the exponential phase, thus maintaining a higher q(mAb) through the stationary phase. The trend of q(mAb) during the batch cultures studied is further confirmed by t-test. Osmotic stress is statistically shown to be able to alter significantly the hybridoma-specific mAb secretion kinetics during batch culture. Determination of the variance of specific secretion rate using the weighted jackknife method offers a powerful approach for establishing the confidence limits of specific protein secretion rate between cell cultures in different nutritional or osmotic environments. (c) 1996 John Wiley & Sons, Inc.     

Effects of glutamine supply on growth and metabolism of mammalian cells in chemostat culture

Glutamine is a major source of energy, carbon, and nitrogen for mammalian cells. The amount of glutamine present in commercial mammalian cell media is, however, not necessarily balanced with cell requirements. Therefore, the effects of glutamine limitation on the physiology of two mammalian cell lines were studied in steady-state chemostat cultures fed with IMDM medium with 5% serum. The cell lines used were MN12, a mouse-mouse hybridoma, and SP2/0-Ag14, a mouse myeloma often used in hybridoma fusions. Cultures, grown at a fixed dilution rate of 0.03 h(-1), were fed with media containing glutamine concentrations ranging from 0.5 to 4 mmol L(-1). Biomass dry weight and cell number were linearly proportional to the glutamine concentrations fed, between 0.5 and 2 mmol L(-1), and glutamine was completely consumed by both cell lines. From this it was concluded that glutamine was the growth-limiting substrate in this concentration range and that the standard formulation of IMDM medium contains a twofold excess of glutamine. In glutamine-limited cultures, the specific rates of ammonia and alanine production were low compared to glutamine-excess cultures containing 4 mmol L(-1) glutamine in the feed medium. The specific consumption rates of nearly all amino acids decreased with increasing glutamine feed, indicating that, in their metabolic function, they may partially be replaced by glutamine. Both cell lines reacted similarly to differences in glutamine feeding in all aspects investigated, except for glucose metabolism, In SP2/0-Ag14 glutamine feed concentrations did not affect the specific glucose consumption, whereas in MN12 this parameter increased with increasing amounts of glutamine fed. This systematic study using controlled culture conditions together with a detailed analysis of culture data shows that, although cells may react similarly in many aspects, cell-line-specific characteristics may be encountered even with respect to fundamental physiological responses like the interaction of the glutamine and glucose metabolism. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 272-286, 1997.     

Evaluation of insulin-mimetic trace metals as insulin replacements in mammalian cell cultures

Insulin is involved in a number of cellular functions, including the stimulation of cell growth, cell cycle progression and glucose uptake and is a common protein supplement in serum-free mammalian cell culture media. However, several trace metals have previously been reported to exhibit insulin-like effects on specific cell types. As a step towards developing chemically-defined, protein-free media for mammalian cells, we tested the effectiveness of five trace metals (cadmium, nickel, lithium, vanadium and zinc) as a replacement for insulin. Four cell lines of biotechnological relevance were used, including the hybridoma CRL1606, the myeloma NS0, and the Chinese hamster ovary cell lines CHO-IFN and CHO-K1. Zinc was found to be an effective insulin replacement for the hybridoma, myeloma and CHO-K1 cells. Cell growth, cell cycle progression and antibody production was not affected by the substitution. Furthermore, no adaptation procedure was required.     

Characterization of the stability of recombinant protein production in the GS-NS0 expression system

The GS-NS0 system is an important mammalian expression system used largely within industry for the high-level expression of recombinant proteins for therapeutic use. It is essential that the productivity of this system remains stable throughout culture expansion for the successful long-term production of recombinant proteins. Here we present a study of the stability of recombinant protein production from unamplified GS-NS0 cell lines over extended period of continuous culture. The cell lines used in this study were generated by the transfection of NS0 cells with DNA encoding for a secreted recombinant protein and by two subsequent rounds of limiting dilution cloning prior to analysis of stability. The stability of recombinant protein production was assessed at intervals over a period of 134 days using repeated batch culture in shake flasks. Heterogeneous stability was identified. The productivity of some clones remained consistent throughout 134 days of continuous culture. Others exhibit rapid and progressive loss of productivity. Analysis of the causal relationships underlying stability indicates that the initial transfectant determines the susceptibility to loss or retention of productivity. Selection of production clones on the basis of growth and productivity alone will not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.     

