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A fundamental understanding of the protein retention mechanism in preparative ion exchange (IEX) chromatography columns is essential for a model-based process development approach. For the past three decades, the mechanistic description of protein retention has been based predominantly on the steric mass action (SMA) model. In recent years, however, retention profiles of proteins have been reported more frequently for preparative processes that are not consistent with the mechanistic understanding relying on the SMA model. In this work, complex elution behavior of proteins in preparative IEX processes is analyzed using a colloidal particle adsorption (CPA) model. The CPA model is found to be capable of reproducing elution profiles that cannot be described by the traditional SMA model. According to the CPA model, the reported complex behavior can be ascribed to a strong compression and concentration of the elution front in the lower unsaturated part of the chromatography column. As the unsaturated part of the column decreases with increasing protein load density, exceeding a critical load density can lead to the formation of a shoulder in the peak front. The general applicability of the model in describing preparative IEX processes is demonstrated using several industrial case studies including multiple monoclonal antibodies on different IEX adsorber systems. In this context, the work covers both salt controlled and pH-controlled protein elution., Competing Interests: Declaration of Competing Interest The authors affiliated to GoSilico declare that the software ChromX used in this study is a commercial product of GoSilico. The presented research does not depend on the usage of ChromX, the model can be implemented in any kind of modeling software., (Copyright © 2021. Published by Elsevier B.V.)

Cation exchange chromatography (CEX) is a widely used technique for the removal of monoclonal antibody (mAb) aggregates. At present, resins are mainly used for this purpose, as convective types of adsorbents such as membrane adsorbers (MAs) have often not demonstrated overall comparable performance for this particular application. Fiber-based adsorbents can overcome the current limitations of MAs with respect to permeability, binding capacity, and adsorbent cost, and could therefore be a viable alternative to resins for the removal of mAb aggregates. It has not been evaluated, however, whether and under which conditions the use of such adsorbents is feasible for this purpose. In the present study, the use of fiber-based CEX adsorbents for mAb aggregate removal was examined. Two types of fiber-based adsorbents, an uncontrolled grafted and a controlled grafted fiber-based adsorbent, were evaluated with respect to permeability, dynamic mAb binding capacity (DBC), resolution capabilities, and the performance in bind and elute (B/E) and frontal chromatography mode with respect to typical performance indicators. The permeabilities of the fiber-based adsorbents ranged from 200 to 1700 mD, making it possible to use the fiber-based adsorbents at larger bed heights than membrane adsorbers with fast mobile phase velocities. Antibody DBCs ranged from 20 to 41 g/L at 150 cm/h, and at higher mobile phase velocities exceeded the DBC of an existing resin material, Poros 50 HS, which has frequently been used for aggregate removal. Both fiber types showed good resolution capabilities of monomer and aggregates, and provided better resolution per column length than Poros 50 HS. Typical purity and yield constraints were fulfilled for both fiber types in both B/E and frontal chromatography mode for mobile phase velocities ranging up to 480 cm/h and 1060 cm/h. The overall performance of the controlled grafted fibers was found to be superior to the performance of uncontrolled grafted fiber-based adsorbents due to higher productivity and lower buffer consumption. The overall performance of the fiber-based adsorbents was found to be comparable to the performance of Poros 50 HS at typical operating conditions. The results in this study indicate that the use of fiber-based adsorbents for mAb aggregate removal is feasible with a performance that is comparable to the performance of an existing resin material. Depending on the cost of the adsorbents and the use scenario, the usage of such adsorbents could be beneficial., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing-scale can be affected by variability in feed composition, mobile phase and resin properties, or dispersion effects depending on the chromatography system at hand. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. In this study, a multicomponent steric mass-action (SMA) adsorption model was applied to the scale-up of a CEX polishing step. Based on chromatograms and elution pool data ranging from laboratory- to manufacturing-scale, the proposed modeling workflow enabled early identification of differences between scales, for example, system dispersion effects or ionic capacity variability. A multistage model qualification approach was introduced to measure the model quality and to understand the model's limitations across scales. The experimental SDM and the in silico model were qualified against large-scale data using the identical state of the art equivalence testing procedure. The mechanistic chromatography model avoided limitations of the SDM by capturing effects of bed height, loading density, feed composition, and mobile phase properties. The results demonstrate the applicability of mechanistic chromatography models as a possible alternative to conventional SDM approaches., (© 2020 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

