Filling the gaps in peptide maps with a platform assay for top-down characterization of purified protein samples.

Liquid chromatography-mass spectrometry (LC-MS) intact mass analysis and LC-MS/MS peptide mapping are decisional assays for developing biological drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We leveraged the strengths of flow-programmed (fp) denaturing online buffer exchange (dOBE) chromatography, including robust automation, relatively high ESI sensitivity, and long MS/MS window time, to support a TDMS platform for industrial protein characterization. We tested data-dependent (DDA) and targeted strategies using 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large, focused dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. A DDA-based workflow provided objective identification of αLac truncation proteoforms with a two-termini clipping search. A targeted TDMS workflow facilitated the characterization of αLac oxidation positional isomers. This strategy relied on using sliding window-based fragment ion deconvolution to generate composite proteoform spectral match (cPrSM) results amenable to fragment noise filtering, which is a fundamental enhancement relevant to TDMS applications generally.

Impact of Excipient Extraction and Buffer Exchange on Recombinant Monoclonal Antibody Stability.

The foundation of a biosimilar manufacturer's regulatory filing is the demonstration of analytical and functional similarity between the biosimilar product and the pertinent originator product. The excipients in the formulation may interfere with characterization using typical analytical and functional techniques during this biosimilarity exercise. Consequently, the producers of biosimilar products resort to buffer exchange to isolate the biotherapeutic protein from the drug product formulation. However, the impact that this isolation has on the product stability is not completely known. This study aims to elucidate the extent to which mAb isolation via ultrafiltration-diafiltration-based buffer exchange impacts mAb stability. It has been demonstrated that repeated extraction cycles do result in significant changes in higher-order structure (red-shift of 5.0 nm in fluorescence maxima of buffer exchanged samples) of the mAb and also an increase in formation of basic variants from 19.1 to 26.7% and from 32.3 to 36.9% in extracted innovator and biosimilar Tmab samples, respectively. It was also observed that under certain conditions of tertiary structure disruptions, Tmab could be restabilized depending on formulation composition. Thus, mAb isolation through extraction with buffer exchange impacts the product stability. Based on the observations reported in this paper, we recommend that biosimilar manufacturers take into consideration these effects of excipients on protein stability when performing biosimilarity assessments.

Micro simulated moving bed chromatography-mass spectrometry as a continuous on-line process analytical tool.

Continuous manufacturing is becoming increasingly important in the (bio-)pharmaceutical industry, as more product can be produced in less time and at lower costs. In this context, there is a need for powerful continuous analytical tools. Many established off-line analytical methods, such as mass spectrometry (MS), are hardly considered for process analytical technology (PAT) applications in biopharmaceutical processes, as they are limited to at-line analysis due to the required sample preparation and the associated complexity, although they would provide a suitable technique for the assessment of a wide range of quality attributes. In this study, we investigated the applicability of a recently developed micro simulated moving bed chromatography system (µSMB) for continuous on-line sample preparation for MS. As a test case, we demonstrate the continuous on-line MS measurement of a protein solution (myoglobin) containing Tris buffer, which interferes with ESI-MS measurements, by continuously exchanging this buffer with a volatile ammonium acetate buffer suitable for MS measurements. The integration of the µSMB significantly increases MS sensitivity by removing over 98% of the buffer substances. Thus, this study demonstrates the feasibility of on-line µSMB-MS, providing a versatile PAT tool by combining the detection power of MS for various product attributes with all the advantages of continuous on-line analytics.

Raman and NIR spectroscopy-based real-time monitoring of the membrane filtration process of a recombinant protein for the diagnosis of SARS-CoV-2.

This research shows the detailed comparison of Raman and near-infrared (NIR) spectroscopy as Process Analytical Technology tools for the real-time monitoring of a protein purification process. A comprehensive investigation of the application and model development of Raman and NIR spectroscopy was carried out for the real-time monitoring of a process-related impurity, imidazole, during the tangential flow filtration of Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike protein. The fast development of Raman and NIR spectroscopy-based calibration models was achieved using offline calibration data, resulting in low calibration and cross-validation errors. Raman model had an RMSEC of 1.53 mM, and an RMSECV of 1.78 mM, and the NIR model had an RMSEC of 1.87 mM and an RMSECV of 2.97 mM. Furthermore, Raman models had good robustness when applied in an inline measurement system, but on the contrary NIR spectroscopy was sensitive to the changes in the measurement environment. By utilizing the developed models, inline Raman and NIR spectroscopy were successfully applied for the real-time monitoring of a process-related impurity during the membrane filtration of a recombinant protein. The results enhance the importance of implementing real-time monitoring approaches for the broader field of diagnostic and therapeutic protein purification and underscore its potential to revolutionize the rapid development of biological products.

