Protein-free Conditions Research Articles

Design principles of PI(4,5)P2 clustering under protein-free conditions: Specific cation effects and calcium-potassium synergy

Phosphatidylinositol 4,5-bisphosphate (PIP2) clustering is a key component in cell signaling, yet little is known about the atomic-level features of this phenomenon. Network-theoretic analysis of multimicrosecond atomistic simulations of PIP2 containing asymmetric bilayers under protein-free conditions, presented here, reveals how design principles of PIP2 clustering are determined by the specific cation effects. Ca2+ generates large clusters (6% are pentamer or larger) by adding existing PIP2 dimers formed by strong O‒Ca2+‒O bridging interactions of unprotonated P4/P5 phosphates. In contrast, monovalent cations (Na+ and K+) form smaller and less-stable clusters by preferentially adding PIP2 monomers. Despite having the same net charge, the affinity to P4/P5 is higher for Na+, while affinity toward glycerol P1 is higher for K+. Consequently, a mixture of K+ and Ca2+ (as would be produced by Ca2+ influx) synergistically yields larger and more stable clusters than Ca2+ alone due to the different binding preferences of these cations.

Multiscale Molecular Dynamics Studies Reveal Different Modes of Receptor Clustering by Gb3-Binding Lectins

The recognition of carbohydrate receptors on host cell membranes by pathogenic lectins is a crucial step in the microbial invasion. Two bacterial lectins, the B-subunit of Shiga toxin from Shigella dysenteria (StxB) and lectin I from Pseudomonas aeruginosa (LecA), are specific to the same galactolipid-globotriaosylceramide (Gb3). In this study we present a coarse-grained (cg) model of Gb3, which we further apply to unravel the molecular details of glycolipid binding by two lectins on the surface of a DOPC/cholesterol/Gb3 bilayer. In cg molecular dynamics simulations with time scales of dozens of microseconds, Gb3 was randomly distributed. The binding of both StxB or LecA is accompanied by Gb3 clustering in a cholesterol environment and with exclusion of DOPC in protein vicinity. StxB being bound by all 15 binding sites induced membrane bending, while LecA interacted with two out of four binding sites for most of the time causing a smaller inward curvature of the model membrane. Stable interactions occurred preferably when LecA was normal to the membrane surface. Furthermore, all-atom simulations revealed that LecA bound Gb3's headgroup at only one out of two possible conformations of the carbohydrate moiety observed at protein-free conditions. The results shed light on the mechanism of interactions between two lectins and Gb3 on the membrane surface and offer a coarse-grained model to study more complex systems at large spatiotemporal scales.

Serum Lowers Bioactivity and Uptake of Synthetic Amorphous Silica by Alveolar Macrophages in a Particle Specific Manner.

Various cell types are compromised by synthetic amorphous silica (SAS) if they are exposed to SAS under protein-free conditions in vitro. Addition of serum protein can mitigate most SAS effects, but it is not clear whether this is solely caused by protein corona formation and/or altered particle uptake. Because sensitive and reliable mass spectrometric measurements of SiO2 NP are cumbersome, quantitative uptake studies of SAS at the cellular level are largely missing. In this study, we combined the comparison of SAS effects on alveolar macrophages in the presence and absence of foetal calf serum with mass spectrometric measurement of 28Si in alkaline cell lysates. Effects on the release of lactate dehydrogenase, glucuronidase, TNFα and H2O2 of precipitated (SIPERNAT® 50, SIPERNAT® 160) and fumed SAS (AEROSIL® OX50, AEROSIL® 380 F) were lowered close to control level by foetal calf serum (FCS) added to the medium. Using a quantitative high resolution ICP-MS measurement combined with electron microscopy, we found that FCS reduced the uptake of particle mass by 9.9% (SIPERNAT® 50) up to 83.8% (AEROSIL® OX50). Additionally, larger particle agglomerates were less frequent in cells in the presence of FCS. Plotting values for lactate dehydrogenase (LDH), glucuronidase (GLU) or tumour necrosis factor alpha (TNFα) against the mean cellular dose showed the reduction of bioactivity with a particle sedimentation bias. As a whole, the mitigating effects of FCS on precipitated and fumed SAS on alveolar macrophages are caused by a reduction of bioactivity and by a lowered internalization, and both effects occur in a particle specific manner. The method to quantify nanosized SiO2 in cells is a valuable tool for future in vitro studies.

