Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes.

The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO2 concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO2 in batch and fed-batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO2 values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO2. The presented results are especially interesting for large-scale and perfusion processes where increased pCO2 concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO2 accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO2 might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.

Prediction of filamentous process performance attributes by CSL quality assessment using mid-infrared spectroscopy and chemometrics.

Every biopharmaceutical production process aims for control strategies to achieve process robustness in order to ensure consistent product quality. Process variability can origin from process parameters, the biological nature as well as from high lot-to-lot variability of raw materials. In filamentous processes raw materials with very complex matrices, such as corn steep liquor (CSL), are used, which are especially challenging to characterize. In this study, CSL was characterized in detail for its ingredients presenting an overall composition of its matrix of 50 analyzed components (19 amino acids, 5 organic acids, 8 reducing sugars, 7 water-soluble vitamins and 11 trace elements/minerals) in order to facilitate analytical reduction to fingerprinting methods FT-MIR was evaluated as fast and non-destructive spectroscopic fingerprinting method for adequate assessment of CSL quality. Feasibility of this method was shown by the correlation of certain bands in the spectra to substance groups present in CSL, such as the Amide I and II band and amino acids, respectively. Additionally, applicability of FT-MIR could be shown for classification of different CSL lots differing in provider and corn quality as well as for predictability of process performance attributes. The latter was demonstrated on a fed-batch filamentous fungi process for the production of antibiotics. By multivariate data analysis, it could be shown that CSL quality assessment via FT-MIR can be used for the prediction of maximal biomass generated in the process, with a correlation coefficient R2 of 0.964, as well as for the prediction of an unwanted impurity. The combination of a fast and easy method for CSL quality assessment and correlations of this quality with process performance attributes may facilitate the establishment of a risk-based acceptance criteria for raw material quality release of CSL. As CSL is a frequent used raw material, we believe that this method will also be useful for other processes and that CSL quality assessment is of high relevance in academia and industry.

Workflow for Target-Oriented Parametrization of an Enhanced Mechanistic Cell Culture Model.

The goal of this study is to develop a macroscopic mechanistic model describing growth and production within fed-batch cultivations of CHO cells. The model should be used for process characterization as well as for process monitoring including real-time parameter adaptations. The model proved to be able to describe a data-set of 40 processes differing in clones, scales, and process conditions with a normalized root mean square error of approximately 10%. However, due to limited parameter identifiability and limited knowledge about physiologically meaningful parameter values, a broad range of parameters could describe the data with similar quality. This hampered comparison of the model parameters as well as their real-time estimation. Therefore an iterative workflow combining techniques like sensitivity and identifiability analysis, analysis of the specific rates as well as structural adaptations of the parameter space is developed. By applying it the parameter variability could be reduced by 80% with similar predictive power as the original parameters. Summing up, based on a mechanistic CHO model, a generic and transferrable workflow is created for target-oriented parameter estimation in case of limited parameter identifiability. Finally, we suggest a methodology, which fits ideally into the frame of Process Analytical Technology aiming to increase process understanding.

Impact of Glycerol as Carbon Source onto Specific Sugar and Inducer Uptake Rates and Inclusion Body Productivity in E. coli BL21(DE3).

The Gram-negative bacterium E. coli is the host of choice for a multitude of used recombinant proteins. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures using isopropyl ß-d-1 thiogalactopyranoside as inducer are often referred to cause stress reactions, leading to a phenomenon known as "metabolic" or "product burden". These high expressions of recombinant proteins mainly result in decreased growth rates and cell lysis at elevated induction times. Therefore, approaches tend to use "soft" or "tunable" induction with lactose and reduce the stress level of the production host. The usage of glucose as energy source in combination with lactose as induction reagent causes catabolite repression effects on lactose uptake kinetics and as a consequence reduced product titer. Glycerol-as an alternative carbon source-is already known to have positive impact on product formation when coupled with glucose and lactose in auto-induction systems, and has been referred to show no signs of repression when cultivated with lactose concomitantly. In recent research activities, the impact of different products on the lactose uptake using glucose as carbon source was highlighted, and a mechanistic model for glucose-lactose induction systems showed correlations between specific substrate uptake rate for glucose or glycerol (qs,C) and the maximum specific lactose uptake rate (qs,lac,max). In this study, we investigated the mechanistic of glycerol uptake when using the inducer lactose. We were able to show that a product-producing strain has significantly higher inducer uptake rates when being compared to a non-producer strain. Additionally, it was shown that glycerol has beneficial effects on viability of cells and on productivity of the recombinant protein compared to glucose.

Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry-the fast alternative to microscopic image analysis.

An important parameter in filamentous bioreactor cultivations is the morphology of the fungi, due to its interlink to productivity and its dependency on process conditions. Filamentous fungi show a large variety of morphological forms in submerged cultures. These range from dispersed hyphae, to interwoven mycelial aggregates, to denser hyphal aggregates, the so-called pellets. Depending on the objective function of the bioprocess, different characteristics of the morphology are favorable and need to be quantified accurately. The most common method to quantitatively characterize morphology is image analysis based on microscopy. This method is work intensive and time consuming. Therefore, we developed a faster, at-line applicable, alternative method based on flow cytometry. Within this contribution, this novel method is compared to microscopy for a penicillin production process. Both methods yielded in comparable distinction of morphological sub-populations and described their morphology in more detail. In addition to the appropriate quantification of size parameters and the description of the hyphal region around pellets, the flow cytometry method even revealed a novel compactness parameter for fungal pellets which is not accessible via light microscopy. Hence, the here presented flow cytometry method for morphological analysis is a fast and reliable alternative to common tools with some new insights in the pellet morphology, enabling at-line use in production environments.

A combination of HPLC and automated data analysis for monitoring the efficiency of high-pressure homogenization.

BACKGROUND: Cell disruption is a key unit operation to make valuable, intracellular target products accessible for further downstream unit operations. Independent of the applied cell disruption method, each cell disruption process must be evaluated with respect to disruption efficiency and potential product loss. Current state-of-the-art methods, like measuring the total amount of released protein and plating-out assays, are usually time-delayed and involve manual intervention making them error-prone. An automated method to monitor cell disruption efficiency at-line is not available to date. RESULTS: In the current study we implemented a methodology, which we had originally developed to monitor E. coli cell integrity during bioreactor cultivations, to automatically monitor and evaluate cell disruption of a recombinant E. coli strain by high-pressure homogenization. We compared our tool with a library of state-of-the-art methods, analyzed the effect of freezing the biomass before high-pressure homogenization and finally investigated this unit operation in more detail by a multivariate approach. CONCLUSION: A combination of HPLC and automated data analysis describes a valuable, novel tool to monitor and evaluate cell disruption processes. Our methodology, which can be used both in upstream (USP) and downstream processing (DSP), describes a valuable tool to evaluate cell disruption processes as it can be implemented at-line, gives results within minutes after sampling and does not need manual intervention.

The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion.

Due to high mixing times and base addition from top of the vessel, pH inhomogeneities are most likely to occur during large-scale mammalian processes. The goal of this study was to set-up a scale-down model of a 10-12 m3 stirred tank bioreactor and to investigate the effect of pH perturbations on CHO cell physiology and process performance. Short-term changes in extracellular pH are hypothesized to affect intracellular pH and thus cell physiology. Therefore, batch fermentations, including pH shifts to 9.0 and 7.8, in regular one-compartment systems are conducted. The short-term adaption of the cells intracellular pH are showed an immediate increase due to elevated extracellular pH. With this basis of fundamental knowledge, a two-compartment system is established which is capable of simulating defined pH inhomogeneities. In contrast to state-of-the-art literature, the scale-down model is included parameters (e.g. volume of the inhomogeneous zone) as they might occur during large-scale processes. pH inhomogeneity studies in the two-compartment system are performed with simulation of temporary pH zones of pH 9.0. The specific growth rate especially during the exponential growth phase is strongly affected resulting in a decreased maximum viable cell density and final product titer. The gathered results indicate that even short-term exposure of cells to elevated pH values during large-scale processes can affect cell physiology and overall process performance. In particular, it could be shown for the first time that pH perturbations, which might occur during the early process phase, have to be considered in scale-down models of mammalian processes.

Physiological capacities decline during induced bioprocesses leading to substrate accumulation.