In biopharmaceutical process development, resin-based high throughput screening (HTS) is well known for overcoming experimental limitations by permitting automated parallel processing at miniaturized scale, which results in fast data generation and reduced feed consumption. For membrane adsorber (MA), HTS solutions have so far only been available to a partial extent. Three case studies were performed with the aim of aligning HTS applications for MAs with those established for column chromatography: Process parameter range determination, mechanistic modeling (MM), and scalability. In order to exploit the MA typically features, such as high mass transfer and easy scalability, for scalable high throughput process development, a scale-down device (SDD) for MA was developed. Its applicability is confirmed for a monoclonal antibody aggregate removal step. The first case study explores the experimental application of the SDD developed. It uses bind and elute mode and variations of pH and salt concentration to obtain process operation windows for ion-exchange MAs Sartobind® S and Q. In the second case study, we successfully developed a mechanistic model based on parameters obtained from the SDD-HTS setup. The results proved to validate the use of the SDD developed for parameter estimation and thus model-based process development. The third case study shows the transferability and scalability of data from the SDD-HTS setup using both a direct scale factor and MM. Both approaches show good applicability with a deviation below 20% in the prediction of 10% dynamic breakthrough capacity and reliable scale-up from 0.42 to 800 ml., (© 2020 American Institute of Chemical Engineers.)

Mechanistic modeling of chromatography processes is one of the most promising techniques for the digitalization of biopharmaceutical process development. Possible applications of chromatography models range from in silico process optimization in early phase development to in silico root cause investigation during manufacturing. Nonetheless, the cumbersome and complex model calibration still decelerates the implementation of mechanistic modeling in industry. Therefore, the industry demands model calibration strategies that ensure adequate model certainty in a limited amount of time. This study introduces a directed and straightforward approach for the calibration of pH-dependent, multicomponent steric mass action (SMA) isotherm models for industrial applications. In the case investigated, the method was applied to a monoclonal antibody (mAb) polishing step including four protein species. The developed strategy combined well-established theories of preparative chromatography (e.g. Yamamoto method) and allowed a systematic reduction of unknown model parameters to 7 from initially 32. Model uncertainty was reduced by designing two representative calibration experiments for the inverse estimation of remaining model parameters. Dedicated experiments with aggregate-enriched load material led to a significant reduction of model uncertainty for the estimates of this low-concentrated product-related impurity. The model was validated beyond the operating ranges of the final unit operation, enabling its application to late-stage downstream process development. With the proposed model calibration strategy, a systematic experimental design is provided, calibration effort is strongly reduced, and local minima are avoided., (© 2020 American Institute of Chemical Engineers.)

Mechanistic modeling of protein adsorption has gained increasing importance in the development of ion-exchange (IEX) chromatography processes. The most common adsorption models use a stoichiometric representation of the adsorption process based on the law of mass action. Despite the importance of these models in model-based development, the stoichiometric representation of the adsorption process is not accurate for the description of long-range electrostatic interactions in IEX chromatography, limiting the application and mechanistic extension of these models. In this work an adsorption model is introduced describing the non-stoichiometric electrostatic interaction in IEX chromatography based on the linear Poisson-Boltzmann equation and a simplified colloidal representation of the protein. In contrast to most recent non-stoichiometric models, the introduced model accounts for charge regulation during the adsorption process. Its capability of describing the adsorption equilibrium is demonstrated by simulating partitioning coefficients of multiple proteins on different adsorber systems as a function of ionic strength and pH. Despite model simplifications the physical meaning and predictive value of the model could be preserved. By transferring model parameters of a monoclonal antibody (mAb) from one adsorber system to another, it could be demonstrated that protein parameters are theoretically not only valid on a specific adsorber system but freely transferable to other adsorbers. The predictive value of the mechanistic model on the new adsorber system was highlighted by predicting the elution behavior of charge variants of the mAb., (Copyright © 2019 Elsevier B.V. All rights reserved.)