Physiological changes during the adaptation of hybridoma cells to low serum and serum-free media

Two murine hybridoma cell lines (167.4G5.3 and S3H5/gamma2bA2) were adapted to grow in low-serum and serum-free media by a weaning procedure. The changes in cell growth, metabolic, and antibody production rates with adaptation were examined using biochemical and flow cytometric analyses. After adaptation to a particular serum level, the short-term serum response of the cells was experimentally determined. Specific growth rates, glucose and glutamine uptake and lactate and ammonia production rates, and specific antibody production rates were evaluated from the data. For both cell lines, an improvement in cell growth was observed after adaptation, and both higher growth rates and higher cell concentrations were obtained. The specific glucose and glutamine uptake rates and the lactate and ammonia production rates changed insignificantly with adaptation. Conversely, changes in the specific antibody production rate of the two cell lines differed. Cell line 167.4G5.3 showed a loss in antibody productivity at low serum levels, while the S3H5/gamma2bA2 kept its original productivity in low-serum-containing media. The intracellular antibody content for S3H5/gamma2bA2 cells remained unaltered by adaptation, but a low antibody containing cell population appeared in the 167.4G5.3 culture. The loss of specific antibody productivity in this cell line was due to the appearance of this population.     

Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cells.     

Bioactive recombinant human lactoferrin,derived from rice,stimulates mammalian cell growth

Today there is a concern about the use of animal source proteins and peptides in cell culture applications due to potential contamination by adventitious infectious pathogens. Recombinant production of these proteins using a plant host provides a safe and cost effective alternative. In this paper, we tested the effect of rice-derived recombinant human lactoferrin (rhLF) on mammalian cell growth. The purified rhLF was partially (about 50%) iron-saturated (pis-rhLF). Chemical modification of pis-rhLF generated apo-rhLF (<10% iron saturation) or holo-rhLF (>90% iron saturation). All three forms of rhLF (pis, apo, holo) promoted growth of intestinal cells (HT-29) measured as [³H]-thymidine incorporation or viable cell count, but holo-rhLF was most effective. Holo-rhLF was further tested on hybridoma, osteoblast, and human embryonic kidney cells. Results showed that holo-rhLF promoted cell growth and reduced cell doubling time. The concentration of holo-rhLF in media was critical in promoting cell growth and each cell line had different concentration dependence with the most effective range from 5 to 200 mg/L. The effect of rhLF on antibody production was determined using a hybridoma cell line. Significantly, more antibodies were produced by cells grown with holo-rhLF than cells grown without holo-rhLF. We also compared the effect of holo-rhLF to that of human transferrin, a component commonly used in cell culture media as an iron source. Holo-rhLF was as effective as human transferrin in promoting cell growth and antibody production. Considering all the data obtained, we conclude that rhLF from rice is effective in promoting mammalian cell growth and increasing cell productivity.     

Suspension culture process of MethA tumor cell for the production of heat-shock protein glycoprotein 96: process optimization in spinner flasks