Automated high-throughput buffer exchange platform enhances rapid flow analysis of antibody drug conjugates by high resolution mass spectrometry.

Antibody drug conjugates (ADCs) are an increasingly important therapeutic class of molecules for the treatment of cancer. Average drug-to-antibody ratio (DAR) and drug-load distribution are critical quality attributes of ADCs with the potential to impact efficacy and toxicity of the molecule and need to be analytically characterized and understood. Several platform methods including hydrophobic interaction chromatography (HIC) and native size-exclusion chromatography-mass spectrometry (nSEC-MS) have been developed for that purpose; however, each presents some limitations. In this work, we assessed a new sample preparation and buffer exchange platform coupled with high-resolution mass spectrometry for characterizing the drug-load and distribution of several cysteine-linked ADCs conjugated with a variety of chemotypes. Several criteria were evaluated during the optimization of the buffer exchange-mass spectrometry system performance and the data generated with the system were compared with results from nSEC-MS and HIC. The results indicated that the platform enables automated and high throughput quantitative DAR characterization for antibody-drug conjugates with high reproducibility and offers several key advantages over existing approaches that are used for chemotype-agnostic ADC characterization.

Purification and Quality Control of Recombinant Proteins Expressed in Mammalian Cells: A Practical Review.

In the recent years, there has been a rapid development of new technologies and strategies when it comes to protein purification and quality control (QC), but the basic technologies for these processes go back a long way, with many improvements over the past few decades. The purpose of this chapter is to review these approaches, as well as some other topics such as the advantages and disadvantages of various purification methods for intracellular or extracellular proteins, the most effective and widely used genetically engineered affinity tags, solubility-enhancing tags, and specific proteases for removal of nontarget sequences. Affinity chromatography (AC), like Protein A or G resins for the recovery of antibodies or Fc fusion proteins or immobilized metals for the recovery of histidine-tagged proteins, will be discussed along with other conventional chromatography techniques: ion exchange (IEC), hydrophobic exchange (HEC), mixed mode (MMC), size exclusion (SEC), and ultrafiltration (UF) systems. How to select and combine these different technologies for the purification of any given protein and the minimal criteria for QC characterization of the purity, homogeneity, identity, and integrity of the final product will be presented.

In-line buffer exchange in the coupling of Protein A chromatography with weak cation exchange chromatography for the determination of charge variants of immunoglobulin G derived from chinese hamster ovary cell cultures.

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.

Development of a 3D printed micro simulated moving bed chromatography system.

In the 1960s, chromatography processes were revolutionized by the invention of simulated moving bed chromatography. This method not only enhances the separation performance and resin utilization in comparison to batch-chromatography, it has also a much lower buffer consumption. While simulated moving bed chromatography nowadays is applied for a wide range of industrial applications, it was never transferred to the micro-scale (in regards to column and system volume). In our opinion a micro simulated moving bed chromatography system (µSMB) would be a useful tool for many applications, ranging from early process development and long term studies to downstream processing of speciality products. We implemented such a µSMB with a 3D printed central rotary valve and a microfluidic flow controller as flow source. We tested the system with a four zone open loop setup for the separation of bovine serum albumin and ammonium sulfate with size exclusion chromatography. We used four process points and could achieve desalting levels of BSA ranging from 94% to 99%, with yields ranging form 65% to 88%. Thus, we were able to achieve comparable results to common lab scale processes. With a total dead volume of 358 µL, including all sensors, connections and the valve, this is, to the best of our knowledge, the smallest SMB system that was ever built and we were able to perform experiments with feed flow rates reaching as low as 15 µL/min.

Rapid and Simple Buffer Exchange Using Cation-Exchange Chromatography to Improve Point-of-Care Detection of Pharmacological Agents.