PEGylation of biological macromolecules is a well-established strategy to increase circulation half-life, decrease renal clearance and improve biocompatibility. PEGylation is a process in which polyethylene glycol (PEG) is covalently attached to a target molecule. The production of PEGylated biopharmaceuticals is usually executed by first producing and purifying the base molecule followed by the PEGylation reaction and purification of the modified molecule. Most PEGylated pharmaceuticals are produced by random PEGylation in batch mode and need to be purified as mainly the mono-PEGylated form is the desired drug product. In this work we propose a method to estimate the degree of PEGylation (DOP) of modified protein eluting from a chromatography column in near real-time. extended multiplicative signal correction (EMSC) is used in conjunction with asymmetric least squares (aaLS) to alleviate the influence of a salt gradient during ion exchange chromatography (IEX) on the spectral data. To convert the raw data obtained from spectral data to the actual DOP additional information obtained from off-line measurements is utilized. Once the signal correction is applied to in-line spectral data the DOP can be estimated without further use of off-line analytics. As the prerequisites for the application of this method are relatively easy to obtain it may also find use to speed up process development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the past decades, research was carried out to find cost-efficient alternatives to Protein A chromatography as a capture step in monoclonal antibody (mAb) purification processes. In this work, polyethylene glycol (PEG) precipitation has shown promising results in the case of mAb yield and purity. Especially with respect to continuous processing, PEG precipitation has many advantages, like low cost of goods, simple setup, easy scalability, and the option to handle perfusion reactors. Nevertheless, replacing Protein A has the disadvantage of renouncing a platform unit operation as well. Furthermore, PEG precipitation is not capable of reducing high molecular weight impurities (HMW) like aggregates or DNA. To overcome these challenges, an integrated process strategy combining PEG precipitation with cation-exchange chromatography (CEX) for purification of a mAb is presented. This work discusses the process strategy as well as the associated fast, easy, and material-saving process development platform. These were implemented through the combination of high-throughput methods with empirical and mechanistic modeling. The strategy allows the development of a common batch process. Additionally, it is feasible to develop a continuous process. In the presented case study, a mAb provided from cell culture fluid (HCCF) was purified. The precipitation and resolubilization conditions as well as the chromatography method were optimized, and the mutual influence of all steps was investigated. A mAb yield of over 95.0% and a host cell protein (HCP) reduction of over 99.0% could be shown. At the same time, the aggregate level was reduced from 3.12% to 1.20% and the DNA level was reduced by five orders of magnitude. Furthermore, the mAb was concentrated three times to a final concentration of 11.9mg/mL., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent studies highlighted the potential of PEGylated proteins to improve stabilities and pharmacokinetics of protein drugs. Ion-exchange chromatography (IEX) is among the most frequently used purification methods for PEGylated proteins. However, the underlying physical mechanisms allowing for a separation of different PEGamers (proteins with a varying number of attached PEG molecules) are not yet fully understood. In this work, mechanistic chromatography modeling is applied to gain a deeper understanding of the mass transfer and adsorption/desorption mechanisms of mono-PEGylated proteins in IEX. Using a combination of the general rate model (GRM) and the steric mass action (SMA) isotherm, simulation results in good agreement with the experimental data are achieved. During linear gradient elution of proteins attached with PEG of different molecular weight, similar peak heights, and peak shapes at constant gradient length are observed. A superimposed effect of increased desorption rate and reduced diffusion rate as a function of the hydrodynamic radius of PEGylated proteins is identified to be the reason of this anomaly. That is why the concept of the diffusion-desorption-compensation effect is proposed. In addition to the altered elution orders, PEGylation results in a considerable decrease of maximum binding capacity. By using the SMA model in a kinetic formulation, the adsorption behavior of PEGylated proteins in the highly concentrated state is described mechanistically. An exponential increase in the steric hindrance effect with increasing PEG molecular weight is observed. This suggests the formation of multiple PEG layers in the interstitial space between bound proteins and an associated shielding of ligands on the adsorber surface to be the cause of the reduced maximum binding capacity. The presented in silico approach thus complements the hitherto proposed theories on the binding mechanisms of PEGylated proteins in IEX., (© 2017 The Authors Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Chromatographic separation of biopharmaceuticals in general and monoclonal antibodies (mAbs) specifically is the bottleneck in terms of cost and throughput in preparative purification. Still, generalized platform processes are used, neglecting molecule specific characteristics, defining protein-resin interaction terms. Currently used in silico modeling approaches do not consider the orientation of the molecule towards the chromatographic resins as a result of the structural features on an atomic level. This paper describes a quantitative structure-activity relationship (QSAR) approach to model the orientation of mAbs on ion exchange chromatographic matrices as a function of property distribution and mobile phase characteristics. 6 mAbs were used to build a predictive QSAR model and to investigate the preferred binding orientations and resulting surface shielding on resins. Thereby different dominating orientations, caused by composition of F ab fragments of the mAbs, could be identified. The presented methodology is suitable to gain extended insight in molecule orientation on chromatographic resins and to tailor purification strategies based on molecule structure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A main requirement for the implementation of model-based process development in industry is the capability of the model to predict high protein load densities. The frequently used steric mass action isotherm assumes a thermodynamically ideal system and, hence constant activity coefficients. In this manuscript, an industrial antibody purification problem under high load conditions is considered where this assumption does not hold. The high protein load densities, as commonly applied in industrial downstream processing, may lead to complex elution peak shapes. Using Mollerup's generalized ion-exchange isotherm (GIEX), the observed elution peak shapes could be modeled. To this end, the GIEX isotherm introduced two additional parameters to approximate the asymmetric activity coefficient. The effects of these two parameters on the curvature of the adsorption isotherm and the resulting chromatogram are investigated. It could be shown that they can be determined by inverse peak fitting and conform with the mechanistic demands of model-based process development., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Quantitative structure-activity relationship (QSAR) modeling for prediction of biomolecule parameters has become an established technique in chromatographic purification process design. Unfortunately available descriptor sets fail to describe the orientation of biomolecules and the effects of ionic strength in the mobile phase on the interaction with the stationary phase. The literature describes several special descriptors used for chromatographic retention modeling, all of these do not describe the screening of electrostatic potential by the mobile phase in use. In this