During the cultivation of E. coli for recombinant protein production, substrate accumulation is often observed in induction phase. Uncontrolled substrate accumulation leads to difficulties in transferring or scaling processes and even to failed batches. The phenomenon of metabolite/substrate accumulation occurs as a result of exceeding the physiological capacity to metabolize substrate (qScrit ). In contrast to the common understanding of qScrit as "static" value, we hypothesize that qScrit essentially has a dynamic nature. Following the state of the art approach of physio logical strain characterization, substrate pulse experiments were used to quantify qScrit in induction phase. The qScrit was found to be temperature and time dependent. Subsequently, qScrit was expressed through a linear equation, to serve as boundary for physiologically controlled experiments. Nevertheless, accumulation was observed within a physiologically controlled verification experiment, although the qScrit boundary was not exceeded. A second set of experiments was conducted, by oscillating the qS set point between discrete plateaus during physiologically controlled experiments. From the results, we deduced a significant interrelation between the metabolic activity and the timely decline of qScrit. This finding highlights the necessity of a comprehensive but laborious physiological characterization for each strain or alternatively, to use physio logical feedback control to facilitate real time monitoring of qScrit , in order to effectively avoid substrate accumulation.

Impact of cell lysis on the description of cell growth and death in cell culture.

The primary task of process development is a process design that guarantees product quality and maximizes product quantity. One part of the process development is the identification of critical process parameters. Especially in cell culture processes, unwanted cell damage as critical process parameter is still challenging in stirred tank reactors and needs therefore to be considered. Nevertheless, this topic and its effects on process performance are currently not well discussed and not verified in literature until now. The process of cell damage or lysis can be defined as the loss of integrity of the cells. For the investigation of this phenomenon, a model-based designed fed-batch cultivation with Chinese hamster ovary cells was performed. Besides measurements of viable and dead cell concentration, lysed cell count was determined by DNA measurements. Based on these analytics, different hypotheses, characterizing the cell death, were compared. From this, four main statements could be derived: (i) consideration of lysis in cell culture processes is of great importance for the description of the living biomass population in terms of growth and dying; (ii) a higher effort in process monitoring facilitates significantly model development; (iii) in contradiction to existing models from literature, our verification approach demonstrated a direct correlation of lysis with viable cell concentration and therefore with productivity; and (iv) lysis could be effectively described by only one model parameter, the specific lysis rate from viable cells to lysed cells. All these statements could be accurately proven by statistical methods. Our results enable future detailed investigations of the causes of cell damage and the influences of cell lysis on product quality and quantity. Potential application fields are scale-up issues, process optimization, and impurity screening.

Investigation of the interactions of critical scale-up parameters (pH, pO2 and pCO2) on CHO batch performance and critical quality attributes.

Understanding process parameter interactions and their effects on mammalian cell cultivations is an essential requirement for robust process scale-up. Furthermore, knowledge of the relationship between the process parameters and the product critical quality attributes (CQAs) is necessary to satisfy quality by design guidelines. So far, mainly the effect of single parameters on CQAs was investigated. Here, we present a comprehensive study to investigate the interactions of scale-up relevant parameters as pH, pO2 and pCO2 on CHO cell physiology, process performance and CQAs, which was based on design of experiments and extended product quality analytics. The study used a novel control strategy in which process parameters were decoupled from each other, and thus allowed their individual control at defined set points. Besides having identified the impact of single parameters on process performance and product quality, further significant interaction effects of process parameters on specific cell growth, specific productivity and amino acid metabolism could be derived using this method. Concerning single parameter effects, several monoclonal antibody (mAb) charge variants were affected by process pCO2 and pH. N-glycosylation analysis showed positive correlations between mAb sialylation and high pH values as well as a relationship between high mannose variants and process pH. This study additionally revealed several interaction effects as process pH and pCO2 interactions on mAb charge variants and N-glycosylation pattern. Hence, through our process control strategy and multivariate investigation, novel significant process parameter interactions and single effects were identified which have to be taken into account especially for process scale-up.

At-line determining spore germination of Penicillium chrysogenum bioprocesses in complex media.

