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The development of biopharmaceutical downstream processes relies on exhaustive experimental studies. The root cause is the poorly understood relationship between the protein structure of monoclonal antibodies (mAbs) and their macroscopic process behavior. Especially the development of preparative chromatography processes is challenged by the increasing structural complexity of novel antibody formats and accelerated development timelines. This study introduces a multiscale in silico model consisting of homology modeling, quantitative structure-property relationships (QSPR), and mechanistic chromatography modeling leading from the amino acid sequence of a mAb to the digital representation of its cation exchange chromatography (CEX) process. The model leverages the mAbs' structural characteristics and experimental data of a diverse set of 21 therapeutic antibodies to predict elution profiles of two mAbs that were removed from the training data set. QSPR modeling identified mAb-specific protein descriptors relevant for the prediction of the thermodynamic equilibrium and the stoichiometric coefficient of the adsorption reaction. The consideration of two discrete conformational states of IgG4 mAbs enabled prediction of split-peak elution profiles. Starting from the sequence, the presented multiscale model allows in silico development of chromatography processes before protein material is available for experimental studies., (© 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Real-time monitoring and control of protein A capture steps by process analytical technologies (PATs) promises significant economic benefits due to the improved usage of the column's binding capacity, by eliminating time-consuming off-line analytics and costly resin lifetime studies, and enabling continuous production. The PAT method proposed in this study relies on ultraviolet (UV) spectroscopy with a dynamic background subtraction based on the leveling out of the conductivity signal. This point in time can be used to collect a reference spectrum for removing the majority of spectral contributions by process-related contaminants. The removal of the background spectrum facilitates chemometric model building and model accuracy. To demonstrate the benefits of this method, five different feedstocks from our industry partner were used to mix the load material for a case study. To our knowledge, such a large design space, which covers possible variations in upstream condition besides the product concentration, has not been disclosed yet. By applying the conductivity-based background subtraction, the root mean square error of prediction (RMSEP) of the partial least squares (PLS) model improved from 0.2080 to 0.0131 g L - 1 . Finally, the potential of the background subtraction method was further evaluated for single wavelength-based predictions to facilitate implementation in production processes. An RMSEP of 0.0890 g L - 1 with univariate linear regression was achieved, showing that by subtraction of the background better prediction accuracy is achieved then without subtraction and a PLS model. In summary, the developed background subtraction method is versatile, enables accurate prediction results, and is easily implemented into existing chromatography setups with typically already integrated sensors., (© 2020 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC.)

The load phase in preparative Protein A capture steps is commonly not controlled in real-time. The load volume is generally based on an offline quantification of the monoclonal antibody (mAb) prior to loading and on a conservative column capacity determined by resin-life time studies. While this results in a reduced productivity in batch mode, the bottleneck of suitable real-time analytics has to be overcome in order to enable continuous mAb purification. In this study, Partial Least Squares Regression (PLS) modeling on UV/Vis absorption spectra was applied to quantify mAb in the effluent of a Protein A capture step during the load phase. A PLS model based on several breakthrough curves with variable mAb titers in the HCCF was successfully calibrated. The PLS model predicted the mAb concentrations in the effluent of a validation experiment with a root mean square error (RMSE) of 0.06 mg/mL. The information was applied to automatically terminate the load phase, when a product breakthrough of 1.5 mg/mL was reached. In a second part of the study, the sensitivity of the method was further increased by only considering small mAb concentrations in the calibration and by subtracting an impurity background signal. The resulting PLS model exhibited a RMSE of prediction of 0.01 mg/mL and was successfully applied to terminate the load phase, when a product breakthrough of 0.15 mg/mL was achieved. The proposed method has hence potential for the real-time monitoring and control of capture steps at large scale production. This might enhance the resin capacity utilization, eliminate time-consuming offline analytics, and contribute to the realization of continuous processing. Biotechnol. Bioeng. 2017;114: 368-373. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc., (© 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