work we introduce two new approaches of descriptor calculations, namely surface patches and plane projection, which capture an oriented binding to charged surfaces and steric hindrance of the interaction with chromatographic ligands with regard to electrostatic potential screening by mobile phase ions. We present the use of the developed descriptor sets for predictive modeling of Langmuir isotherms for proteins at different pH values between pH 5 and 10 and varying ionic strength in the range of 10-100mM. The resulting model has a high correlation of calculated descriptors and experimental results, with a coefficient of determination of 0.82 and a predictive coefficient of determination of 0.92 for unknown molecular structures and conditions. The agreement of calculated molecular interaction orientations with both, experimental results as well as molecular dynamic simulations from literature is shown. The developed descriptors provide the means for improved QSAR models of chromatographic processes, as they reflect the complex interactions of biomolecules with chromatographic phases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Fibers are prominent among novel stationary phase supports for preparative chromatography. Several recent studies have highlighted the potential of fiber-based adsorbents for high productivity downstream processing in both batch and continuous mode, but so far the development of these materials and of processes employing these materials has solely been based on experimental data. In this study we assessed whether mechanistic modeling can be performed on fiber-based adsorbents. With a column randomly filled with short cut hydrogel grafted anion exchange fibers, we tested whether tracer, linear gradient elution, and breakthrough data could be reproduced by mechanistic models. Successful modeling was achieved for all of the considered experiments, for both non-retained and retained molecules. For the fibers used in this study the best results were obtained with a transport-dispersive model in combination with a steric mass action isotherm. This approach accurately accounted for the convection and dispersion of non-retained tracers, and the breakthrough and elution behaviors of three different proteins with sizes ranging from 6 to 160kDa were accurately modeled, with simulation results closely resembling the experimental data. The estimated model parameters were plausible both from their physical meaning, and from an analysis of the underlying model assumptions. Parameters were determined within good confidence levels; the average confidence estimate was below 7% for confidence levels of 95%. This shows that fiber-based adsorbents can be modeled mechanistically, which will be valuable for the future design and evaluation of these novel materials and for the development of processes employing such materials., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mechanistic models are successfully used for protein purification process development as shown for ion-exchange column chromatography (IEX). Modeling and simulation of hydrophobic interaction chromatography (HIC) in the column mode has been seldom reported. As a combination of these two techniques is often encountered in biopharmaceutical purification steps, accurate modeling of protein adsorption in HIC is a core issue for applying holistic model-based process development, especially in the light of the Quality by Design (QbD) approach. In this work, a new mechanistic isotherm model for HIC is derived by consideration of an equilibrium between well-ordered water molecules and bulk-like ordered water molecules on the hydrophobic surfaces of protein and ligand. The model's capability of describing column chromatography experiments is demonstrated with glucose oxidase, bovine serum albumin (BSA), and lysozyme on Capto™ Phenyl (high sub) as model system. After model calibration from chromatograms of bind-and-elute experiments, results were validated with batch isotherms and prediction of further gradient elution chromatograms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recombinant protein-based virus-like particles (VLPs) are steadily gaining in importance as innovative vaccines against cancer and infectious diseases. Multiple VLPs are currently evaluated in clinical phases requiring a straightforward and rational process design. To date, there is no generic platform process available for the purification of VLPs. In order to accelerate and simplify VLP downstream processing, there is a demand for novel development approaches, technologies, and purification tools. Membrane adsorbers have been identified as promising stationary phases for the processing of bionanoparticles due to their large pore sizes. In this work, we present the potential of two strategies for designing VLP processes following the basic tenet of 'quality by design': High-throughput experimentation and process modeling of an anion-exchange membrane capture step. Automated membrane screenings allowed the identification of optimal VLP binding conditions yielding a dynamic binding capacity of 5.7 mg/mL for human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A mechanistic approach was implemented for radial ion-exchange membrane chromatography using the lumped-rate model and stoichiometric displacement model for the in silico optimization of a VLP capture step. For the first time, process modeling enabled the in silico design of a selective, robust and scalable process with minimal experimental effort for a complex VLP feedstock. The optimized anion-exchange membrane chromatography process resulted in a protein purity of 81.5%, a DNA clearance of 99.2%, and a VLP recovery of 59%., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The performance of functionalized materials, e.g., ion exchange resins, depends on multiple resin characteristics, such as type of ligand, ligand density, the pore accessibility for a molecule, and backbone characteristics. Therefore, the screening and identification process for optimal resin characteristics for separation is very time and material consuming. Previous studies on the influence of resin characteristics have focused on an experimental approach and to a lesser extent on the mechanistic understanding of the adsorption mechanism. In this in silico study, a previously developed molecular dynamics (MD) tool is used, which simulates any given biomolecule on resins with varying ligand densities. We describe a set of simulations and experiments with four proteins and six resins varying in ligand density, and show that simulations and experiments correlate well in a wide range of ligand density. With this new approach simulations can be used as pre-experimental screening for optimal adsorber characteristics, reducing the actual number of screening experiments, which results in a faster and more knowledge-based development of custom-tailored adsorbers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The accelerating growth of the market for proteins and the growing interest in new, more complex molecules are bringing new challenges to the downstream process development of these proteins. This results in a demand for faster, more cost efficient, and highly understood downstream processes. Screening procedures based on high-throughput methods are widely applied nowadays to develop purification processes for proteins. However, screening highly complex biotechnological feedstocks, such as complete cell lysates containing target proteins often expressed with a low titre, is still very challenging. In this work we demonstrate a multidimensional, analytical screening approach based on pH gradient ion exchange chromatography (IEC), gel electrophoresis and protein identification via mass spectrometry to rationally characterize a biotechnological feedstock for the purpose of purification process development. With this very simple characterization strategy a two-step purification based on consecutive IEC operations was rapidly laid out for the purification of a diagnostic protein from a cell lysate reaching a purity of ∼80%. The target protein was recombinantly produced using an insect cell expression system., (Copyright © 2013 Elsevier B.V. All rights reserved.)

