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Monitoring the protein concentration and buffer composition during the Ultrafiltration/Diafiltration (UF/DF) step enables the further automation of biopharmaceutical production and supports Real-time Release Testing (RTRT). Previously, in-line Ultraviolet (UV) and Infrared (IR) measurements have been used to successfully monitor the protein concentration over a large range. The progress of the diafiltration step has been monitored with density measurements and Infrared Spectroscopy (IR). Raman spectroscopy is capable of measuring both the protein and excipient concentration while being more robust and suitable for production measurements in comparison to Infrared Spectroscopy (IR). Regardless of the spectroscopic sensor used, the low concentration of excipients poses a challenge for the sensors. By combining sensor measurements with a semi-mechanistic model through an Extended Kalman Filter (EKF), the sensitivity to determine the progress of the diafiltration can be improved. In this study, Raman measurements are combined with an EKF for three case studies. The advantages of Kalman-filtered Raman measurements for excipient monitoring are shown in comparison to density measurements. Furthermore, Raman measurements showed a higher measurement speed in comparison to Variable Pathlength (VP) UV measurement at the trade-off of a slightly worse prediction accuracy for the protein concentration. However, the Raman-based protein concentration measurements relied mostly on an increase in the background signal during the process and not on proteinaceous features, which could pose a challenge due to the potential influence of batch variability on the background signal. Overall, the combination of Raman spectroscopy and EKF is a promising tool for monitoring the UF/DF step and enables process automation by using adaptive process control. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Monitoring of ultra- and diafiltration processes by Kalman-filtered Raman measurements." The authors acknowledge that there was a flaw in the sign convention in the original publication, specifically in the description of the extended Kalman filter. They provide the corrected definitions for the state vectors and note that the sign of both state variables was changed. The authors assure readers that the code developed for the project was not affected by these errors and that all visualizations and conclusions in the paper remain valid. They also provide a Python-based version of the code for future readers. The document includes acknowledgements and supplementary information. [Extracted from the article]

The protein cloud-point temperature (TCloud) is a known representative of protein–protein interaction strength and provides valuable information during the development and characterization of protein-based products, such as biopharmaceutics. A high-throughput low volume TCloud detection method was introduced in preceding work, where it was concluded that the extracted value is an apparent TCloud (TCloud,app). As an understanding of the apparent nature is imperative to facilitate inter-study data comparability, the current work was performed to systematically evaluate the influence of 3 image analysis strategies and 2 experimental parameters (sample volume and cooling rate) on TCloud,app detection of lysozyme. Different image analysis strategies showed that TCloud,app is detectable by means of total pixel intensity difference and the total number of white pixels, but the latter is also able to extract the ice nucleation temperature. Experimental parameter variation showed a TCloud,app depression for increasing cooling rates (0.1–0.5 °C/min), and larger sample volumes (5–24 μL). Exploratory thermographic data indicated this resulted from a temperature discrepancy between the measured temperature by the cryogenic device and the actual sample temperature. Literature validation confirmed that the discrepancy does not affect the relative inter-study comparability of the samples, regardless of the image analysis strategy or experimental parameters. Additionally, high measurement precision was demonstrated, as TCloud,app changes were detectable down to a sample volume of only 5 μL and for 0.1 °C/min cooling rate increments. This work explains the apparent nature of the TCloud detection method, showcases its detection precision, and broadens the applicability of the experimental setup. [ABSTRACT FROM AUTHOR]

Formulation conditions have a significant influence on the degree of freeze/thaw (FT) stress-induced protein instabilities. Adding cryoprotectants might stabilize the induced FT stress instabilities. However, a simple preservation of protein stability might be insufficient and further methods are necessary. This study aims to evaluate the addition of a heat cycle following FT application as a function of different cryoprotectants with lysozyme as exemplary protein. Sucrose and glycerol were shown to be the most effective cryoprotectants when compared to PEG200 and Tween20. In terms of heat-induced reversibility of aggregates, glycerol showed the best performance followed by sucrose, NaCl and Tween20 systems. The analysis was performed using a novel approach to visualize complex interplays by a clustering and data reduction scheme. In addition, solubility and structural integrity were measured and confirmed the obtained results. [ABSTRACT FROM AUTHOR]

As competition in the biopharmaceutical market gets keener due to the market entry of biosimilars, process analytical technologies (PATs) play an important role for process automation and cost reduction. This article will give a general overview and address the recent innovations and applications of spectroscopic methods as PAT tools in the downstream processing of biologics. As data analysis strategies are a crucial part of PAT, the review discusses frequently used data analysis techniques and addresses data fusion methodologies as the combination of several sensors is moving forward in the field. The last chapter will give an outlook on the application of spectroscopic methods in combination with chemometrics and model predictive control (MPC) for downstream processes. [ABSTRACT FROM AUTHOR]

Ultrafiltration/diafiltration (UF/DF) plays an important role in the manufacturing of biopharmaceuticals. Monitoring critical process parameters and quality attributes by process analytical technology (PAT) during those steps can facilitate process development and assure consistent quality in production processes. In this study, a lab-scale cross-flow filtration (CFF) device was equipped with a variable pathlength (VP) ultraviolet and visible (UV/Vis) spectrometer, a light scattering photometer, and a liquid density sensor (microLDS). Based on the measured signals, the protein concentration, buffer exchange, apparent molecular weight, and hydrodynamic radius were monitored. The setup was tested in three case studies. First, lysozyme was used in an UF/DF run to show the comparability of on-line and off-line measurements. The corresponding correlation coefficients exceeded 0.97. Next, urea-induced changes in protein size of glucose oxidase (GOx) were monitored during two DF steps. Here, correlation coefficients were ≥ 0.92 for static light scattering (SLS) and dynamic light scattering (DLS). The correlation coefficient for the protein concentration was 0.82, possibly due to time-dependent protein precipitation. Finally, a case study was conducted with a monoclonal antibody (mAb) to show the full potential of this setup. Again, off-line and on-line measurements were in good agreement with all correlation coefficients exceeding 0.92. The protein concentration could be monitored in-line in a large range from 3 to 120 g L− 1. A buffer-dependent increase in apparent molecular weight of the mAb was observed during DF, providing interesting supplemental information for process development and stability assessment. In summary, the developed setup provides a powerful testing system for evaluating different UF/DF processes and may be a good starting point to develop process control strategies. [ABSTRACT FROM AUTHOR]

Short-term parameters correlating to long-term protein stability, such as the protein cloud point temperature (Tcloud), are of interest to improve efficiency during protein product development. Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput (HT) manner. This study presents a low volume HT detection method for (sub-zero) Tcloud determination of lysozyme, as such an experimental method is not available yet. The setup consists of a cryogenic device with an automated imaging system. Measurement reproducibility (median absolute deviation of 0.2 °C) and literature-based parameter validation (Pearson correlation coefficient of 0.996) were shown by a robustness and validation study. The subsequent case study demonstrated a partial correlation between the obtained apparent Tcloud parameter and long-term protein stability as a function of lysozyme concentration, ion type, ionic strength, and freeze/thaw stress. The presented experimental setup demonstrates its ability to advance short-term strategies for efficient protein formulation development. [ABSTRACT FROM AUTHOR]

The influence of process parameters during freeze/thaw (FT) operations is essential for the preservation of the protein stability/activity during production and storage processes in the biopharmaceutical industry. Process parameters, such as FT ramps, the final storage time and temperature, affect the occurring FT stress onto the target protein in different ways. FT stress includes cold denaturation, freeze concentration, and ice crystal formation which can result in protein aggregation. To visualize the impact of variations in FT ramps, descriptors such as solubility, phase behavior and crystal morphology were evaluated. The phase diagram-based toolbox in combination with an HTS-compatible cryo-device allowed the identification of suitable ramping schemes during FT operations. It could be clearly shown that rapid operations are needed above the glass transition temperature of the target protein to circumvent precipitation during FT cycles. Finally, a stability index is introduced which allows ranking of the systems investigated. [ABSTRACT FROM AUTHOR]

Preparative protein precipitation is known as a cost-efficient and easy-to-use alternative to chromatographic purification steps. This said, at the moment, there is no process for monoclonal antibodies (mAb) on the market, although especially polyethylene glycol-induced precipitation has shown great potential. One reason might be the highly complex behavior of each component of a crude feedstock during the precipitation process. For different investigated mAbs, significant variations in the host cell protein (HCP) reduction are observed. In contrast to the precipitation behavior of single components, the interactions and interplay in a complex feedstock are not fully understood yet. This work discusses the influence of contaminants on the precipitation behavior of two different mAbs, an IgG1, and an IgG2. By spiking the mAbs with mock solution, a complex feedstock could successfully be mimicked. Spiking contaminants influenced the yield and purity of the mAbs after the precipitation step, compared to the precipitation behavior of the single components. The mixture showed a decrease in the contaminant and mAb solubility. By re-buffering the mock solution prior to spiking, special salts, small molecules like amino acids, vitamins, or sugars could be depleted while larger ones like HCP or DNA were still present. Therefore, it was possible to distinguish the influence of small molecules and larger ones. Hence, mAb–macromolecular interaction could be identified as a possible reason for the observed higher precipitation propensity, while small molecules of the cell culture medium were identified as solubilisation factors during the precipitation process. [ABSTRACT FROM AUTHOR]

The continuous availability of cells with defined cell characteristics represents a crucial issue in the biopharmaceutical and cell therapy industry. Here, development of cell banks with a long-term stability is essential and ensured by a cryopreservation strategy. The strategy needs to be optimized for each cell application individually and usually comprises controlled freezing, storage at ultra-low temperature, and fast thawing of cells. This approach is implemented by the development of master and working cell banks. Currently, empirical cryopreservation strategy development is standard, but a knowledge-based approach would be highly advantageous. In this article, we report the development of a video-based tool for the characterisation of freezing and thawing behaviour in cryopreservation process to enable a more knowledge-based cryopreservation process development. A successful tool validation was performed with a model cryopreservation process for the β-cell line INS-1E. Performance was evaluated for two working volumes (1.0 mL and 2.0 mL), based on freezing-thawing rates (20 °C to − 80 °C) and cell recovery and increase of biomass, to determine tool flexibility and practicality. Evaluation confirmed flexibility by correctly identifying a delay in freezing and thawing for the larger working volume. Further more, a decrease in cell recovery from 0.94 (± 0.14) % using 1.0 mL working volume to 0.61 (± 0.05) % using a 2.0 mL working volume displays tool practicality. The video-based tool proposed in this study presents a powerful tool for cell-specific optimisation of cryopreservation protocols. This can facilitate faster and more knowledge-based cryopreservation process development In this study, a video-based analytical tool was developed for the characterisation of freezing and thawing behaviour in cryopreservation process development. Evaluation of the practicality and flexibility of the developed tool was done based on a scale-up case study with the cell line INS-1E. Here, the influence of sample working volume on process performance was investigated. Increasing the volume from 1to 2 mL led to a delay in freezing and thawing behaviour which caused cell recovery loss. We believe that the developed tool will facilitate more directed and systematic cryopreservation process development. [ABSTRACT FROM AUTHOR]

For the purification of biopharmaceutical proteins, liquid chromatography is still the gold standard. Especially with increasing product titers, drawbacks like slow volumetric throughput and high resin costs lead to an intensifying need for alternative technologies. Selective preparative protein precipitation is one promising alternative technique. Although the capability has been proven, there has been no precipitation process realized for large-scale monoclonal antibody (mAb) production yet. One reason might be that the mechanism behind protein phase behavior is not completely understood and the precipitation process development is still empirical. Mechanistic modeling can be a means for faster, material-saving process development and a better process understanding at the same time. In preparative chromatography, mechanistic modeling was successfully shown for a variety of applications. Lately, a new isotherm for hydrophobic interaction chromatography (HIC) under consideration of water molecules as participants was proposed, enabling an accurate description of HIC. In this work, based on similarities between protein precipitation and HIC, a new precipitation model was derived. In the proposed model, the formation of protein-protein interfaces is thought to be driven by hydrophobic effects, involving a reorganization of the well-ordered water structure on the hydrophobic surfaces of the protein-protein complex. To demonstrate model capability, high-throughput precipitation experiments with pure or prior to the experiments purified proteins lysozyme, myoglobin, bovine serum albumin, and one mAb were conducted at various pH values. Polyethylene glycol (PEG) 6000 was used as precipitant. The precipitant concentration as well as the initial protein concentration was varied systematically. For all investigated proteins, the initial protein concentrations were varied between 1.5 mg/mL and 12 mg/mL. The calibrated models were successfully validated with experimental data. This mechanistic description of protein precipitation process offers mathematical explanation of the precipitation behavior of proteins at PEG concentration, protein concentration, protein size, and pH. [ABSTRACT FROM AUTHOR]

Biosurfactants are surface-active molecules produced principally by microorganisms. They are a sustainable alternative to chemically-synthesized surfactants, having the advantages of being non-toxic, highly functional, eco-friendly and biodegradable. However they are currently only used in a few industrial products due to costs associated with production and purification, which exceed those for commodity chemical surfactants. DAMP4, a member of a four-helix bundle biosurfactant protein family, can be produced in soluble form and at high yield in Escherichia coli, and can be recovered using a facile thermal phase-separation approach. As such, it encompasses an interesting synergy of biomolecular and chemical engineering with prospects for low-cost production even for industrial sectors. DAMP4 is highly functional, and due to its extraordinary thermal stability it can be purified in a simple two-step process, in which the combination of high temperature and salt leads to denaturation of all contaminants, whereas DAMP4 stays stable in solution and can be recovered by filtration. This study aimed to characterize and understand the fundamental drivers of DAMP4 stability to guide further process and surfactant design studies. The complementary use of experiments and molecular dynamics simulation revealed a broad pH and temperature tolerance for DAMP4, with a melting point of 122.4 °C, suggesting the hydrophobic core as the major contributor to thermal stability. Simulation of systematically created in silico variants of DAMP4 showed an influence of number and location of hydrophilic mutations in the hydrophobic core on stability, demonstrating a tolerance of up to three mutations before a strong loss in stability occurred. The results suggest a consideration of a balance of stability, functionality and kinetics for new designs according to their application, aiming for maximal functionality but at adequate stability to allow for cost-efficient production using thermal phase separation approaches. [ABSTRACT FROM AUTHOR]

A continuous aerosol-based process is described for the encapsulation of nanoparticles with a thin polymer shell. The process is essentially based on directed binary collisions between gas-borne core particles and liquid monomer droplets carrying opposite electrical charges, followed by photo-initiated polymerization. Once the two streams are mixed together, the process runs to completion on a time scale of about 2 min or less, required for coagulation and polymerization. Gold, silica, and sodium chloride nanoparticles were successfully coated by this technique with PHDDA [poly(hexanediol diacrylate)] and/or crosslinked PMMA [poly(methyl methacrylate)]. It was found that all core materials as well as agglomerates were wettable at room temperature and that the spreading kinetics of the monomer were fast enough to cover the core particles uniformly within the time scale provided for coagulation. The shell thickness depends on the volume ratio between core particles and monomer droplets. This was demonstrated for a combination of monodisperse silica spheres ( d = 241 nm) and polydisperse methyl methacrylate droplets, resulting in a theoretical shell thickness of 18 nm. There was very good agreement between measurements by TEM and electrical mobility spectroscopy. The results revealed that about 90 % or more of the core-shell structures were formed from 1:1 collisions between a core particle and a single monomer droplet. [ABSTRACT FROM AUTHOR]

The technique of aerosol-photopolymerization is employed for the generation of organic-inorganic spherical polymer-matrix nanocomposites (PMNCs). The loading amount of well-distributed ZnO nanoparticles in polymer networks is varied in a broad range up to 40 wt.%. Similar hybrid particles are produced without the addition of a conventional photoinitiator by making use of the UV absorptivity of ZnO nanoparticles only. Highlights of the process are the continuous, aerosol-based setup with a flow-through photoreactor operated at ambient temperature and atmospheric pressure. Aerosol-photopolymerization possesses great potential of incorporating various materials in situ into a polymer matrix, resulting in hybrid materials for diverse applications. Furthermore, the process can be integrated with further unit operations for the design of smart materials. [ABSTRACT FROM AUTHOR]