Mammalian Cell Culture Media Research Articles

Influence of medium composition on oxygen transfer rate in animal cell culture

AbstractExperiments were conducted in a 0.25 m diameter bubble column to investigate the effect of medium composition on oxygen transfer rate. Aqueous solutions, the composition of which mimics a mammalian cell culture medium, are used. The effect on oxygen transfer rate of additives used to protect cells against local hydrodynamic stresses induced by bubble coalescence and bursting is addressed, in the range of operating conditions (aeration rates) met in animal cell cultures.The mass transfer coefficient, the liquid viscosity and surface tension, and the bubble size distribution are measured as a function of liquid composition and of gas superficial velocity, allowing to decouple the effects of the different additive on kL and on a.

Binding site on human immunoglobulin G for the affinity ligand HWRGWV.

Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383-Asn389 (SNGQPEN) located in the C(H)3 domain. Subsequent modeling has suggested a possible three-dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG.

Purification of human immunoglobulin G via Fc-specific small peptide ligand affinity chromatography

Chromatographic resins of a family of linear Fc-binding hexamer peptides (HWRGWV, HYFKFD, and HFRRHL) exhibited the ability to selectively adsorb and isolate human IgG (hIgG) from complete mammalian cell culture medium (cMEM). Among them, the HWRGWV resin with a peptide density of 0.08 mequiv./g of resin was able to purify hIgG from cMEM with both purity and yield as high as 95%, comparable to Protein A and A2P agarose gels. The influences of N-terminal acetylation of the HWRGWV resin, ligand density on the resin, initial hIgG concentration, and temperature on IgG isolation were also investigated. The results indicate that these small peptide ligands, especially HWRGWV, offer a potential alternative to the use of Protein A or Protein G for large scale affinity chromatography.

Prediction of Key Analytes in Mammalian Cell Culture Media with On-Line near Infrared Spectroscopy

Prediction of Key Analytes in Mammalian Cell Culture Media with On-Line near Infrared Spectroscopy

Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography

Humanized monoclonal antibodies (mAbs) hold significant promise as biopharmaceuticals. One of the main challenges faced in the purification of mAbs is their separation from bovine serum albumin, which is the main protein present in most mammalian cell culture media. This paper discusses the purification of humanized mAb hIgG1-CD4 from CHO cell culture media by hydrophobic interaction membrane chromatography using a stack of microporous synthetic membranes. The effects of solution conditions on mAb solubility and binding on the membrane were first studied. The separation of a simulated mixture of bovine albumin and the mAb was then carried out to examine the feasibility of mAb purification. Separation experiments carried out under optimized conditions demonstrated that this membrane-based technique could be used for mAb purification from cell culture media. High purity (97%) and recovery (in excess of 97%) were obtained.

Design of Adsorptive Columns for Specific Pathogen Removal: Application to Staphylococcal Enterotoxin B

The removal of pathogens such as toxins, viruses, bacteria, and prions in human blood, mammalian cell culture media, fermentation broths, food items, and water streams has gained increasing importance in ensuring product safety and in combatting acts of terrorism. Adsorption processes can play an important role in removing such pathogens from solution without affecting other desirable components. Adsorptive columns that can remove specific families of pathogens would need to achieve a reduction of several logs in pathogen concentration. This requirement is much more stringent than the normal yield requirements associated with adsorptive separations aimed at product recovery and purification in a process stream. This paper considers the design of an adsorptive column aimed at reducing the concentration of infectious agents from a known volume of solution by several logs in a fixed amount of time. The general rate (GR) model of chromatography is used in the analysis, including all major transport and kinetic steps in the adsorption process. The theory, with no adjustable parameters, is shown to predict with great accuracy the effect of residence time on the log removal of staphylococcal enterotoxin B (SEB) from solution using an affinity resin with a small peptide (YYWLHH) that has been found to bind specifically to this toxin.

Hexamer peptide affinity resins that bind the Fc region of human immunoglobulin G

Abstract: A family of linear hexapeptides composed of histidine on the N‐terminus followed by aromatic amino acid(s) and positively charged amino acid(s) has been identified through a three‐step screening of a synthetic solid phase library. These peptides were able to recognize human immunoglobulin G (HIgG) through its Fc region, and their selectivity to Fc is comparable to Protein A. This is the first known report of short peptides that are able to bind HIgG by recognizing its Fc portion. One of the ligands from the identified family, HWRGWV, was examined for its ability to isolate HIgG from complex mixtures. It was found that HWRGWV possessed the potential to purify HIgG from complete mammalian cell culture medium containing 10% fetal calf serum with purity comparable to commercially available resins, but using milder elution conditions. HWRGWV bound all HIgG subclasses and IgGs from bovine, mouse, goat, and rabbit. The broad affinity spectrum as well as its Fc recognition ability may be useful in capturing and detecting both polyclonal and monoclonal antibodies.