pH gradient protein separations are widely used techniques in the field of protein analytics, of which isoelectric focusing is the most well known application. The chromatographic variant, based on the formation of pH gradients in ion exchange columns is only rarely applied due to the difficulties to form controllable, linear pH gradients over a broad pH range. This work describes a method for the systematic generation of buffer compositions with linear titration curves, resulting in well controllable pH gradients. To generate buffer compositions with linear titration curves an in silico method was successfully developed. With this tool, buffer compositions for pH gradient ion exchange chromatography with pH ranges spanning up to 7.5 pH units were established and successfully validated. Subsequently, the buffer systems were used to characterize the elution behavior of 22 different model proteins in cation and anion exchange pH gradient chromatography. The results of both chromatographic modes as well as isoelectric focusing were compared to describe differences in between the methods., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Covalent attachment of PEG to proteins, known as PEGylation, is currently one of the main approaches for improving the pharmacokinetics of biopharmaceuticals. However, the separation and characterization especially of positional isoforms of PEGylated proteins are still challenging tasks. A common purification strategy uses ion exchange chromatography with increasing ionic strength by shallow salt gradients. This paper presents a method which applies a linear pH gradient chromatography to separate five of six possible isoforms of mono-PEGylated lysozyme, modified with 5 kDa and 10 kDa mPEG-aldehyde. To identify the corresponding PEGylation sites a comparison of elution pH values and calculated isoelectric points of each isoform, was used. The resulting correlation showed an R(2)>0.99. Fractionation, tryptic digestion and subsequent MALDI-MS analysis of each peak, verified the predicted elution order. Based on UV areas the N-terminal amine at lysine 1 exhibited the highest reactivity, followed by the lysine 33 residue., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In the last decade, high-throughput downstream process development techniques have entered the biopharmaceutical industry. As chromatography is the standard downstream purification method, several high-throughput chromatographic methods have been developed and applied including miniaturized chromatographic columns for utilization on liquid handling stations. These columns were used to setup a complete downstream process on a liquid handling station for the first time. In this article, a monoclonal antibody process was established in lab-scale and miniaturized afterwards. The scale-down methodology is presented and discussed. Liquid handling in miniaturized single and multicolumn processes was improved and applicability was demonstrated by volume balances. The challenges of absorption measurement are discussed and strategies were shown to improve volume balances and mass balances in 96-well microtiter plates. The feasibility of miniaturizing a complete downstream process was shown. In the future, analytical bottlenecks should be addressed to gain the full benefit from miniaturized complete process development., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