The current COVID-19 pandemic has highlighted the power, speed, and simplicity of point-of-care (POC) diagnostics. POC diagnostics are available for a wide range of targets, including both drugs of abuse as well as performance-enhancing drugs. For pharmacological monitoring, minimally invasive fluids such as urine and saliva are commonly sampled. However, false positives or negatives caused by interfering agents excreted in these matrices may confound results. For example, false positives have, in most cases, prevented the use of POC diagnostics for pharmacological agent detection; the consequence is that centralized labs are instead tasked to perform these screenings, resulting in significant delays between sampling and testing. Thus, a rapid, simple, and inexpensive methodology for sample purification is required for the POC to reach a field-deployable tool for the pharmacological human health and performance assessments. Buffer exchange is a simple, rapid approach to remove interfering agents, but has traditionally been difficult to perform on small pharmacological molecules. Therefore, in this communication, we use salbutamol, a performance-enhancing drug, as a case example to demonstrate the efficacy of ion-exchange chromatography as a technique to perform buffer exchange for charged pharmacological agents. This manuscript demonstrates the efficacy of this technique leveraging a commercial spin column to remove interfering agents found in simulant urines, such as proteins, creatinine, and urea, while retaining salbutamol. The utility and efficacy of the method was then confirmed in actual saliva samples. The eluent was then collected and run on the lateral flow assays (LFAs), improving the reported limit of detection by over 5× (new lower limit of detection of 10 ppb compared to reported 60 ppb by the manufacturer) while simultaneously removing noise due to background interfering agents.

Use of tandem affinity-buffer exchange chromatography online with native mass spectrometry for optimizing overexpression and purification of recombinant proteins.

Purification of recombinant proteins typically entails overexpression in heterologous systems and subsequent chromatography-based isolation. While denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is routinely used to screen a variety of overexpression conditions (e.g., host, medium, inducer concentration, post-induction temperature and/or incubation time) and to assess the purity of the final product, its limitations, including aberrant protein migration due to compositional eccentricities or incomplete denaturation, often preclude firm conclusions regarding the extent of overexpression and/or purification. Therefore, we recently reported an automated liquid chromatography-mass spectrometry-based strategy that couples immobilized metal affinity chromatography (IMAC) with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS) to directly analyze cell lysates for the presence of target proteins. IMAC-OBE-nMS can be used to assess whether target proteins (1) are overexpressed in soluble form, (2) bind and elute from an IMAC resin, (3) oligomerize, and (4) have the expected mass. Here, we use four poly-His-tagged proteins to demonstrate the potential of IMAC-OBE-nMS for expedient optimization of overexpression and purification conditions for recombinant protein production.

Integrated system for temperature-controlled fast protein liquid chromatography. III. Continuous downstream processing of monoclonal antibodies.

Three different applications of travelling heating zone reactor (THZR) chromatography for the downstream processing of monoclonal antibodies (mAbs) are described. mAb containing feedstocks were applied to a fixed bed of the thermoresponsive rProtein A matrix, Byzen Pro™, contained in a bespoke column (held at 15 °C) fitted with a travelling heating (42 °C) device encircling a narrow section of the column. For the demonstration of continuous concentration, uninterrupted loading of 1.0 g/L mAb in a pH 8 binding buffer was synchronized with 5 repeated movements of the heating zone along the column's full length at a velocity of 0.1 mm/s. Elution of mAbs was induced solely by the travelling heating zone's action, each full movement generating a sharp concentrated elution peak accompanied by a small transient mAb concentration-dependent dip in conductivity. Quasi-steady-state operation occurred from the third elution onwards, delivering a mean mAb concentration of 4.9 g/L and process yield >93%. Quasi-continuous separation of the target mAb (1.41 g/L) from bovine serum albumin, BSA (1.0 g/L), was achieved by cyclically alternating the feeding of the mAb + BSA feedstock, with that of the binding buffer alone; supply of the latter was timed to coincide with movement of the heating zone. Accurate coordination of the heating zone's travel and switching from feed to buffer permitted quasi-steady-state collection (elutions 3-6) of sharp peaks of mAb in high purity (98.7%) and yield (88.7%) in 4.5-fold concentrated form, with BSA exiting in the flow through fractions between successive mAb elution peaks. Fully automated THZR-mediated quasi-continuous buffer exchange of 1.34 g/L mAb from a phosphate buffer pH 8 into a HEPES buffer pH 8 of slightly lower conductivity was performed over a 19 h period by carefully timed switching from one feed solution to the other and back again, whilst synchronising movement of the heating zone with feeding of the exchange buffer. Quasi-steady-state operation (elutions 2-9) resulted in an average eluted mAb yield of 94.5% and concentration of 4.8 g/L. Triggering movement of the heating zone slightly ahead of the switch from mAb feed to exchange buffer permitted the positioning of mAb elution peaks in 9 mL volume segments with the lowest recorded conductivity. Measurements of buffer exchange performance conducted with two 'protein-free' systems demonstrated that compared to tangential flow filtration in diafiltration mode, which represents the 'state-of-the-art' technology for buffer exchange, the THZR chromatography based approach affords a >60% saving in minimum volume of exchange buffer required to remove 99.9% of the original buffer. Combined far and near UV circular dichroism, intrinsic fluorescence and thermal melting experiments showed that, unlike conventional Protein A/G affinity chromatography, the conditions for THZR Protein A chromatography respect maintenance of a favourable structural profile for mAbs.