Structure and assembly of PEO-PPO-PEO co-polymers in mammalian cell-culture media

We investigate the structure of a poly(ethylene oxide)-poly(propylene oxide)-poly (ethylene oxide) co-polymer, Pluronic® F127, in mammalian cell-culture media. It is well known that aqueous solutions of F127 gel at physiological temperatures with this transition, which corresponds to the formation of a close-packed cubic assembly of spherical micelles. Previous work has shown that in both mammalian cell minimum essential medium (MEM) and MEM with added fetal bovine serum, the gel phase boundary shifts to lower temperatures and concentrations as compared to pure water. Using DLS, we have found that at 25°C the critical micelle concentration (CMC) decreases in the presence of MEM. Our SANS studies at 25°C indicate that F127 in MEM-D2O also forms a close-packed cubic micellar gel, suggesting that the mechanism of gelation is the same in both pure water and MEM. Fits to the neutron spectra on 2 wt% F127 in D2O and MEM-D2O show a large difference in the micelle aggregation number (N agg) and a small difference in the micelle size, with both N agg and the micelle size larger in the presence of MEM. In addition, our SANS spectra in MEM-D2O indicate repulsive interactions between micelles at 2 wt% polymer, whereas no correlation peak is observed for this concentration in water. Finally, moderately concentrated samples (5–18 wt% polymer) in MEM exhibit slightly stronger ordering and sharper peaks, perhaps indicating stronger intermicellar interactions in the presence of MEM. This stronger repulsive interaction may be the cause of the shift in the liquid–gel phase boundary that is observed.

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.

Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan

Several excellent procedures for trapping tagged proteins have been devised, but many of these are expensive, cannot be used outside a limited pH range, fail to work in the presence of chaotropic agents, or are difficult to use. The chitin binding domain (CBD) of Bacillus circulans chitinase, which binds to chitin matrices prepared from inexpensive reagents isolated from crab shells, is an alternative tag that can be used under a variety of pH and denaturing conditions. Kits based on the interaction between the CBD and the chitin beads are available commercially. Here, we show that simultaneous treatment of microtiter plates with chitosan, a deacetylated form of chitin, and acetic anhydride produces a surface-bound film of chitin that also interacts tightly with the CBD. Chitin-coated microtiter well plates captured a CBD-tagged heterodimeric human glycoprotein hormone analog directly from mammalian cell culture media, even when present in trace amounts. Binding to the surface was stable in sodium dodecylsulfate and reversed only partially at low pH or in 8 M urea at 37 °C. This technique appears well suited to surface attachment and permits biochemical or other analyses of molecules that can be tagged with a CBD.

Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection

Cell culture and fermentation broth media are used in the manufacture of biotherapeutics and many other biological materials. Characterizing the amino acid composition in cell culture and fermentation broth media is important because deficiencies in these nutrients can reduce desired yields or alter final product quality. Anion-exchange (AE) chromatography using sodium hydroxide (NaOH) and sodium acetate gradients, coupled with integrated pulsed amperometric detection (IPAD), determines amino acids without sample derivatization. AE-IPAD also detects carbohydrates, glycols, and sugar alcohols. The presence of these compounds, often at high concentrations in cell culture and fermentation broth media, can complicate amino acid determinations. To determine whether these samples can be analyzed without sample preparation, we studied the effects of altering and extending the initial NaOH eluent concentration on the retention of 42 different carbohydrates and related compounds, 30 amino acids and related compounds, and 3 additional compounds. We found that carbohydrate retention is impacted in a manner different from that of amino acid retention by a change in [NaOH]. We used this selectivity difference to design amino acid determinations of diluted cell culture and fermentation broth media, including Bacto yeast extract–peptone–dextrose (yeast culture medium) broth, Luria–Bertani (bacterial culture medium) broth, and minimal essential medium and serum-free protein-free hybridoma medium (mammalian cell culture media). These media were selected as representatives for both prokaryotic and eukaryotic culture systems capable of challenging the analytical technique presented in this paper. Glucose up to 10 mM (0.2%, w/w) did not interfere with the chromatography, or decrease recovery greater than 20%, for the common amino acids arginine, lysine, alanine, threonine, glycine, valine, serine, proline, isoleucine, leucine, methionine, histidine, phenylalanine, glutamate, aspartate, cystine, and tyrosine.