A comparative study on weak anion exchangers was performed to investigate the pH dependence, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included: DEAE Sepharose FF, Poros 50 D, Fractogel EMD DEAE (M), MacroPrep DEAE Support, DEAE Ceramic HyperD 20, and Toyopearl DEAE 650 M. Testing was performed with five different model proteins: Anti-FVII mAb (immunoglobulin G), aprotinin, bovine serum albumin (BSA), Lipolase (Novozymes), and myoglobin. Retention showed an expected increasing trend as a function of pH for proteins with low pI. A decrease in retention was observed for some resins at pH 9 likely due to initiation of deprotonation of the weak anion-exchange ligands. Expected particle size distribution was obtained for all resins compared to previous studies. Binding strength to weak anion-exchange resins as a function of ionic strength depends on the specific protein. Binding and elution at low salt concentration may be performed with Toyopearl DEAE 650 M, while binding and elution at high salt concentration may be performed with MacroPrep DEAE Support. Highest binding capacities were generally obtained with Poros 50 D followed by DEAE Ceramic HyperD 20. A general good agreement was obtained between this study and data obtained by the suppliers. Verification of binding strength trends with model proteins was achieved with human growth hormone (hGH) and a hGH variant on the same resins with different elution salts, sodium chloride, sodium hydrogenphosphate, sodium sulphate, and sodium acetate. Static capacity measurements obtained in the traditional experimental set-up were compared with high-throughput screening (HTS) technique experiments with reasonable agreement. Isotherm data obtained from HTS techniques and pulse experiments were successfully combined with mathematical modelling to simulate, develop and optimise the separation process of two model proteins, Lipolase and BSA. The data presented in this paper may be used for selection of resins for testing in process development.