In Vitro Evaluation of a Peptide-Mesoporous Silica Nanoparticle Drug Release System against HIV-1

It has been shown that the optimized VIR-576 derivative of the natural HIV-1 entry inhibitor targeting the viral gp41 fusion peptide is safe and effective in infected individuals. However, high doses of this peptide were required, and stability, as well as delivery, must be improved for clinical application. Here, we examined the loading and release of VIR-576 into/from mesoporous silica nanoparticles (MSNs) in vitro. We found that a moderately high peptide loading of 11.5 wt % could be achieved by adsorption from PBS buffer (pH 7.2), i.e., under mild, fully peptide-compatible conditions. The release rate of peptide into the same buffer was slow and the equilibrium concentration as indicated by the adsorption isotherm could not be reached even within 50 h at the particle concentrations studied. However, a faster release was observed at lower particle concentrations, indicating that partial particle dissolution had a positive influence on peptide release. To determine the antiviral activity of VIR-576-loaded MSNs, TZM-bl indicator cells were exposed to HIV-1 and the infection rates were followed as a function of time and VIR-576 concentration. The inhibitory activity observed for VIR-576 released from the MSNs was virtually identical to that of free VIR-576 at the 48 h time point, indicating that (a) VIR-576 was released in an active form from the MSNs, and (b) the release rate in the presence of serum proteins was clearly higher than that observed under protein-free conditions. These observations are discussed based on competitive peptide/protein adsorption, as well as potential influences of serum proteins on the dissolution-reprecipitation of silica under conditions where the total silica concentration is above the saturation level for dissolved silica. Our results highlight the need for studying drug release kinetics in the presence of serum proteins, in order to allow for a better extrapolation of in vitro data to in vivo conditions. Furthermore, due to the high peptide loadings that can be achieved using MSNs as carriers, such a formulation appears promising for local release applications. For systemic administration, however, peptides with a higher potency would be needed, due to their high molar masses limiting the drug loading in terms of moles per gram carrier.

Effects of Ultrasonic Dispersion Energy on the Preparation of Amorphous SiO2 Nanomaterials for In Vitro Toxicity Testing

Synthetic amorphous silica (SAS) constitute a large group of industrial nanomaterials (NM). Based on their different production processes, SAS can be distinguished as precipitated, fumed, gel and colloidal. The biological activity of SAS, e.g., cytotoxicity or inflammatory potential in the lungs is low but has been shown to depend on the particle size, at least for colloidal silica. Therefore, the preparation of suspensions from highly aggregated or agglomerated SAS powder materials is critical. Here we analyzed the influence of ultrasonic dispersion energy on the biologic activity of SAS using NR8383 alveolar macrophage (AM) assay. Fully characterized SAS (7 precipitated, 3 fumed, 3 gel, and 1 colloidal) were dispersed in H2O by stirring and filtering through a 5 µm filter. Aqueous suspensions were sonicated with low or high ultrasonic dispersion (USD) energy of 18 or 270 kJ/mL, respectively. A dose range of 11.25–90 µg/mL was administered to the AM under protein-free conditions to detect particle-cell interactions without the attenuating effect of proteins that typically occur in vivo. The release of lactate dehydrogenase (LDH), glucuronidase (GLU), and tumor necrosis factor α (TNF) were measured after 16 h. Hydrogen peroxide (H2O2) production was assayed after 90 min. The overall pattern of the in vitro response to SAS (12/14) was clearly dose-dependent, except for two SAS which showed very low bioactivity. High USD energy gradually decreased the particle size of precipitated, fumed, and gel SAS whereas the low adverse effect concentrations (LOECs) remained unchanged. Nevertheless, the comparison of dose-response curves revealed slight, but uniform shifts in EC50 values (LDH, and partially GLU) for precipitated SAS (6/7), gel SAS (2/3), and fumed SAS (3/3). Release of TNF changed inconsistently with higher ultrasonic dispersion (USD) energy whereas the induction of H2O2 was diminished in all cases. Electron microscopy and energy dispersive X-ray analysis showed an uptake of SAS into endosomes, lysosomes, endoplasmic reticulum, and different types of phagosomes. The possible effects of different uptake routes are discussed. The study shows that the effect of increased USD energy on the in vitro bioactivity of SAS is surprisingly small. As the in vitro response of AM to different SAS is highly uniform, the production process per se is of minor relevance for toxicity.