Quantitation of underivatized free amino acids in mammalian cell culture media using matrix assisted laser desorption ionization time-of-flight mass spectrometry.

In this investigation, a quantitative matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) method was developed for the analysis of underivatized free amino acids in mammalian cell culture media. Calibration curves were developed for 12 amino acids over the linear range of 1-100 microM with coefficients of determination ranging from r2 = 0.9220 to r2 = 0.9973. An aerospray method was utilized for the sample deposition method, and the matrix, alpha-cyano-4-hydroxycinnamic acid, served as the internal standard. This assay was used to analyze bioreactor samples from five time points in the process. Concentrations determined through interpolation of the calibration curves were comparable to those obtained via reversed-phase HPLC based analysis with an average percent difference of 19.71%. Repeatability and intermediate precision studies were also performed, and the relative standard deviations ranged from 0.5943 to 21.41 and 3.157 to 18.97, respectively.

Optimizing expression and purification from cell culture medium of trispecific recombinant antibody derivatives

Antibody fragments offer the possibility to build multifunctional manifolds tailored to meet a large variety of needs. We optimized the production of a manifold consisting of one (bibody) or two (tribody) single-chain variable fragments coupled to the C-terminus of Fab chains. Different strong mammalian promoters were compared and the influence of expression media on production and recovery was investigated. Since the physical and chemical nature of these molecules largely depends on the nature of the antibody building blocks incorporated, a generally applicable process for the purification of recombinant antibody derivatives from serum containing mammalian cell culture medium was designed. To this end we compared protein L, hydroxyapatite, immobilized metal affinity chromatography, cation-exchange chromatography and hydrophobic charge induction chromatography.

Effect of mammalian cell culture medium on the gelation properties of Pluronic ® F127

We investigate the gelation of a poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) copolymer, Pluronic ® F127, in mammalian cell culture medium for applications in tissue engineering and cell encapsulation. In both minimum essential medium (MEM) and MEM with added fetal bovine serum (MEM-FBS), the gel-phase boundary shifts to lower temperatures and concentrations as compared to pure water. The thermodynamics of gel formation are similar in MEM, MEM-FBS, and pure water, suggesting that the mechanism of gelation is similar in all three solvents. The shift of the sol–gel boundary to lower concentrations is particularly significant for development of cell encapsulation protocols using Pluronics ® and applications where copolymer concentration must be minimized due to toxicity concerns.

Economic analysis of immunoadsorption systems

Immunoadsorption possesses an attractive combination of selectivity and yield, and in some cases may replace many conventional purification steps with a single step. The economic performance of immunoadsorption is dependent on apparatus configuration (e.g. Sepharose ® bed, perfusive bed and membrane), operating conditions (e.g. bed depth, flow rate and concentration of adsorbate in the effluent at breakthrough) and antibody binding kinetics. The solution space for the lowest operating cost is potentially very large and is prohibitive to exhaustively investigate in the laboratory. A mathematical model of a generic immunoadsorption process is useful in identifying the most promising configurations, operating conditions and antibodies, without having to resort to experiments that explore every possibility. The model system was chosen to represent the purification of a valuable biochemical product (1000 mg) from mammalian cell culture media or milk where the protein is present at low concentration (2 mg l −1). The antigen–antibody kinetic rate constants for a bovine serum albumin–monoclonal antibody system are available in the literature and were representative for this study. A modified chromatographic model was developed to predict the dimensionless operating cost of the generic immunoadsorption system subject to different apparatus configurations, operating conditions and antibody kinetics. Optimal operating conditions for each of the three configurations were identified. Under these conditions, the operating costs for a perfusive bed and a membrane were comparable and 28% lower than for a packed bed. The antibody binding kinetics significantly affected the dimensionless operating cost of the system and, therefore, the model can help select the most suitable antibody from a panel of prospective antibodies.