Heat-shock proteins (HSPs) act like "chaperones", making sure that the cell's proteins are in the right shape and in the right place at the right time. Heat-shock protein glycoprotein 96 (gp96) is a member of the HSP90 protein family, which chaperones a number of molecules in protein folding and transportation. Heat-shock protein gp96 serves as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. Currently, heat-shock protein gp96 was only isolated from murine and human tissues and cell lines. An animal cell suspension culture process for the production of heat-shock protein gp96 by MethA tumor cell was developed for the first time in spinner flasks. Effects of culture medium and condition were studied to enhance the MethA tumor cell density and the production and productivity of heat-shock protein gp96. Initial glucose concentration had a significant effect on the heat-shock protein gp96 accumulation, and an initial glucose level of 7.0 g/L was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Cultures at an initial glutamine concentration of 3 and 6 mM were nutritionally limited by glutamine. At an initial glutamine concentration of 6 mM, the maximal viable cell density of 19.90 x 10(5) cells/mL and the maximal heat-shock protein gp96 production of 4.95 mg/L was obtained. The initial concentration of RPMI 1640 and serum greatly affected the MethA tumor cell culture process. Specifically cultures with lower initial concentration of RPMI 1640 resulted in lower viable cell density and lower heat-shock protein gp96 production. At an initial serum concentration of 8%, the maximal viable cell density of 19.18 x 10(5) cells/mL and the maximal heat-shock protein gp96 production of 5.67 mg/L was obtained. The spin rate significantly affected the cell culture process in spinner flasks, and a spin rate of 150 rpm was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Not only the cell density but also the production and productivity of heat-shock protein gp96 attained in this work are the highest reported in the culture of MethA tumor cell. This work offers an effective approach for producing heat-shock protein glycoprotein 96 from the cell culture process. The fundamental information obtained in this study may be useful for the efficient production of heat-shock protein by animal cell suspension culture on a large scale.     

Real-time monitoring of protein secretion in mammalian cell fermentation: measurement of monoclonal antibodies using a computer-controlled HPLC system (BioCad/RPM)

On-line, "real-time" monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG(1), without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer-controlled 15-L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell-free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real-time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement. (c) 1995 John Wiley & Sons, Inc.     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modulation of hybridoma cell growth and antibody production by coating cell culture material with extracellular matrix proteins

The influence of coating polystyrene tissue culture plates with different proteins on murine hybridoma cell growth and antibody production was investigated. Fibronectin, collagen I, bovine serum albumin and laminin were used to coat NUNC^® and COSTAR^® cell culture plates. Cell number and antibody concentration in culture fluids were quantified as indicators for cell viability, proliferation and productivity. Adhesive behaviour, morphology, expression of surface receptors of hybridoma cells and the presence of tyrosine-phosphorylated proteins in cell lysates were characterized by cell adhesion experiments, microscopy, flow cytometry and Western Blot analysis.It was shown that coatings with fibronectin (0.2 μg/ml) lead to a substantial improvement of cell growth by 50–70% and an increase of monoclonal antibody production by 100–120%.Collagen I coatings showed an improvement in cell growth by 30–70% and by 60% for the production of monoclonal antibodies. Coatings with BSA and laminin had minor effects on these parameters. It was found that the hybridoma cell lines used in this study did not express the α₂-chain of the α₂β₁-integrin, which is responsible for binding to collagen and laminin.However, the presence of β₁-integrin on the cell surface was shown, which should enable hybridoma cells to bind fibronectin. We propose, therefore, that fibronectin adsorption to cell culture materials may be a promising approach to enhance the production of monoclonal antibodies by cultivated hybridoma cells.     

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.     

A mathematical model of the cell cycle of a hybridoma cell line

A one-dimensional age-based population balance model of the cell cycle is proposed for a mouse-mouse hybridoma cell line (mm321) producing immunoglobulin G antibody to paraquat. It includes the four conventional cell cycle phases, however, G1 is divided into two parts (G1a and G1b). Two additional phases have been added, a non-cycling state G1', and a pre-death phase D. The duration of these additional phases is determined by cumulative glutamine content and ammonia concentration, respectively. It is assumed that glutamine is only consumed during G1 and antibody is only produced during G1b and S, the kinetics are assumed to be zero-order. Glucose is consumed throughout the cell cycle at a rate that is dependent upon its prevalent concentration. Ammonia and lactate are produced in direct proportion to glutamine and glucose consumption, respectively. Parameters in the model have been determined from experimental data or from fitting the model to post-synchronisation data. The model thus fitted has been used to successfully predict this cell lines behaviour in conventional batch culture at different initial glutamine concentrations, and in chemostat culture at steady-state and in response to a glutamine pulse. The model predicts viable cell, glutamine, glucose and lactate kinetics well, but there are some discrepancies in the prediction for ammonia and antibody. Overall, the results obtained support the assumptions made in the model relating to the regulation of cell cycle progression. It is concluded that this approach has the potential to be exploited with other cell lines and used in a model-based control scheme.