Mass Balance Model with Donnan Equilibrium Accurately Describes Unusual pH and Excipient Profiles during Diafiltration of Monoclonal Antibodies.

There is extensive experimental data showing that the final pH and buffer composition after protein diafiltration (DF), particularly with monoclonal antibodies, can be considerably different than that in the DF buffer due to electrostatic interactions between the charged protein and the charged ions. Previous models for this behavior have focused on the final (equilibrium) partitioning and are unable to explain the complex pH and concentration profiles during the DF process. The objective of this study is to develop a new model for antibody DF based on solution of the transient mass balance equations, with the permeate concentrations of the charged species evaluated assuming Donnan equilibrium across the semipermeable membrane in combination with electroneutrality constraints. Model predictions are in excellent agreement with experimental data obtained during DF of both acidic and basic monoclonal antibodies, with the protein charge determined from independent electrophoretic mobility measurements. The model is able to predict the entire pH/histidine concentration profiles during DF, providing a framework for the development of DF processes that yield the desired antibody formulation.

Hollow fiber countercurrent dialysis for continuous buffer exchange of high-value biotherapeutics.

Buffer exchange, desalting, and formulation of high-value biotherapeutics are currently performed using batch diafiltration (DF); however, this type of tangential flow filtration process may be difficult to implement as part of a fully continuous biomanufacturing process. The objective of this study was to explore the potential of using countercurrent dialysis for continuous protein formulation and buffer exchange. Experiments were performed using concentrated solutions of immunoglobuin G (IgG) with commercially available hollow fiber dialyzers having 1.5 and 1.8 m2 membrane surface area. More than 99.9% buffer exchange was obtained over a range of conditions, as determined from the removal of a model impurity (vitamin B12 ). The dialyzers were able to process more than 0.5 kg of IgG per day in an easily scalable low-cost process. In addition, buffer requirements were less than 0.02 L of buffer per gram IgG, which is several times less than that used in current batch DF processes. These results clearly demonstrate the potential of using low-cost hollow fiber dialyzers for buffer exchange and product formulation in continuous bioprocessing. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2763, 2019.

Multi-step high-throughput conjugation platform for the development of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) form a rapidly growing class of biopharmaceuticals which attracts a lot of attention throughout the industry due to its high potential for cancer therapy. They combine the specificity of a monoclonal antibody (mAb) and the cell-killing capacity of highly cytotoxic small molecule drugs. Site-specific conjugation approaches involve a multi-step process for covalent linkage of antibody and drug via a linker. Despite the range of parameters that have to be investigated, high-throughput methods are scarcely used so far in ADC development. In this work an automated high-throughput platform for a site-specific multi-step conjugation process on a liquid-handling station is presented by use of a model conjugation system. A high-throughput solid-phase buffer exchange was successfully incorporated for reagent removal by utilization of a batch cation exchange step. To ensure accurate screening of conjugation parameters, an intermediate UV/Vis-based concentration determination was established including feedback to the process. For conjugate characterization, a high-throughput compatible reversed-phase chromatography method with a runtime of 7 min and no sample preparation was developed. Two case studies illustrate the efficient use for mapping the operating space of a conjugation process. Due to the degree of automation and parallelization, the platform is capable of significantly reducing process development efforts and material demands and shorten development timelines for antibody-drug conjugates.

pH variations during diafiltration due to buffer nonidealities.

Diafiltration is used for final formulation of essentially all biotherapeutics. Several studies have demonstrated that buffer/excipient concentrations in the final diafiltered product can be different than that in the diafiltration buffer due to interactions between buffer species and the protein product. However, recent work in our lab has shown variations in solution pH that are largely independent of the protein concentration during the first few diavolumes. Our hypothesis is that these pH variations are due to nonidealities in the acid-base equilibrium coefficient. A model was developed for the diafiltration process accounting for the ionic strength dependence of the pKa . Experimental results obtained using phosphate and histidine buffers were in excellent agreement with model predictions. A decrease in ionic strength leads to an increase in the pKa for the phosphate buffer, causing a shift in the solution pH, even under conditions where the initial feed and the diafiltration buffer are at the same pH. This effect could be eliminated by matching the ionic strength of the feed and diafiltration buffer. The experimental data and model provide new insights into the factors controlling the pH profile during diafiltration processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1555-1560, 2017.

Method for Folding of Recombinant Prion Protein to Soluble ß-Sheet Secondary Structure.

A key event in the pathogenesis of prion diseases is the change in structure of the normal cellular form of the prion protein from a predominantly α-helix form to the ß-sheet-rich prion protein found in disease-associated tissue. To allow more detailed structural research into PrP misfolding, it is necessary to have techniques which enable enrichment of the ß-sheet content in recombinant PrP.This method describes the procedure for inducing ß-folding of recombinant PrP to resemble a disease-associated structure and ultimately produce soluble ß-folded recombinant PrP.

Semiautomated Sample Preparation for Protein Stability and Formulation Screening via Buffer Exchange.

A novel semiautomated buffer exchange process workflow was developed to enable efficient early protein formulation screening. An antibody fragment protein, BMSdab, was used to demonstrate the workflow. The process afforded 60% to 80% cycle time and scientist time savings and significant material efficiencies. These efficiencies ultimately facilitated execution of this stability work earlier in the drug development process, allowing this tool to inform the developability of potential candidates for development from a formulation perspective. To overcome the key technical challenges, the protein solution was buffer-exchanged by centrifuge filtration into formulations for stability screening in a 96-well plate with an ultrafiltration membrane, leveraging automated liquid handling and acoustic volume measurements to allow several cycles of exchanges. The formulations were transferred into a vacuum manifold and sterile filtered into a rack holding 96 glass vials. The vials were sealed with a capmat of individual caps and placed in stability stations. Stability of the samples prepared by this process and by the standard process was demonstrated to be comparable. This process enabled screening a number of formulations of a protein at an early pharmaceutical development stage with a short sample preparation time.

Aggregation kinetic dataset to determine the stability of the purified and refolded recombinant ppTvCP4 protein of Trichomonas vaginalis.

The recombinant ppTvCP4 (ppTvCP4r) protein, a specific inhibitor of the proteolytic activity and virulence properties of Trichomonas vaginalis, depending on cathepsin L-like cysteine proteinases (CPs) (http:dx.doi.org/ 10.1016/j.biocel.2014.12.001[1], http:dx.doi.org/ 10.1016/j.micinf.2013.09.002[2], http:dx.doi.org/ 10.1155/2015/946787[3]) was stable in the elution buffer up to two months at 4 °C. However, it was prone to aggregate in PBS (functional assay buffer) [1]. Therefore, before functional assays, the aggregation kinetic of refolded ppTvCP4r was determined after the exchange to PBS. Samples of purified and refolded ppTvCP4r (0.15 mg/ml) in PBS were incubated for 0-24 h at 4 and 25 °C, spun down, measured the protein concentration in the supernatant and checked for the presence of aggregated protein in the pellet. The concentration of protein progressively decreased in the supernatant through time at both temperatures as the protein aggregated. Data in this article are related to the research paper [1].

Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance.

In situ microfluidic dialysis for biological small-angle X-ray scattering.

Owing to the demand for low sample consumption and automated sample changing capabilities at synchrotron small-angle X-ray (solution) scattering (SAXS) beamlines, X-ray microfluidics is receiving continuously increasing attention. Here, a remote-controlled microfluidic device is presented for simultaneous SAXS and ultraviolet absorption measurements during protein dialysis, integrated directly on a SAXS beamline. Microfluidic dialysis can be used for monitoring structural changes in response to buffer exchange or, as demonstrated, protein concentration. By collecting X-ray data during the concentration procedure, the risk of inducing protein aggregation due to excessive concentration and storage is eliminated, resulting in reduced sample consumption and improved data quality. The proof of concept demonstrates the effect of halted or continuous flow in the microfluidic device. No sample aggregation was induced by the concentration process at the levels achieved in these experiments. Simulations of fluid dynamics and transport properties within the device strongly suggest that aggregates, and possibly even higher-order oligomers, are preferentially retained by the device, resulting in incidental sample purification. Hence, this versatile microfluidic device enables investigation of experimentally induced structural changes under dynamically controllable sample conditions.