Identification of lysine decarboxylase as a mammalian cell growth inhibitor in Eikenella corrodens: possible role in periodontal disease

The pathogenesis of inflammatory periodontal disease was studied by examining the mechanism of HeLa and HL60 cell growth inhibition by cell-free saline-soluble extracts of Eikenella corrodens and bacterial plaque. Previous studies identified a protein (p80) as causing growth inhibition by E. corrodens extracts. After purification by two-dimensional SDS-PAGE, p80 was digested with protease lysC. Amino acid sequences were obtained and backtranslated for use as PCR primers. A 5840 nucleotide sequence containing a lysine decarboxylase gene was obtained from a Sau3 A1 genomic library ofE. corrodens DNA. Lysine decarboxylase activity was present at physiologic pH in the E. corrodens extracts containing p80, and also in bacterial plaque. Both extracts caused growth inhibition by depleting lysine from cell culture media through conversion to cadaverine. Adding lysine, or immune goat IgG to a peptide derived from the active site sequence of E. corrodens lysine decarboxylase, retarded lysine depletion and growth inhibition. -Amino caproic acid specifically enhanced lysine decarboxylase activity at the low lysine concentration in HL60 cell culture media, and also increased the growth inhibition. Thus, lysine decarboxylases such as p80 inhibit growth by removing lysine from mammalian cell culture media. A new role for lysine decarboxylase activity in the microbial aetiology of periodontal disease is discussed.

Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media

Ammonium ion accumulation in mammalian cell culture media causes toxicity which inhibits cell growth and productivity. To reduce the level of the accumulated ammonium ion, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC) were used, which catalyze the first and second steps of the urea cycle in the liver. To examine the effects of overexpressed CPS I and OTC genes on the concentration of the ammonium ion in culture media, the two genes were introduced into Chinese hamster ovary (CHO) dhfr-cells. The CPS I expressing cell lines (CPS I-CHO) and both CPS I and OTC expressing cell lines (CPS I/OTC-CHO) were confirmed at the mRNA level and analyzed in terms of the cell growth and the accumulation of ammonium ion in culture media. The accumulation of ammonium ion was ≈25–33% less in CPS I/OTC-CHO than in either CPS I-CHO or the vector-control cell lines. Interestingly however, the cell growth was ≈15–30% faster in both CPS I-CHO and CPS I/OTC-CHO than in the control cell lines. Forced expression of urea cycle enzymes in the CHO cells revealed that both the expression of CPS I and OTC can reduce the accumulation of ammonium ion in the culture media.

Analyzing mixtures of amino acids and carbohydrates using bi-modal integrated amperometric detection

Described in this work is a new detection methodology – bi-modal integrated amperometric detection – for identifying peaks and as a tool for solving difficult separation problems. Bi-modal integrated amperometry makes it possible to selectively detect amino acids, amino sugars, and carbohydrates following their separation by anion-exchange. Selectivity is gained by two different methods of integrating anodic current on an otherwise identical waveform. As with the single-mode integrated amperometry reported previously, the limits of detection are in the femtomole range and linear calibration plots are possible over three orders of magnitude. This new detection method does not require analyte derivatization. The practical utility of this new technique is demonstrated in the analysis of amino acids and sugars in a recombinant mammalian cell culture medium.

Enzyme-Based Flow Injection Analysis System for Glutamine and Glutamate in Mammalian Cell Culture Media

We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the technical setup are presented in this paper.

Ammonia measurements in mammalian cell culture media with a diffuse reflectance-based fiberoptic ammonia sensor.

A solid-state, diffuse reflectance-based fiberoptic sensor is described for quantifying ammonia. This sensor is constructed by immobilizing chlorophenol red, a weak acid chromophoric indicator dye, in a microporous polypropylene membrane. A flow-injection analysis system is used to carry a 10-μL aliquot of the sample across the treated membrane. Ammonia in the sample diffuses through the air-filled pores within the membrane structure before reacting with the indicator dye. A reversible acid-base reaction between ammonia and chlorophenol red results in a measurable change in the reflectance at 560 nm. Response characteristics include a peak response within 20 s, a limit of detection of 0.2 ± 0.1 mM ammonia, and a dynamic range of up to 60 mM ammonia. The analytical utility of this sensor is demonstrated by measuring ammonia levels in 48 individual samples collected during the growth of PC3 human prostate cancer cells in a typical serum-containing growth medium. Accuracy of the proposed sensor is verified by a comparison of results from this sensor to those from a conventional enzyme assay.