Molecular genetic approaches to decrease the uncontrolled misincorporation of non-canonical branched chain amino acids into recombinant mini-proinsulin expressed in Escherichia coli.

The uncontrolled incorporation of non-canonical branched chain amino acids (ncBCAAs) such as norleucine, norvaline and ß-methylnorleucine into recombinant proteins in E. coli production processes is a crucial problem in the pharmaceutical industry, since it can lead to the production of altered proteins with non-optimal characteristics. Despite various solutions, to date there are no engineered strains that exhibit a reduced accumulation of these ncBAAs. In this study, novel E. coli K-12 BW25113 strains with exogenous tunable expression of target genes of the BCAA biosynthetic pathway were developed. For this purpose, single gene knock-outs for thrA, ilvA, leuA, ilvIH, ilvBN, ilvGM and ilvC were complemented with plasmids containing the respective genes under control of an arabinose promoter. These clones were screened in a mL-bioreactor system in fed-batch mode under both standard cultivation conditions and with pyruvate pulses, and induction of a min-proinsulin. Screening was performed by comparing the impurity profile of the recombinant mini-proinsulin expressed of each clone with the E. coli BW25113 WT strain, and the most promising clones were cultivated in a 15L Screening showed that up-regulation of ilvC, ilvIH and ilvGM, and downregulation of leuA and ilvBN trigger a reduction of norvaline and norleucine accumulation and misincorporation into mini-proinsulin. The stirred tank bioreactor cultivations confirmed that up-regulation of ilvIH and ilvGM were most effective to reduce the ncBCAA misincorporation. This novel approach for a reduced ncBCAA misincorporation may be solution to this old challenging problem in the large-scale production of human therapeutics.

A model-based framework for parallel scale-down fed-batch cultivations in mini-bioreactors for accelerated phenotyping.

Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities to reduce the risk of failure during process scale-up. Although there are various scale-down techniques to study these effects, scale-down strategies are rarely applied in the early developmental phases of a bioprocess, as they have not yet been implemented on small-scale parallel cultivation devices. In this study, we combine mechanistic growth models with a parallel mini-bioreactor system to create a high-throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scaled-down approach, a model-based glucose pulse feeding scheme is applied and compared with a continuous feed profile to study the influence of glucose and dissolved oxygen gradients on both cell physiology and incorporation of noncanonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the noncanonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depends on the pulse frequency and is negligible with continuous feeding, confirming observations from large-scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale-down scheme, compared with the reference cultivations. In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale-down studies to select the most robust strains for bioprocess scale-up.

Glucose-Limited Fed-Batch Cultivation Strategy to Mimic Large-Scale Effects in Escherichia coli Linked to Accumulation of Non-Canonical Branched-Chain Amino Acids by Combination of Pyruvate Pulses and Dissolved Oxygen Limitation.

Insufficient mixing in large-scale bioreactors provokes gradient zones of substrate, dissolved oxygen (DO), pH, and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and ß-methylnorleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids (ncBCAA), but also that pyruvate pulses produce the same effect. Therefore, we combined the enzyme-based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor (STR). To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

Model-based monitoring of industrial reversed phase chromatography to predict insulin variants.

Downstream processing in the manufacturing biopharmaceutical industry is a multistep process separating the desired product from process- and product-related impurities. However, removing product-related impurities, such as product variants, without compromising the product yield or prolonging the process time due to extensive quality control analytics, remains a major challenge. Here, we show how mechanistic model-based monitoring, based on analytical quality control data, can predict product variants by modeling their chromatographic separation during product polishing with reversed phase chromatography. The system was described by a kinetic dispersive model with a modified Langmuir isotherm. Solely quality control analytical data on product and product variant concentrations were used to calibrate the model. This model-based monitoring approach was developed for an insulin purification process. Industrial materials were used in the separation of insulin and two insulin variants, one eluting at the product peak front and one eluting at the product peak tail. The model, fitted to analytical data, used one component to simulate each protein, or two components when a peak displayed a shoulder. This monitoring approach allowed the prediction of the elution patterns of insulin and both insulin variants. The results indicate the potential of using model-based monitoring in downstream polishing at industrial scale to take pooling decisions.

Wideband impedance spectrum analyzer for process automation applications.

For decades impedance spectroscopy is used in technical laboratories and research departments to investigate effects or material characteristics that affect the impedance spectrum of the sensor. Establishing this analytical approach for process automation and stand-alone applications will deliver additional and valuable information beside traditional measurement techniques such as the measurement of temperature, flow rate, and conductivity, among others. As yet, most of the current impedance analysis methods are suited for laboratory applications only since they involve stand-alone network analyzers that are slow, expensive, large, or immobile. Furthermore, those systems offer a large range of functionality that is not being used in process control and other fields of application. We developed a sensor interface based on high speed direct digital signal processing offering wideband impedance spectrum analysis with high resolution for frequency adjustment, excellent noise rejection, very high measurement rate, and convenient data exchange to common interfaces. The electronics has been implemented on two small circuit boards and it is well suited for process control applications such as monitoring phase transitions, characterization of fluidal systems, and control of biological processes. The impedance spectrum analyzer can be customized easily for different measurement applications by adapting the appropriate sensor module. It has been tested for industrial applications, e.g., dielectric spectroscopy and high temperature gas analysis.

Optimization of buffer rod geometry for ultrasonic sensors with reference path.

Several applications of ultrasonic techniques are limited by the signal-to-noise ratio (SNR). Transducers in these applications usually operate in the pulse-echo mode. Many transducers, especially those for high temperatures, use buffer rods. Often a reference path is used to eliminate electrical and transducer drift. Interference of echo signals and noise causes errors of both amplitude and phase measurement of the detected echoes. In this paper we discuss the influence of major noise sources as a function of geometry and operating environment. The effects are studied using both experimental results and models. Although the results are applied to an ultrasonic density sensor operating in the pulse-echo mode, they are applicable to other pulse-echo mode transducers comprising homogeneous cylindrical buffer rods. This paper will show how the SNR of the density transducer was improved in a special time window from 34 to 72 dB by careful design.

Is an oscillator-based measurement adequate in a liquid environment?

Oscillator-based measurements with quartz crystal resonators will be analyzed. The investigations have shown that classical thickness monitors as well as many chemical vapor sensors based on a quartz crystal microbalance (QCM) work properly, even with simple oscillators. It was demonstrated that, for applications in a liquid environment, more sophisticated electronics are necessary. Also a comparison between the experimental results in liquids and the theoretical predictions is hardly possible without the knowledge of the oscillator behavior. As our solution, we present an automatic gain-controlled oscillator with two output signals, the oscillator frequency, and a signal that represents the damping of the quartz resonator. A calibration method is introduced, which allows one to calculate the series resonance frequency fs and the series resistance Rs from these oscillator signals.

Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

Acoustic microsensors--the challenge behind microgravimetry.

Acoustic microsensors are commonly known as high-resolution mass-sensitive devices. This is a restricted view in many chemical and biosensor applications, especially in liquids. Sensitivity to non-gravimetric effects is a challenging feature of acoustic sensors. In this review we give an overview of recent developments in resonant sensors including micromachined devices and also list recent activity relating to the (bio)chemical interface of acoustic sensors. Major results from theoretical analysis of quartz crystal resonators, descriptive for all acoustic microsensors are summarized, and non-gravimetric contributions to the sensor signal from viscoelasticity and interfacial effects are discussed. We finally conclude with some future perspectives.

Ultrasonic concentration measurement of aqueous solutions using PLS regression.

This work demonstrates the use of a multivariate statistical technique called partial least squares (PLS) to extract material related data by analyzing spectra of ultrasonic pulses. We show how PLS can be used to estimate the concentration of sodium chloride in an aqueous solution. The paper describes the use of PLS and discusses pre-processing of ultrasonic data, the PLS algorithm as well as model validation. The measured concentrations are compared to reference values. The influence of disturbances and parameter changes is highlighted. The proposed method is easily adaptable to similar applications and permits a cost-saving implementation using existing and approved hardware.

Defects in N-glycosylation induce apoptosis in yeast.

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Defects of N-glycosylation in humans lead to congenital disorders. The pivotal step of this pathway is the transfer of the evolutionarily conserved lipid-linked core-oligosaccharide to the nascent polypeptide chain, catalysed by the oligosaccharyltransferase. One of its nine subunits, Ost2, has homology to DAD1, originally characterized in hamster cells as a defender against apoptotic death. Here we show that ost mutants, such as ost2 and wbp1-1, display morphological and biochemical features of apoptosis upon induction of the glycosylation defect. We observe nuclear condensation, DNA fragmentation as well as externalization of phosphatidylserine. We also demonstrate induction of caspase-like activity, both determined by flow cytometric analysis and in cell-free extracts. Similarly, the N-glycosylation inhibitor tunicamycin in combination with elevated temperature is able to challenge the apoptotic cascade. Heterologous expression of anti-apoptotic human Bcl-2 diminishes caspase activation, improves survival of cells and suppresses the temperature-sensitive growth defect of wbp1-1. Furthermore, accumulation of reactive oxygen species occurs in response to defective glycosylation. As deletion of the metacaspase YCA1 does not seem to abrogate glycosylation-induced apoptosis, we postulate a different proteolytic process to be involved in this death pathway.