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.

A systematic approach for the development of a column model for protein purification is introduced. The approach includes phenomenological investigations of mass transfer and adsorption behaviour applying confocal laser scanning microscopy (CLSM). Insights from CLSM measurements are then implemented mathematically into a single particle model which is coupled afterwards with a column model (i.e. general rate model). Finally, the general rate model is used to predict the results from pulse and frontal analysis in a chromatographic column. The applied exemplary chromatographic system is bovine serum albumin (BSA) on strong anion-exchanger Source30Q at neutral pH value.

Expanded bed adsorption (EBA) is an integrative step in downstream processing allowing the direct capture of target proteins from cell-containing feedstocks. Extensive co-adsorption of biomass, however, may hamper the application of this technique. The latter is especially observed at anion exchange processes as cells or cell debris are negatively charged under common anion exchange conditions. The restrictions observed under these conditions are, however, directly related to processing steps prior to fluidised bed application. In this study, it could be shown that the effective surface charge of cell debris obtained during homogenisation is closely related to the debris size and thus to the homogenisation method and conditions. The amount and thus effect of cells binding to the adsorbent could be significantly decreased when optimising the homogenisation step not only towards optimal product release but towards a reduction of debris size and charge. The lower size and charge of the debris results not only in a reduced retention probability but also, in a lower collision probability between debris and adsorbent. The applicability was shown in an example where the homogenisation conditions of E. coli were optimised towards EBA applications. In a previous report (Reichert et al., 2001) studying the suitability of EBA for the capture of formate dehydrogenate from E. coli homogenate the pseudo affinity resin Streamline Red was identified as the only suitable adsorbent. The new approach, however, led to a system where anion exchange as capture step became possible, however, to the cost of binding capacity., (2006 Wiley Periodicals, Inc.)

Expanded bed adsorption is an innovative chromatographic technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed. Provided a perfectly classified fluidized bed (termed expanded bed) is formed in the crude feedstock and the biomass is not influencing protein transport towards the adsorbent surface, a sorption performance comparable to packed beds is found. The influence of biomass on the hydrodynamic stability of expanded beds is essential and was investigated systematically in this article. Residence-time distribution analyses were performed using model systems and a yeast suspension under various fluid-phase conditions. It is demonstrated that three factors (biomass/adsorbent interactions, biomass concentration, and flow rate) play an interdependent role disturbing the classified fluidization of an expanded bed. A clear correlation between the degree of aggregative fluidization--obtained by PDE modeling of RTD data--and the expansion behavior of the fluidized bed has been found. Thus, combining three analytical methods, namely cell transmission index analysis, expansion analysis, and RTD analysis provides a solid base for understanding and control of the fluidization behavior and thus further process design during the initial phase of process development.

An experimental study on the interplay of sorbent structure and fluid phase conditions (pH) has been carried out examining adsorption and transport of bovine serum albumin (BSA) and a monoclonal antibody (IgG 2a) on SP Sepharose Fast Flow and SP Sepharose XL. SP Sepharose Fast Flow is characterised by a relatively open pore network, while SP Sepharose XL is a composite structure with ligand-carrying dextran chains filling the pore space. Both adsorbents have similar ionic capacity. Protein transport and adsorption profiles were evaluated using confocal laser scanning microscopy. Under all investigated conditions, BSA uptake could be adequately explained by a pore diffusion mechanism. The adsorption profiles obtained for IgG 2a, however, indicated that changes in fluid phase conditions as well as a change in the solid phase structure could result in a more complex uptake mechanism as compared to pore diffusion alone. This mechanism results in a fast transport of proteins into the adsorbent, followed by an overshoot of protein in the center of the sorbent and a setback towards a homogeneous adsorption profile.

The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent.