In-line pH Research Articles

Real-Time Adaptive Inline Acidification Enhances Continuous pH Control for Viral Inactivation.

Existing low pH viral inactivation methods for continuous downstream processing of biologics typically rely on predictive models to estimate the necessary pH adjustments. However, these methods are of limited use during the process development stage due to the dynamic nature of capture chromatography, where batch variations can alter the eluted protein titer. This study introduces an inline viral inactivation system (IVIS) that utilizes real-time adaptive control and inline sensor readings to precisely regulate the pH manipulation for inline acidification and continuous viral inactivation. The IVIS, which includes a coiled flow inversion reactor (CFIR), is integrated with a multicolumn capture chromatography system to demonstrate a fully continuous process from protein capture chromatography to inline pH manipulation. The system achieved precise inline pH manipulation within ±0.15 and a narrow residence time distribution of 13.5min with a relative width of 0.7. The introduction of real-time inline pH manipulation with the IVIS signifies a notable advancement in managing critical process parameters (CPPs) and ensuring consistent product quality across varied production environments for continuous downstream bioprocessing.

Validation of an Optical Technology for the Determination of pH in Milk during Yogurt Manufacture.

Current systems that allow inline pH control in the fermented dairy industry have drawbacks, such as protein adhesion on the non-glass pH probes, measurement distortion, frequent recalibration needs, and sensitivity to extreme pH conditions encountered during clean-in-place operations. Therefore, the objective of this study was to validate the feasibility of estimating the pH of milk during the yogurt making process by using a NIR light backscatter sensor measuring under different fermentation temperatures and milk protein concentrations using a mathematical model that correlates the light scatter signal with pH. Three replications of the experiment with two protein concentrations (3.5 and 4.0%) and two fermentation temperatures (43 and 46 °C) were used to validate this inline pH prediction model. Continuous and discontinuous measurements of pH were collected as a reference during fermentation, simultaneously with the light backscatter data acquisition. Also, the effect of adjusting the initial voltage gain of the light scatter device on the accuracy of the pH prediction model was evaluated. Temperature and initial voltage were the main factors affecting the fitting accuracy of the model. The adjustment of the initial voltage gain improved the pH prediction model fit. The model has been successfully validated for both continuous and discontinuous measurements of pH, with SEP values < 0.09 pH units and CV < 1.78%. The proposed optical inline and non-destructive method was feasible for inline pH monitoring of milk fermentation, avoiding traditional manual pH measurement.

3D Printed, Single-Use Bioreactor with Integrated Inline Sensors for Microbial and Mammalian Cell Cultivation—A Case Study

The development of upstream bioprocesses necessitates small, instrumented bioreactors for investigating and optimizing production processes in a cost-effective manner. Due to advances in both the equipment and the materials used in additive manufacturing, 3D printing of customized bioreactors is now in the realm of possibilities. In this study, a small-scale 3D printed bioreactor suitable for mammalian and microbial cultivations was developed, featuring a working volume of 90 mL, inline pH and dissolved oxygen probes and a levitating magnetic stirrer. Aeration channels and a sampling port were printed directly into the vessel walls. Additionally, the vessel was equipped with a 3D printed customizable optical biomass-sensor. The bioreactor’s performance was evaluated through technical characterization and proof of concept cultivations, demonstrating that mixing time and oxygen mass transfer were sufficient for cultivating mammalian as well as microbial cells at high cell densities. Specifically, an Escherichia coli fed-batch cultivation achieved a maximum OD600 of 204. Furthermore, a fed-batch cultivation of an IgG antibody-producing Chinese hamster ovary cell line reached a peak viable cell density of 10.2 × 106 cells mL−1 and a maximum product titer of 2.75 g L−1. Using a three-parameter fit, the inline biomass signal could be correlated to the corresponding offline values with satisfactory accuracy, making it possible to monitor cell growth in real-time.

Studying the effect of nickel, copper, and zinc on the precipitation kinetics and the composition of Ca-carbonates for the integrated process of CO[formula omitted] mineralization and brine mining

Brine mining of energy-relevant elements (EREs) requires the separation of transition metals from major cations in order to create high-purity streams. Precipitation of carbonates could be used as a separation method leaving EREs in the supernatant, therefore opening the possibility to integrate carbon dioxide (CO2) mineralization with brine mining. Major challenges are that the reaction should reach completion in few days and the inclusion of transition metals in the carbonates should be avoided.In this paper, we report an experimental and modeling work on the precipitation of calcium (Ca) carbonate in the presence of nickel (Ni), copper (Cu), and zinc (Zn). Brines of various compositions were tested to simulate brine enrichment. Atmospheric conditions were applied to study the effect of the metal concentration on the precipitation kinetics and the composition of the carbonates produced. The experiments were run in batch and monitored with inline pH and conductivity probes. Results show that Ni, Cu, and Zn may retard or even hinder Ca-carbonate precipitation. This effect can be observed at concentrations between 10 and 500 ppm, and the specific values depend on the type of metal, Cu<Zn<Ni. At low metal concentrations, SEM-EDS and XRD analyses confirm the formation of pure calcite. At large metal concentrations amorphous products were obtained with significant metal inclusions. A mathematical model coupled with geochemistry was developed to describe the process. It was calibrated with pH and conductivity measurements, and used to determine reaction kinetics.Overall, the results suggest that the precipitation of high-purity Ca-carbonates from a supersaturated brine enriched with transition metals can be fast, but transition metal concentrations must be below certain thresholds. If brine enrichment is required to favor mineral recovery optimal process control is necessary.

Measuring pH of skim milk and milk permeate at ultra-high temperatures at laboratory and pilot scale

Changes in the pH of skim milk and skim milk ultrafiltration permeate on heating from 25 to 140 °C were examined. Results showed that the decrease in pH with increase in temperature up to 140 °C was not linear. Hydrogen ion release due to changes in the milk mineral balance were responsible for the reduction in pH with increase in temperature. The presence of milk proteins offered little buffering against the drop in pH. The precipitation of calcium phosphate resulted in sediment in milk permeate heated above ∼70 °C, but this did not occur in skim milk, with the pH remaining lower in milk permeate after heat treatment when measured at 25 °C. This study has shown that in-line pH measurements of milk at ultra-high temperatures is feasible, and could prove useful at laboratory and pilot-scale for studying interactions within, and stability of, more complex formulations with added minerals.

Programmable Millifluidic Platform Integrating Automatic Electromembrane Extraction Cleanup and In-Line Electrochemical Detection: A Proof of Concept.

A fully automatic millifluidic sensing platform coupling in-line nonsupported microelectromembrane extraction (μ-EME) with electrochemical detection (ECD) is herein proposed for the first time. Exploiting the features of the second generation of flow analysis, termed sequential injection (SI), the smart integration of SI and μ-EME-ECD enables (i) the repeatable formation of microvolumes of phases for the extraction step in a membrane-less (nonsupported) arrangement, (ii) diverting the acceptor plug to the ECD sensing device, (iii) in-line pH adjustment before the detection step, and (iv) washing of the platform for efficient removal of remnants of wetting film solvent, all entirely unsupervised. The real-life applicability of the miniaturized sensing system is studied for in-line sample cleanup and ECD of diclofenac as a model analyte after μ-EME of urine as a complex biological sample. A comprehensive study of the merits and the limitations of μ-EME solvents on ECD is presented. Under the optimal experimental conditions using 14 μL of unprocessed urine as the donor, 14 μL of 1-nonanol as the organic phase, and 14 μL of 25 mM NaOH as the acceptor in a 2.4 mm ID PTFE tubing, an extraction voltage of 250 V, and an extraction time of 10 min, an absolute (mass) extraction recovery of 48% of diclofenac in urine is obtained. The proposed flow-through system is proven to efficiently remove the interfering effect of predominantly occurring organic species in human urine on ECD with RSD% less than 8.6%.

Process analytical technology in continuous processing: Model-based real time control of pH between capture chromatography and viral inactivation for monoclonal antibody production

A real time mechanistic model-based control strategy is demonstrated for in-line pH adjustment post-capture chromatography and prior to viral inactivation for continuous processing of monoclonal antibodies. At this point in the process, tight control of pH is essential, as pH fluctuations above 3.5 can result in incomplete viral inactivation, while fluctuations below 3.5 can lead to significant aggregate formation. The present approach predicts the pH profile during the transition phase between chromatography wash and elution steps by modelling the process stream at the column outlet as a mixture of two independent buffer systems. Control of pH in this transition phase is a critical consideration in capture chromatography as a significant amount of mAb material is eluted at this time. The model inputs are buffer concentrations, flow rates, and theoretical pKa values, along with cleaning step conductivity profiles which are readily available from a typical process chromatography equipment. The utilization of the most recent cleaning cycle data as an input to the model allows sensitive calibration to the individual process at hand on a column-to-column basis. The model is able to accurately predict the pH profile throughout the elution, as well as calculate the flow rate of the acid (titrant) required at each time point to maintain the pH consistently at 3.5±0.2. The strategy is demonstrated for various buffers, columns, operating conditions, and process deviations in a three-column continuous process, and is a useful and simple approach for achieving robust control of pH at this critical point in the continuous train.

Proof-of-Concept Design of an In-Line pH Neutralization System with Coarse and Fine Adjustments for the Continuous Manufacturing of Pharmaceuticals

Proof-of-Concept Design of an In-Line pH Neutralization System with Coarse and Fine Adjustments for the Continuous Manufacturing of Pharmaceuticals

Polishing approach with fully connected flow-through purification for therapeutic monoclonal antibody.

The biopharmaceutical industry is evolving toward process intensification that can offer increased productivity and improved economics without sacrificing process robustness. A semi-continuous downstream process linking purification/polishing unit operations in series can reduce or eliminate intermediate holding tanks and reduce overall processing time. Accordingly, we have developed a therapeutic monoclonal antibody polishing template comprised of a connected flow-through polishing technologies that include activated carbon, cation exchange, and anion-exchange chromatography. In this report, we evaluated fully-connected pool-less polishing with three flow-through technologies, operating as a single skid to streamline and improve an mAb purification platform. Laboratory-scale pool-less processing was achieved without utilizing in-line pH adjustment and conductivity dilution based on the previously optimized single process parameter. Two connected flow-through configurations of polishing steps were evaluated: a two-step process using anion exchange and cation exchange and a three step process using activated carbon, anion exchange and cation exchange chromatography. Laboratory-scale proof of concept studies showed comparable performance between the batch purification process and the pool-less process configuration. Three step polishing highly intensified the processes and provided higher process loading and achieved bulk drug specification with higher impurity clearance (>95%) and high overall mAb yield (>95%).

Automated preconcentration of Fe, Zn, Cu, Ni, Cd, Pb, Co, and Mn in seawater with analysis using high-resolution sector field inductively-coupled plasma mass spectrometry

A rapid, automated, high-throughput analytical method capable of simultaneous analysis of multiple elements at trace and ultratrace levels is required to investigate the biogeochemical cycle of trace metals in the ocean. Here we present an analytical approach which uses a commercially available automated preconcentration device (SeaFAST) with accurate volume loading and in-line pH buffering of the sample prior to loading onto a chelating resin (WAKO) and subsequent simultaneous analysis of iron (Fe), zinc (Zn), copper (Cu), nickel (Ni), cadmium (Cd), lead (Pb), cobalt (Co) and manganese (Mn) by high-resolution inductively-coupled plasma mass spectrometry (HR-ICP-MS). Quantification of sample concentration was undertaken using isotope dilution for Fe, Zn, Cu, Ni, Cd and Pb, and standard addition for Co and Mn. The chelating resin is shown to have a high affinity for all analyzed elements, with recoveries between 83 and 100% for all elements, except Mn (60%) and Ni (48%), and showed higher recoveries for Ni, Cd, Pb, Co and Mn in direct comparison to an alternative resin (NOBIAS Chelate-PA1). The reduced recoveries for Ni and Mn using the WAKO resin did not affect the quantification accuracy. A relatively constant retention efficiency on the resin over a broad pH range (pH 5–8) was observed for the trace metals, except for Mn. Mn quantification using standard addition required accurate sample pH adjustment with optimal recoveries at pH 7.5 ± 0.3. UV digestion was necessary to increase recovery of Co and Cu in seawater by 15.6% and 11.4%, respectively, and achieved full break-down of spiked Co-containing vitamin B12 complexes. Low blank levels and detection limits could be achieved (e.g., 0.029 nmol L−1 for Fe and 0.028 nmol L−1 for Zn) with the use of high purity reagents. Precision and accuracy were assessed using SAFe S, D1, and D2 reference seawaters, and results were in good agreement with available consensus values. The presented method is ideal for high throughput simultaneous analysis of trace elements in coastal and oceanic seawaters. We present a successful application of the analytical method to samples collected in June 2014 in the Northeast Atlantic Ocean.

Metabolic Control in Mammalian Fed-Batch Cell Cultures for Reduced Lactic Acid Accumulation and Improved Process Robustness

Biomass and cell-specific metabolic rates usually change dynamically over time, making the “feed according to need” strategy difficult to realize in a commercial fed-batch process. We here demonstrate a novel feeding strategy which is designed to hold a particular metabolic state in a fed-batch process by adaptive feeding in real time. The feed rate is calculated with a transferable biomass model based on capacitance, which changes the nutrient flow stoichiometrically in real time. A limited glucose environment was used to confine the cell in a particular metabolic state. In order to cope with uncertainty, two strategies were tested to change the adaptive feed rate and prevent starvation while in limitation: (i) inline pH and online glucose concentration measurement or (ii) inline pH alone, which was shown to be sufficient for the problem statement. In this contribution, we achieved metabolic control within a defined target range. The direct benefit was two-fold: the lactic acid profile was improved and pH could be kept stable. Multivariate Data Analysis (MVDA) has shown that pH influenced lactic acid production or consumption in historical data sets. We demonstrate that a low pH (around 6.8) is not required for our strategy, as glucose availability is already limiting the flux. On the contrary, we boosted glycolytic flux in glucose limitation by setting the pH to 7.4. This new approach led to a yield of lactic acid/glucose (Y L/G) around zero for the whole process time and high titers in our labs. We hypothesize that a higher carbon flux, resulting from a higher pH, may lead to more cells which produce more product. The relevance of this work aims at feeding mammalian cell cultures safely in limitation with a desired metabolic flux range. This resulted in extremely stable, low glucose levels, very robust pH profiles without acid/base interventions and a metabolic state in which lactic acid was consumed instead of being produced from day 1. With this contribution, we wish to extend the basic repertoire of available process control strategies, which will open up new avenues in automation technology and radically improve process robustness in both process development and manufacturing.

In-line high-temperature pH control during hot-water extraction of wood

An in-line pH control system for hot-water extraction of wood was developed and tested for optimal extraction of high-molar-mass hemicelluloses from spruce wood. Automated alkali addition was achieved by combining a controller and HPLC pump with a solid ZrO2-based high-temperature and high-pressure pH electrode and a high-temperature and high-pressure Ag/AgCl reference electrode. Three different set points were tested; 600mV, 575mV and 550mV corresponding to a pH at 170°C of 4.3, 4.6, and 4.85, respectively. An extraction with no pH control was conducted for reference. The extracts were analyzed for total dissolved solids, non-cellulosic carbohydrates, monomeric sugars, average weight molar mass of the hemicelluloses and free acetic acid. The results show that higher extraction pH increases the molar mass but decreases the total yield of dissolved hemicelluloses and also inhibit sugar monomer formation.

High-temperature pH measuring during hot-water extraction of hemicelluloses from wood

A high-temperature pH measuring system was developed, calibrated, and validated for measuring pH during hot-water extraction of hemicelluloses from wood. The aim was to measure in-line pH during extraction in order to control the extraction pH. An yttria-stabilized Zr/ZrO2 electrode was used for measuring the potential. Phthalate and phosphate buffers were used to calibrate the system at 160, 170, and 180°C. Also, different buffer concentrations were tested for stability. Tap water and different salt solutions were tested to investigate the system response to pure water and water solutions. Calibration curves were acquired from the calibration results and Nernst equation was used to calculate the in-line pH during wood extractions. The setup showed stable and reliable potential readings during both calibration tests and wood extractions. The in-line pH was found to be 0.35 pH units higher than when measured at room temperature with a conventional pH meter.

On-line speciation analysis of inorganic arsenic in complex environmental aqueous samples by pervaporation sequential injection analysis

A proof of concept of a novel pervaporation sequential injection (PSI) analysis method for automatic non-chromatographic speciation analysis of inorganic arsenic in complex aqueous samples is presented. The method is based on hydride generation of arsine followed by its on-line pervaporation-based membrane separation and CCD spectrophotometric detection. The concentrations of arsenite (As(III)) and arsenate (As(V)) are determined sequentially in a single sample zone. The leading section of the sample zone merges with a citric acid/citrate buffer solution (pH 4.5) for the selective reduction of As(III) to arsine while the trailing section of the sample zone merges with hydrochloric acid solution to allow the reduction of both As(III) and As(V) to arsine at pH lower than 1. Virtually identical analytical sensitivity is obtained for both As(III) and As(V) at this high acidity. The flow analyzer also accommodates in-line pH detector for monitoring of the acidity throughout the sample zone prior to hydride generation. Under optimal conditions the proposed PSI method is characterized by a limit of detection, linear calibration range and repeatability for As(III) of 22 μg L(-1) (3sblank level criterion), 50-1000 μg L(-1) and 3.0% at the 500 μg L(-1) level and for As(V) of 51 μg L(-1), 100-2000 μg L(-1) and 2.6% at the 500 μg L(-1) level, respectively. The method was validated with mixed As(III)/As(V) standard aqueous solutions and successfully applied to the determination of As(III) and As(V) in river water samples with elevated content of dissolved organic carbon and suspended particulate matter with no prior sample pretreatment. Excellent relative recoveries ranging from 98% to 104% were obtained for both As(III) and As(V).

In-line mixer pH system for the identification of titration curve

An in-line mixer pH system is proposed to identify the titration curve before changes in the input stream affect the main pH neutralization reactor. It is operated under integral control and shows oscillatory responses for a wide range of controller integral gain. The bifurcation diagram for the change of the controller integral gain is very similar to that of the quadratic map which is often used in illustrating the chaos phenomena of nonlinear processes. Various bifurcation diagrams are obtained as operating conditions of the in-line mixer system such as the set point of pH and components of the input stream change. The integral control keeps oscillations be around the set point, providing plentiful information about the pH process. The oscillations can be used to identify the titration curve of the input acid stream. Simulation and experimental results show that the chaotic operations of a small in-line mixer can be positively used for the identification of the titration curve.

Reset Control of an Industrial In-Line pH Process

This work presents a reset/hybrid control application of an in-line pH process. The nonlinear process dynamic is linearized around different operating points, and as a result a second-order plus dead time plant with uncertain gain and delay is obtained for control purposes. A standard PI compensator and reset compensator are designed and tuned. The main aim of this work was to compare the performance of both compensators by practical experimentation. The results show the reset compensator is able to overcome fundamental limitations of linear and time invariant control with a faster tracking response and overall improvement in disturbance rejection.

Distributed parameter model for pH process including distributed continuous and discrete reactant feed

In most cases the pH process is treated as a lumped parameter system for modeling, simulation and control. This approach deals not only with some advantages but also with several limits. After detailed discussion, the distributed parameter model for the pH process is derived for tubular mixers and for the in-line pH process. Although the distributed parameter approach for the pH process is more complicated than classic well-known lumped parameter pH model, it enables several new investigations. Based on the proposed model two research problems are discussed. The first problem deals with the influence of the mixer shape on the pH process dynamics. The second one presents a discussion on the identification possibilities of the reagents concentration in the distributed parameter pH process. The model discussed in the paper was explicated in more detail by the description of imperfect mixing phenomenon.

A silicon‐based multi‐sensor chip for monitoring of fermentation processes

AbstractThis paper describes the design and characterization of a silicon‐based sensor chip for monitoring of fermentation processes. The sensor chip consists of three sensors using different transducer principles. A capacitive electrolyte‐insulator‐semiconductor (EIS) field‐effect structure with Ta2O5 as gate material was utilized as pH sensor. An electrolyte conductivity sensor was realized by measuring the impedance between two interdigitated electrodes (IDE). A platinum thermistor was included for temperature measurements. The EIS sensor was integrated into a bioreactor and successfully used for an inline pH measurement. The layout of the IDE has been optimized with respect to a high cell constant and a wide detectable conductivity range. The integrated platinum thermistor allowed for temperature compensation of the electrolyte conductivity measurement.

A passivity-based approach to reset control systems stability

The stability of reset control systems has been mainly studied for the feedback interconnection of reset compensators with linear time-invariant systems. This work gives a stability analysis of reset compensators in feedback interconnection with passive nonlinear systems. The results are based on the passivity approach to L 2 -stability for feedback systems with exogenous inputs, and the fact that a reset compensator will be passive if its base compensator is passive. Several examples of full and partial reset compensations are analyzed, and a detailed case study of an in-line pH control system is given.

Concept for a solid-state multi-parameter sensor system for cell-culture monitoring

In this study, a concept for a silicon-based modular solid-state sensor system for inline multi-parameter monitoring of cell-culture fermentation processes is presented. The envisaged multi-parameter sensor system consists of two identical sensor modules and is intended for continuous quantification of up to five (bio-)chemical and physical parameters, namely, glucose and glutamine concentration, pH value, electrolyte conductivity and temperature by applying different transducer principles and/or different operation modes. Experimental results for the field-effect electrolyte-insulator-semiconductor (EIS) sterilisable pH sensor and electrolyte conductivity sensor based on interdigitated electrodes are presented. The ongoing autoclaving does not have any significant impact on the pH-sensitive properties of a Ta 2O 5-gate EIS sensor. Even after 30 autoclaving cycles, the pH sensors show a clear pH response and nearly linear calibration curve with a slope of 57 ± 1 mV/pH. Additional scanning electron microscopy and ellipsometric investigations do not show any visible surface degradation or changes in the thickness of the pH-sensitive Ta 2O 5 layer. The preliminary results demonstrate the suitability of the developed EIS sensor for an inline pH measurement during a fermentation process. In addition, interdigitated electrodes of different geometries serving as electrolyte conductivity sensor have been tested for measurements in relatively high ionic-strength solutions.