Dynamic analysis of the interactions between Si/SiO2 quantum dots and biomolecules for improving applications based on nano-bio interfaces

Due to their outstanding properties, quantum dots (QDs) received a growing interest in the biomedical field, but it is of major importance to investigate and to understand their interaction with the biomolecules. We examined the stability of silicon QDs and the time evolution of QDs – protein corona formation in various biological media (bovine serum albumin, cell culture medium without or supplemented with 10% fetal bovine serum-FBS). Changes in the secondary structure of BSA were also investigated over time. Hydrodynamic size and zeta potential measurements showed an evolution in time indicating the nanoparticle-protein interaction. The protein corona formation was also dependent on time, albumin adsorption reaching the peak level after 1 hour. The silicon QDs adsorbed an important amount of FBS proteins from the first 5 minutes of incubation that was maintained for the next 8 hours, and diminished afterwards. Under protein-free conditions the QDs induced cell membrane damage in a time-dependent manner, however the presence of serum proteins attenuated their hemolytic activity and maintained the integrity of phosphatidylcholine layer. This study provides useful insights regarding the dynamics of BSA adsorption and interaction of silicon QDs with proteins and lipids, in order to understand the role of QDs biocorona.

Visualization of RNA structure models within the Integrative Genomics Viewer.

Analyses of the interrelationships between RNA structure and function are increasingly important components of genomic studies. The SHAPE-MaP strategy enables accurate RNA structure probing and realistic structure modeling of kilobase-length noncoding RNAs and mRNAs. Existing tools for visualizing RNA structure models are not suitable for efficient analysis of long, structurally heterogeneous RNAs. In addition, structure models are often advantageously interpreted in the context of other experimental data and gene annotation information, for which few tools currently exist. We have developed a module within the widely used and well supported open-source Integrative Genomics Viewer (IGV) that allows visualization of SHAPE and other chemical probing data, including raw reactivities, data-driven structural entropies, and data-constrained base-pair secondary structure models, in context with linear genomic data tracks. We illustrate the usefulness of visualizing RNA structure in the IGV by exploring structure models for a large viral RNA genome, comparing bacterial mRNA structure in cells with its structure under cell- and protein-free conditions, and comparing a noncoding RNA structure modeled using SHAPE data with a base-pairing model inferred through sequence covariation analysis.

How High Concentrations of Proteins Stabilize the Amorphous State of Calcium Orthophosphate: A Solid-State Nuclear Magnetic Resonance (NMR) Study of the Casein Case.