Spore inoculum quality in filamentous bioprocesses is a critical parameter associated with viable spore concentration (1) and spore germination (2). It influences pellet morphology and, consequently, process performance. The state-of-the-art method to measure viable spore concentration is tedious, associated with significant inherent bias, and not applicable in real-time. Therefore, it is not usable as process analytical technology (PAT). Spore germination has so far been monitored using image analysis, which is hampered by complex medium background often observed in filamentous bioprocesses. The method presented here is based on the combination of viability staining and large-particle flow cytometry which enables measurements in real-time and hence aims to be applicable as a PAT tool. It is compatible with the complex media background and allows the quantification of metabolically active spores and the monitoring of spore germination. A distinction of germinated spores and not germinated spores was based on logistic regression, using multiparameteric data from flow cytometry. In a first step, a significant correlation between colony-forming unit (CFU) counts and viable spore concentration (1) in an industrially relevant model bioprocess was found. Spore germination (2) was followed over the initial process phase with close temporal resolution. The validation of the method showed an error below 5 %. Differences in spore germination for various spore inocula ages and spore inoculum concentrations were monitored. The real-time applicability of the method suggests the implementation as a PAT tool in filamentous bioprocesses.

Clinical features of critically ill patients with Shiga toxin-induced hemolytic uremic syndrome.

OBJECTIVE: In Spring 2011, an unprecedented outbreak of Shiga toxin-producing Escherichia coli serotype O104:H4-associated hemolytic uremic syndrome occurred in Northern Germany. The aim of this study was to describe the clinical characteristics, treatments, and outcomes of critically ill patients with Shiga toxin-producing E. coli-associated hemolytic uremic syndrome during this outbreak. DESIGN, SETTING, AND PATIENTS: Multicenter, retrospective, observational study of critically ill adult patients with Shiga toxin-producing E. coli-associated hemolytic uremic syndrome in six hospitals in Hamburg, Germany, between May 2011 and August 2011. MEASUREMENTS AND MAIN RESULTS: During the study period, 106 patients with Shiga toxin-producing E. coli-associated hemolytic uremic syndrome were admitted to eight ICUs. The median age was 40 years (range, 18-83) with a female:male ratio of 3:1. The median time from onset of clinical symptoms to hospital admission was 3 days and from hospital to ICU admission an additional 3 days. A total of 101 patients (95.3%) had acute renal failure and 78 (73.6%) required renal replacement therapy. Intubation and mechanical ventilation were required in 38 patients (35.8%) and noninvasive ventilation was required in 17 patients (16.0%). The median duration of invasive ventilation was 7 days (range, 1-32 days) and the median ICU stay was 10 days (range, 1-45 days). Fifty-one patients (48.1%) developed sepsis; of these 51 patients, 27 (25.4%) developed septic shock. Seventy patients (66.0%) developed severe neurological symptoms. Ninety-seven patients (91.5%) were treated with plasma exchange and 50 patients (47.2%) received eculizumab (monoclonal anti-C5 antibody). The mortality rate was 4.7%. Mild residual neurological symptoms were present in 21.7% of patients at ICU discharge, and no patient required renal replacement therapy 6 months after ICU admission. CONCLUSIONS: During the 2011 Shiga toxin-producing E. coli-associated hemolytic uremic syndrome outbreak in Germany, critical illness developed rapidly after hospital admission, often in young women. The infection was associated with severe neurological and renal symptoms, requiring mechanical ventilation and renal replacement therapy in a substantial proportion of patients. Overall, recovery was much better than expected.

Designing a fully automated multi-bioreactor plant for fast DoE optimization of pharmaceutical protein production.

The identification of optimal expression conditions for state-of-the-art production of pharmaceutical proteins is a very time-consuming and expensive process. In this report a method for rapid and reproducible optimization of protein expression in an in-house designed small-scale BIOSTAT® multi-bioreactor plant is described. A newly developed BioPAT® MFCS/win Design of Experiments (DoE) module (Sartorius Stedim Systems, Germany) connects the process control system MFCS/win and the DoE software MODDE® (Umetrics AB, Sweden) and enables therefore the implementation of fully automated optimization procedures. As a proof of concept, a commercial Pichia pastoris strain KM71H has been transformed for the expression of potential malaria vaccines. This approach has allowed a doubling of intact protein secretion productivity due to the DoE optimization procedure compared to initial cultivation results. In a next step, robustness regarding the sensitivity to process parameter variability has been proven around the determined optimum. Thereby, a pharmaceutical production process that is significantly improved within seven 24-hour cultivation cycles was established. Specifically, regarding the regulatory demands pointed out in the process analytical technology (PAT) initiative of the United States Food and Drug Administration (FDA), the combination of a highly instrumented, fully automated multi-bioreactor platform with proper cultivation strategies and extended DoE software solutions opens up promising benefits and opportunities for pharmaceutical protein production.

p38 and OGT sequestration into viral inclusion bodies in cells infected with human respiratory syncytial virus suppresses MK2 activities and stress granule assembly.

Respiratory syncytial virus (RSV) forms cytoplasmic inclusion bodies (IBs) that are thought to be sites of nucleocapsid accumulation and viral RNA synthesis. The present study found that IBs also were the sites of major sequestration of two proteins involved in cellular signaling pathways. These are phosphorylated p38 mitogen-activated protein kinase (MAPK) (p38-P), a key regulator of cellular inflammatory and stress responses, and O-linked N-acetylglucosamine (OGN) transferase (OGT), an enzyme that catalyzes the posttranslational addition of OGN to protein targets to regulate cellular processes, including signal transduction, transcription, translation, and the stress response. The virus-induced sequestration of p38-P in IBs resulted in a substantial reduction in the accumulation of a downstream signaling substrate, MAPK-activated protein kinase 2 (MK2). Sequestration of OGT in IBs was associated with suppression of stress granule (SG) formation. Thus, while the RSV IBs are thought to play an essential role in viral replication, the present results show that they also play a role in suppressing the cellular response to viral infection. The sequestration of p38-P and OGT in IBs appeared to be reversible: oxidative stress resulting from arsenite treatment transformed large IBs into a scattering of smaller bodies, suggestive of partial disassembly, and this was associated with MK2 phosphorylation and OGN addition. Unexpectedly, the RSV M2-1 protein was found to localize in SGs that formed during oxidative stress. This protein was previously shown to be a viral transcription elongation factor, and the present findings provide the first evidence of possible involvement in SG activities during RSV infection.

A multi-bioreactor system for optimal production of malaria vaccines with Pichia pastoris.

The successful development of optimal multistage production processes for recombinant products with Pichia pastoris needs to meet three pre-conditions. These pre-conditions are (i) strategies for performing fully automated and observable processes, (ii) characterization of the host cell-specific reaction parameters in order to make an adapted process layout for feeding and aeration strategies, and (iii) knowledge of optimal operation parameter conditions for maximizing the expression productivity of target protein amount and/or quality. In this report, an approach of a fully automated multi-bioreactor plant is described that meets all these requirements. The expression and secretion of a potential malaria vaccine with Pichia pastoris was chosen as an example to demonstrate the quality of the bioreactor system. Methods for the simultaneous identification of reaction kinetics were developed for strain characterization. Process optimization was carried out by applying a sequential/parallel Design of Experiments. In the view of Process Analytical Technology (PAT)-applications and in order to develop fully automated and globally observable production processes, methods for quasi on-line monitoring of recombinant protein secretion titers and the immunological quality of the products are also discussed in detail.

The secreted form of respiratory syncytial virus G glycoprotein helps the virus evade antibody-mediated restriction of replication by acting as an antigen decoy and through effects on Fc receptor-bearing leukocytes.

Respiratory syncytial virus (RSV) readily infects and reinfects during infancy and throughout life, despite maternal antibodies and immunity from prior infection and without the need for significant antigenic change. RSV has two neutralization antigens, the F and G virion glycoproteins. G is expressed in both membrane-bound (mG) and secreted (sG) forms. We investigated whether sG might act as a decoy for neutralizing antibodies by comparing the in vitro neutralization of wild-type (wt) RSV versus recombinant mG RSV expressing only mG. wt RSV indeed was less susceptible than mG RSV to monovalent G-specific and polyvalent RSV-specific antibodies, whereas susceptibility to F-specific antibodies was equivalent. This difference disappeared when the virus preparations were purified to remove sG. Thus, sG appears to function as a neutralization decoy. We evaluated this effect in vivo in mice by comparing the effects of passively transferred antibodies on the pulmonary replication of wt RSV versus mG RSV. Again, wt RSV was less sensitive than mG RSV to G-specific and RSV-specific antibodies; however, a similar difference was also observed with F-specific antibodies. This confirmed that sG helps wt RSV evade the antibody-dependent restriction of replication but indicated that in mice, it is not acting primarily as a decoy for G-specific antibodies, perhaps because sG is produced in insufficient quantities in this poorly permissive animal. Rather, we found that the greater sensitivity of mG versus wt RSV to the antiviral effect of passively transferred RSV antibodies required the presence of inflammatory cells in the lung and was Fc gamma receptor dependent. Thus, sG helps RSV escape the antibody-dependent restriction of replication via effects as an antigen decoy and as a modulator of leukocytes bearing Fc gamma receptors.

Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH.

Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.

Metabolic activity of Corynebacterium glutamicum grown on L: -lactic acid under stress.

Respiration measurement in shake flasks is introduced as a new method to characterize the metabolic activity of microorganisms during and after stress exposure. The major advantage of the new method is the possibility to determine the metabolic activity independent of manual sampling without the necessity to change the culture vessel or the cultivation medium. This excludes stress factors, which may be induced by transferring the microorganisms to plates or respirometers. The negative influence, which interruptions of the shaker during sampling times may have on the growth of microorganisms was demonstrated. The applicability of the method was verified by characterizing the behavior of Corynebacterium glutamicum grown on the carbon source L: -lactic acid under stress factors such as carbon starvation, anaerobic conditions, lactic acid, osmolarity, and pH. The following conditions had no effect on the metabolic activity of C. glutamicum: a carbon starvation of up to 19 h, anaerobic conditions, lactic acid concentrations up to 10 g/l, 3-(N-morpholino) propanesulfonic acid buffer concentrations up to 42 g/l, or pH from 6.4 to 7.4. Lactic-acid concentrations from 10 to 30 g/l lead to a decrease of the growth rate and the biomass substrate yield without effecting the oxygen substrate conversion. Without adaptation, the organism did not grow at pH< or =5 or > or =9.

Processing of a pestivirus protein by a cellular protease specific for light chain 3 of microtubule-associated proteins.

The genome of the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) JaCP contains a cellular insertion coding for light chain 3 (LC3) of microtubule-associated proteins, the mammalian homologue of yeast Aut7p/Apg8p. The cellular insertion induces cp BVDV-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease Aut2p/Apg4p. Three candidate bovine protease genes were identified on the basis of the sequence similarity of their products with the Saccharomyces cerevisiae enzyme. The search for a system for functional testing of these putative LC3-specific proteases revealed that the components involved in this processing have been highly conserved during evolution, so that the substrate derived from a mammalian virus is processed in cells of mammalian, avian, fish, and insect origin, as well as in rabbit reticulocyte lysate, but not in wheat germ extracts. Moreover, two of these proteases and a homologous protein from chickens were able to rescue the defect of a yeast AUT2 deletion mutant. In coexpression experiments with yeast and wheat germ extracts one of the bovine proteases and the corresponding enzyme from chickens were able to process the viral polyprotein containing LC3. Northern blots showed that bovine viral diarrhea virus infection of cells has no significant influence on the expression of either LC3 or its protease, bAut2B2. However, LC3-specific processing of the viral polyprotein containing the cellular insertion is essential for replication of the virus since mutants with changes in the LC3 insertion significantly affecting processing at the LC3/NS3 site were not viable.

Comparison of inflammatory and systemic sources of growth factors in acute and chronic human wounds.

Inflammatory cells are generally assumed to be the primary source of most growth factors/cytokines that participate in wound healing. Correspondingly, most attempts to enhance wound healing have been directed at the wound itself. However, certain key factors such as insulin-like growth factor-I (IGF-I) and related proteins are present in blood in sufficient quantities to suggest significant contributions from outside the wound. Because little is known of the dynamics of IGF family members in wounds, particularly in human wounds, we serially measured the mRNAs and proteins of the IGF family in fluid and tissues taken from acute as well as chronically inflamed human wounds and compared them to their corresponding concentrations in blood. We also measured transforming growth factor-beta1 and -beta3, vascular endothelial growth factor, interleukin-1beta, matrix metalloproteinases and selected isomers/receptors, all of which are associated with inflammation. All IGF proteins reached their highest concentrations immediately after injury. No difference between IGF-I mRNA expression between acute and inflamed wounds was found. As a group, IGF-related proteins, in contrast to transforming growth factor-beta, vascular endothelial growth factor, and interleukin-1beta, are highly correlated to and are generally below their concentrations in blood and are not elevated by inflammation. The IGF family therefore appears to enter wounds, even inflamed wounds, mainly from blood. If blood IGF-I is low, wound levels are lower. This data suggests that healing impairment due to IGF-I deficiency can be readily detected and is, at least in part, easily and safely correctable.