This study aims at defining rheological parameters for the characterization of highly concentrated protein solutions. As a basis for comparing rheological behavior with protein solution characteristics the protein phase behavior of Lysozyme from chicken egg white with concentrations up to 225 mg/mL, changing pH values and additive concentrations was studied in a microbatch scale format. The prepared phase diagrams, scored after 40 days (t40) give insights into the kind and kinetics of the phase transitions that occur. Oscillatory frequency sweep measurements of samples with exactly the same conditions were conducted immediately after preparation (t0). The protein solutions behave viscoelastic and show a characteristic curve shape of the storage modulus (G') and the loss modulus (G″). The graphs provide information about the cross-linking degree of the respective sample. The measured rheological parameters were sensitive concerning solution composition, protein concentration and solution inner structure. The rheological moduli G' and G″ and especially the ratio of these parameters over a frequency range from 100 to 40000 rad/sec give information about the aggregation tendency of the protein under tested conditions. We succeeded to correlate protein phase behavior with the defined rheological key parameter ωCO. This point represents the frequency value of the intersection point from G' and G″. In our study Lysozyme expressed a ωCO threshold value of 20000 rad/sec as a lower limit for stable protein solutions. The predictability of lysozyme aggregation tendency and crystallization by means of squeeze flow rheometry is shown., (© 2015 Wiley Periodicals, Inc.)

Since the first FDA approval of a PEGylated product in 1990, so called random PEGylation reactions are still used to increase the efficacy of biopharmaceuticals and represent the major technology of all approved PEG-modified drugs. However, the great influence of process parameters on PEGylation degree and the PEG-binding site results in a lack of reaction specificity which can have severe impact on the product profile. Consequently, reproducible and well characterized processes are essential to meet increasing regulative requirements resulting from the quality-by-design (QbD) initiative, especially for this kind of modification type. In this study we present a general approach which combines the simple chemistry of random PEGylation reactions with high throughput experimentation (HTE) to achieve a well-defined process. Robotic based batch experiments have been established in a 96-well plate format and were analyzed to investigate the influence of different PEGylation conditions for lysozyme as model protein. With common SEC analytics highly reproducible reaction kinetics were measured and a significant influence of PEG-excess, buffer pH, and reaction time could be investigated. Additional mono-PEG-lysozyme analytics showed the impact of varying buffer pH on the isoform distribution, which allowed us to identify optimal process parameters to get a maximum concentration of each isoform. Employing Micrococcus lysodeikticus based activity assays, PEG-lysozyme33 was identified to be the isoform with the highest residual activity, followed by PEG-lysozyme1 . Based on these results, a control space for a PEGylation reaction was defined with respect to an optimal overall volumetric activity of mono-PEG-lysozyme isoform mixtures., (© 2013 Wiley Periodicals, Inc.)

Chromatography is the method of choice for the separation of proteins, at both analytical and preparative scale. Orthogonal purification strategies for industrial use can easily be implemented by combining different modes of adsorption. Nevertheless, with flexibility comes the freedom of choice and optimal conditions for consecutive steps need to be identified in a robust and reproducible fashion. One way to address this issue is the use of mathematical models that allow for an in silico process optimization. Although this has been shown to work, model parameter estimation for complex feedstocks becomes the bottleneck in process development. An integral part of parameter assessment is the accurate measurement of retention times in a series of isocratic or gradient elution experiments. As high-resolution analytics that can differentiate between proteins are often not readily available, pure protein is mandatory for parameter determination. In this work, we present an approach that has the potential to solve this problem. Based on the uniqueness of UV absorption spectra of proteins, we were able to accurately measure retention times in systems of up to four co-eluting compounds. The presented approach is calibration-free, meaning that prior knowledge of pure component absorption spectra is not required. Actually, pure protein spectra can be determined from co-eluting proteins as part of the methodology. The approach was tested for size-exclusion chromatograms of 38 mixtures of co-eluting proteins. Retention times were determined with an average error of 0.6 s (1.6% of average peak width), approximated and measured pure component spectra showed an average coefficient of correlation of 0.992., (Copyright © 2012 Wiley Periodicals, Inc.)

The application of high throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Recently, a new label-free and non-invasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements was presented. Analytics based on the methodology was shown to increase analytical throughput for selective protein quantification significantly, however this was only demonstrated for one particular protein combination. In this work, the possibilities and limitations of the analytical method are investigated further. Principal component analysis (PCA) was performed on a broad range of absorption spectra to investigate their common characteristics and differences. The PCA was used both for cluster analysis and to define a measure for spectral similarity. For binary protein combinations, the calibration precision was shown to decrease exponentially with the defined spectral similarity factor. Knowledge of this correlation can be used to determine a priori whether a calibration will be successful or not. Calibration robustness was investigated by applying the analytics to liquid chromatography performed in HTE mode. Further it was shown, that a spectral difference of 0.6% was sufficient to sucessfully preform a spectral based calibration of two IgG1 monoclonals., (Copyright © 2012 Wiley Periodicals, Inc.)

Aqueous two-phase systems (ATPSs) as separation technique have regained substantial interest from the biotech industry. Biopharmaceutical companies faced with increasing product titers and stiffening economic competition reconsider ATPS as an alternative to chromatography. As the implementation of an ATPS is material, time, and labor intensive, a miniaturized and automated screening process would be beneficial. In this article such a method, its statistical evaluation, and its application to a biopharmaceutical separation task are shown. To speed up early stage ATPS profiling an automated application of the cloud-point method for binodal determination was developed. PEG4000-PO(4) binodals were measured automatically and manually and were found to be identical within the experimental error. The ATPS screening procedure was applied to a model system and an industrial separation task. PEG4000-PO(4) systems at a protein concentration of 0.75 mg/mL were used. The influence of pH, NaCl addition, and tie line length was investigated. Lysozyme as model protein, two monoclonal antibodies, and a host cell protein pool were used. The method was found to yield partition coefficients identical to manually determined values for lysozyme. The monoclonal antibodies were shifted from the bottom into the upper phase by addition of NaCl. This shift occurred at lower NaCl concentration when the pH of the system was closer to the pI of the distributed protein. Addition of NaCl, increase in PEG4000 concentration and pH led to significant loss of the mAb due to precipitation. Capacity limitations of these systems were thus demonstrated. The chosen model systems allowed a reduction of up to 50% HCP with a recovery of greater than 95% of the target proteins. As these values might not be industrially relevant when compared to current chromatographic procedures, the developed screening procedure allows a fast evaluation of more suitable and optimized ATPS system for a given task., (© 2010 Wiley Periodicals, Inc.)

In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.

We introduce a robust and scale-flexible approach to macromolecule purification employing tailor-made magnetic adsorbents and high-gradient magnetic separation technology adapted from the mineral processing industries. Detailed procedures for the synthesis of large quantities of low-cost defined submicron-sized magnetic supports are presented. These support materials exhibit unique features, which facilitate their large-scale processing using high magnetic field gradients, namely sufficiently high magnetization, a relatively narrow particle size distribution and ideal superparamagnetism. Following systematic optimization with respect to activation chemistry, spacer length and ligand density, conditions for preparation of effective high capacity (Q(max) = 120 mg g(-1)) strongly interacting (Kd < 0.3 microm) trypsin-binding adsorbents based on immobilized benzamidine were established. In small-scale studies approximately 95% of the endogenous trypsin present in a crude porcine pancreatin feedstock was recovered with a purification factor of approximately 4.1 at the expense of only a 4% loss in alpha-amylase activity. Efficient recovery of trypsin from the same feedstock was demonstrated at a vastly increased scale using a high-gradient magnetic separation system to capture loaded benzamidine-linked adsorbents following batch adsorption. With the aid of a simple recycle loop over 80% of the initially adsorbed trypsin was recovered in-line with an overall purification factor of approximately 3.5., (Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 301-313, 2002.)