Understanding how proteins stabilize amorphous calcium ortho-phosphate (ACP) phases is of great importance in biology and for pharmaceutical or food applications. Until now, most of the former investigations about ACP-protein stability and equilibrium were performed under conditions where ACP colloidal nanoclusters are surrounded by low to moderate concentrations of peptides or proteins (15-30 g L-1). As a result, the question of ACP-protein interactions in highly concentrated protein systems has clearly been overlooked, whereas it corresponds to actual industrial conditions such as drying or membrane filtration in the dairy industry for instance. In this study, the structure of an ACP phase is monitored in association with one model phosphorylated protein (casein) using solid-state nuclear magnetic resonance (ssNMR) under two conditions of high protein concentration (300 and 400 g L-1). At both concentrations and at 25 °C, it is found that the caseins maintain the mineral phase in an amorphous form with no detectable influence on its structure or size. Interestingly, and in both cases, a significant amount of the nonphosphorylated side chains interacts with ACP through hydrogen bonds. The number of these interacting side chains is found to be higher at the highest casein concentration. At 45 °C, which is a destabilizing temperature of ACP under protein-free conditions, the amorphous structure of the mineral phase is partially transformed at a casein concentration of 300 g L-1, while it remains almost intact at a casein concentration of 400 g L-1. Therefore, these results clearly indicate that increasing the concentration of proteins favors ACP-protein interactions and stabilizes the ACP clusters more efficiently.

Validation of a total IC50 method which enables in vitro assessment of transporter inhibition under semi-physiological conditions

1. Accurate predictions of clinical transporter-mediated drug–drug interactions (DDI) from in vitro data can be challenging when compounds have poor solubility and/or high nonspecific binding. Additionally, current DDI predictions for compounds with high plasma–protein binding assume that the unbound fraction in plasma is 0.01, if the experimental value is less than 0.01 or cannot be determined. This approach may result in an overestimation of DDI risk. To overcome these challenges, it may be beneficial to conduct inhibition studies under physiologically relevant conditions.2. Here, IC50 values, determined in the presence of 4% bovine serum albumin approximating human plasma albumin concentrations, were successfully used to predict DDI for uptake transporters, OATP1B1/1B3, OCT1/2, OAT1/3 and MATE1/2K.3. The IC50 values of reference inhibitors with 4% bovine serum albumin, considered total IC50, were comparable to the predicted values based on nominal IC50 values determined under protein-free conditions and unbound fraction in plasma. Calculation of R-total and Cmax/IC50,total values using total plasma exposure and total IC50 values explained the clinical DDI or absence of it for these inhibitors.4. These results suggest that IC50 determinations in the presence of 4% albumin can be used, in the context of clinical total exposure, to predict DDI involving uptake transporters.

Adaptation of CHO cells in serum-free conditions for erythropoietin production: Application of EVOP technique for process optimization.

Mammalian cell cultures are the preferred expression systems for the production of biopharmaceuticals requiring posttranslational processing. Usually, cell cultures are cultivated in medium supplemented with serum, which supports cell proliferation, viability, and productivity. However, due to scientific and regulatory concerns, serum-free conditions are required in recombinant protein production. Cell lines that are intended for commercial recombinant protein production have to adapt to serum- or protein-free conditions early in their development. This is a labor- and time-consuming process because of the specific cell requirements related to their adaptation in new microenvironment. In the present study, a Chinese hamster ovary (CHO) cell line producing glycosylated recombinant human erythropoietin (rhEPO) was adapted for growth and rhEPO production in serum- and protein-free conditions. The physiology, growth parameters, and morphology of the CHO cells and rhEPO biosynthesis and structure were closely monitored during the adaptation process to avoid unwanted selection of cell subpopulations. The results showed that the CHO cells were successfully adapted to suspension growth and rhEPO production in the protein-free conditions and that the structure of rhEPO remained nearly unchanged. In addition, during rhEPO production in the protein-free suspension conditions, the agitation rate seem to be significant for optimal process performance in contrast to the initial cell concentration, evaluated through evolutionary operation method.

Surface functionalization affects the zeta potential, coronal stability and membranolytic activity of polymeric nanoparticles

Nano materials are commonly functionalized to boost their physicochemical properties. However, there is little known about the impact of these modifications on cellular systems. Herein, we synthesized eight types of polymeric nanoparticles (NPs) bearing different functional groups, and investigated their effects on interactions with cellular membranes. As models for particle membrane interactions, hemolysis assays using human red blood cells and culture with A549 cells were utilized. Under protein-free conditions, the NPs showed a wide distribution of zeta potentials (ζPs) which showed a good correlation with their hemolytic potential. However, in the presence of serum or lung lining fluid, the ζPs of all NPs coalesced towards a single common negative value and showed neither hemolytic activity nor cytotoxicity to A549 cells. Lipase and protease treatment of the coronated particles did not restore their reactivity. These result simply proves that particle functionalization influences the stability of the particle corona which, if intact, prevents hemolytic activity and membrane disrupture.

First-pass metabolism of peptide drugs in rat perfused liver.

To elucidate the extent and mechanisms of the first-pass metabolism of peptide drugs in the liver after oral administration, a liver perfusion study was performed in rats using metkephamid, a stable analogue of methionine enkephalin, and thyrotropin-releasing hormone (TRH), as model peptides. The fraction of intact metkephamid recovered after single-pass constant perfusion through rat liver reached steady-state very quickly, and it was concluded that metkephamid was hydrolysed enzymatically at the surface of hepatocytes or endothelial cells of microvessels, or both, rather than being taken up by hepatocytes. The fraction of metkephamid recovered intact was approximately 40% under protein-free conditions but increased to 70-75% on addition of bovine serum albumin (BSA) to the perfusate. The fraction of metkephamid bound to BSA was approximately 50% under these conditions, implying that only the free fraction of metkephamid in the plasma was metabolized in the liver. Calculations based on the tube model showed that approximately 30-35% of metkephamid absorbed from the intestine undergoes first-pass metabolism before entering the systemic circulation in-vivo. In contrast, the fraction of TRH metabolized in the liver was less than 10%, indicating a remarkably low contribution of first-pass metabolism to the bioavailability of TRH. These results show that hepatic first-pass metabolism of metkephamid contributes to its low systemic bioavailability. After intestinal absorption free metkephamid is rapidly hydrolysed on the surface of hepatocytes or endothelial cells, rather than being taken up by hepatocytes. This information has important implications in the oral delivery of many kinds of peptide.

Removal of TSE agents by depth or membrane filtration from plasma products

The removal of the abnormal form of prion protein i.e. PrP SC by filtration steps in the plasma fractionation process has been investigated by immuno-Western blotting. Depth filtration has been shown to be capable of removing scrapie by 2–3 log from certain plasma product intermediates. These include cryoprecipitate supernatant, used for the manufacture of immunoglobulin and albumin, and albumin fraction V, by filtration using Pall Seitz or 3m Cuno depth filters respectively. However no significant removal occurred with immunoglobulin Fraction II after Cuno depth filtration. When 0.2 μm PVDF and Nylon membrane filters were tested, the removal of TSEs from 20% albumin was limited i.e. 0.6–1.3 log. However under protein free conditions using phosphate buffered saline, filtration was not effective in the case of a PVDF filter but very effective i.e. >2.9 log in the case of a Nylon filter.

Abstracts

In vitro models predicting the metabolic clearance of new chemical entities have contributed greatly to the selection of lead compounds with favorable pharmacokinetic properties in humans. However, in vitro-in vivo predictions are still not always accurate. An important determinant of the outcome of predictions and, therefore, a potential source of error, is the in vivo free fraction (fu) of a compound in plasma required to extrapolate the intrinsic clearance (obtained in vitro under protein-free conditions) to clearance in vivo. To investigate the relation between metabolic clearance and fu, we determined the metabolic rate of testosterone and methyltestosterone (at substrate concentrations <Km) in precision-cut liver slices in medium with increasing concentrations of plasma (0-100%) and albumin (0-4%) and measured fu by ultracentrifugation in medium supplemented with the same concentrations of plasma or albumin. In theory, at concentrations well below Km, the metabolic rate is proportional to the free concentration of a drug at the enzyme site. At low plasma/albumin concentrations, the metabolic rate was, indeed, found to be proportional to the extracellular free concentration. However, at higher plasma and albumin concentrations, the metabolic rate was higher than predicted from the free concentration. From the difference between the metabolic rate in slices incubated under protein-free conditions and in 100% plasma or 4% w/v albumin, an apparent free fraction of 0.19 and 0.24, respectively, for testosterone and 0.13 and 0.23, respectively, for methyltestosterone was calculated, whereas the measured free fractions for these compounds were 0.02 in 100% plasma and 0.09 in 4% w/v albumin. In conclusion, for both compounds used in this study, the intracellular free drug concentration at the enzyme site does not seem to correlate with the extracellular free fraction at physiological protein concentration. As a consequence, using the fu measured in plasma to calculate in vivo clearance from in vitro data, measured in protein-free medium, is an important source of underprediction of in vivo clearance.

Combined Action of Endothelial and Mesenchymal Niche Cells to Amplify Hematopoietic Progenitor Expansion in a Humanized System

The hematopoietic stem cell (HSC) niche is an anatomically confined space governing HSC proliferation, differentiation and self renewal. Recent research identified distinct compartments described as stromal and vascular niches. This study was initiated to directly compare mesenchymal stromal cell (MSC) and vascular endothelial progenitor cell (EPC) contribution to niche functions in a humanized co-culture system. MSCs and EPCs were propagated under animal protein-free conditions following recently established protocols. Autologous MSC-EPC pairs were established to avoid donor variation. Purified CD34+ hematopoietic progenitor cells (HPCs) and HSCs were used as responders in a cytokine-driven (IL-3, 6ng/mL; Flt-3-L, 50ng/mL; SCF, 20ng/mL) niche cell-regulated HPC co-culture system. Four different standard media were employed to compare fully serum-free conditions with humanized cultures that were supplemented with pooled human platelet lysate (pHPL) as a source of platelet-derived growth factors present within the niche. Primary expansion culture with and without niche cell support was followed by colony forming cell (CFC) assays to determine maintenance of clonogenicity. Humanized liquid cultures supplemented with 10% pHPL without niche cell support were more efficient than parallel serum-free cultures under three of four medium conditions resulting in a mean 16–84-fold increase in nucleated cell progeny within 11 days. Both MSCs and EPCs further amplified HPC proliferation, MSC actually up to 341-fold, confirming recently published results. Interestingly, the supportive effect of MSCs or EPCs was constantly more pronounced in humanized pHPL-supplemented compared to completely serum-free cultures, indicating a peculiar role of the natural platelet-derived growth factors in this process. Surprisingly, the combination of equal cumulative numbers of MSCs + EPCs constantly resulted in the highest HPC proliferation. This produced an up to 567-fold increase indicating at least nine population doublings within only 11 days. Under all tested conditions the HPC progeny mainly comprised differentiating myeloid cells. Consecutive CFC assays revealed that liquid culture supplemented with 10%HPL were more efficient than serum-free cultures resulting in a measurable 1.2–4.5 fold CFC expansion under three of four tested medium conditions. Both MSC and EPC initiated a more than 6-fold increase of CFCs. CFC expansion through combined action of MSCs + EPCs resulted in a maximum of 23.5-fold CFC increase in pHPL-supplemented cultures. We demonstrate for the first time that HPC expansion can be modulated in a fully humanized co-culture system based on the use of pHPL as a natural source of human platelet-derived growth factors. Our data indicate that the combination of MSCs + EPCs is at least as efficient in supporting HPC proliferation and CFC amplification as either niche cell compartment alone. We speculate that human platelet-derived factors may avoid premature HSC exhaustion in this system. The humanized co-culture thus provides a novel model system for subtractive analyses of HPC and HSC-niche cell interactions. It also builds the basis for further developments towards more effective animal serum-free HSC expansion strategies.

Defined Protein-Free NS0 Myeloma Cell Cultures: Stimulation of Proliferation by Conditioned Medium Factors

A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, beta-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 x 10(6) cells mL(-1) in this medium. The antibody yield was 195 mg L(-1) in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 x 10(6) cells mL(-1), had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20-25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes.

Protein‐free transfection of CHO host cells with an IgG‐fusion protein: Selection and characterization of stable high producers and comparison to conventionally transfected clones

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.

Development, amino acid utilization and cell allocation in bovine embryos after in vitro production in contrasting culture systems

The effects of protein-supplemented and protein-free media on amino acid uptake, protein synthesis and cell differentiation in bovine blastocysts were investigated. Four formulations of synthetic oviduct fluid were used. Each formulation was identified by the principal supplement: bovine serum albumin (0.4%, w/v); polyvinyl alcohol (0.3%, w/v); or either of two steer sera (10%, v/v). After zygote culture, blastocyst yields (day 7.5) were lowest in protein-free medium and highest in albumin-supplemented medium. Subsequent 12 h incubation in the presence of both essential and non-essential amino acids was used for the measurement of amino acid flux. All blastocysts released alanine but consumed aspartate (P < 0.001) and the extent was influenced by prior culture conditions. Aspartate uptake was lower in blastocysts produced in protein-free conditions (P < 0.05) than in blastocysts produced in albumin-supplemented conditions. Consumption indices for 16 other amino acids were not influenced by blastocyst source. Cell counts and hatching incidences were highest for albumin-supplemented blastocysts, but were similar among blastocysts from the protein-free and serum-dependent treatments. Crucially, the use of protein-free medium for zygote culture did not compromise resultant blastocysts in terms of either de novo protein synthesis ([3H]phenylalanine incorporation) or trophectoderm function (phenotype based on interferon-tau detection). Thus, although blastocyst yields were compromised after zygote culture in a protein-free (vis-à-vis albumin-supplemented) medium, amino acid flux was qualitatively conserved, and only quantitatively modified in the case of alanine and aspartate. Moreover, vital properties of blastocysts that were produced, including de novo protein synthesis and trophectodermal cell function, apparently were not adversely affected by protein deprivation.

Performance of thiophilic adsorption chromatography in the purification of rat IgG2b monoclonal antibodies from serum- and protein-free culture supernatants.

The usefulness of thiophilic adsorption chromatography for the purification of rat IgG2b monoclonal antibodies has been evaluated. This approach has not shown specificity for immunoglobulins; therefore, to minimize potential interferences, the purification was carried out from supernatants of hybridomas grown in serum- and protein-free conditions. The protein purity of the six final antibody preparations assayed was always >/= 97%. In addition to the easiness of this procedure, which enables one-step antibody purification, the materials employed are rather inexpensive and milligram amounts of antibody can be recovered from 1 L of supernatant. Overall, the purification of rat IgG2b monoclonals under the conditions reported here offers an advantageous alternative to other more expensive and cumbersome methods.

Performance of thiophilic adsorption chromatography in the purification of rat IgG2b monoclonal antibodies from serum‐ and protein‐free culture supernatants

The usefulness of thiophilic adsorption chromatography for the purification of rat IgG2b monoclonal antibodies has been evaluated. This approach has not shown specificity for immunoglobulins; therefore, to minimize potential interferences, the purification was carried out from supernatants of hybridomas grown in serum- and protein-free conditions. The protein purity of the six final antibody preparations assayed was always ≥97%. In addition to the easiness of this procedure, which enables one-step antibody purification, the materials employed are rather inexpensive and milligram amounts of antibody can be recovered from 1 L of supernatant. Overall, the purification of rat IgG2b monoclonals under the conditions reported here offers an advantageous alternative to other more expensive and cumbersome methods. Copyright © 1999 John Wiley & Sons, Ltd.