Buffer Exchange Steps Research Articles

Comparing in silico flowsheet optimization strategies in biopharmaceutical downstream processes.

The challenging task of designing biopharmaceutical downstream processes is initially to select the type of unit operations, followed by optimizing their operating conditions. For complex flowsheet optimizations, the strategy becomes crucial in terms of duration and outcome. In this study, we compared three optimization strategies, namely, simultaneous, top-to-bottom, and superstructure decomposition. Moreover, all strategies were evaluated by either using chromatographic Mechanistic Models (MMs) or Artificial Neural Networks (ANNs). An overall evaluation of 39 flowsheets was performed, including a buffer-exchange step between the chromatography operations. All strategies identified orthogonal structures to be optimal, and the weighted overall performance values were generally consistent between the MMs and ANNs. In terms of time-efficiency, the decomposition method with MMs stands out when utilizing multiple cores on a multiprocessing system for simulations. This study analyses the influence of different optimization strategies on flowsheet optimization and advices on suitable strategies and modeling techniques for specific scenarios.

An unconventional strategy for purifying recombinant SARS-CoV-2 spike protein

The soluble domain of the trimeric SARS-CoV-2 spike protein is a promising candidate for a COVID-19 vaccine. Purification of this protein from mammalian cell culture supernatant using conventional resin-based chromatography is challenging as its large size (∼550 kDa) restricts its access and mobility within the pores of the resin particles. This reduces binding capacity and process robustness very significantly as extremely low flow rates need to be used during purification. Convection-based ion-exchange membrane chromatography has been found to be suitable in this respect. However, the high ionic strength of mammalian cell culture supernatant makes it difficult to bind this protein on charged membranes without dilution with a suitable buffer. An unconventional strategy involving size-exclusion chromatography as the first step, followed by cation exchange membrane chromatography as the second step is proposed in this paper. In the size exclusion chromatography step, the spike protein is excluded from the pores and can therefore be isolated in the void volume fraction. This step removes small molecule impurities and also serves as a desalting and buffer exchange step, making the partially purified material suitable for the cation exchange membrane chromatography step. The proposed process is variant-independent, fast and scalable and addresses some of the challenges associated with the currently used purification methods.

Titer and charge-based heterogeneity multiattribute monitoring of mAbs in cell culture harvest using 2D ProA CEX MS

Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60–90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 μg to 160 μg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.

Thermal-plex: fluidic-free, rapid sequential multiplexed imaging with DNA-encoded thermal channels.

Multiplexed fluorescence imaging is typically limited to three- to five-plex on standard setups. Sequential imaging methods based on iterative labeling and imaging enable practical higher multiplexing, but generally require a complex fluidic setup with several rounds of slow buffer exchange (tens of minutes to an hour for each exchange step). We report the thermal-plex method, which removes complex and slow buffer exchange steps and provides fluidic-free, rapid sequential imaging. Thermal-plex uses simple DNA probes that are engineered to fluoresce sequentially when, and only when, activated with transient exposure to heating spikes at designated temperatures (thermal channels). Channel switching is fast (<30 s) and is achieved with a commercially available and affordable on-scope heating device. We demonstrate 15-plex RNA imaging (five thermal × three fluorescence channels) in fixed cells and retina tissues in less than 4 min, without using buffer exchange or fluidics. Thermal-plex introduces a new labeling method for efficient sequential multiplexed imaging.

Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses.

The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 minutes and plasmid purification <60 minutes. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention.

Novel Surfactant Compatibility with Downstream Protein Bioprocesses

Downstream processing of antibodies consists of a series of steps aimed at purifying the product and ensuring it is delivered to formulators structurally and functionally intact. The process can be complex and time-consuming, involving multiple filtrations, chromatography, and buffer exchange steps that can interfere with product integrity. This study explores the possibility and benefits of adding N-myristoyl phenylalanine polyether amine diamide (FM1000) as a process aid. FM1000 is a nonionic surfactant that is highly effective at stabilizing proteins against aggregation and particle formation and has been extensively explored as a novel excipient for antibody formulations. In this work, FM1000 is shown to stabilize proteins against pumping-induced aggregation which can occur while transporting them between process units and within certain processes. It is also shown to prevent antibody fouling of multiple polymeric surfaces. Furthermore, FM1000 can be removed after some steps and during buffer exchange in ultrafiltration/diafiltration, if needed. Additionally, FM1000 was compared to polysorbates in studies focusing on surfactant retention on filters and columns. While the different molecular entities of polysorbates elute at different rates, FM1000 flows through purification units as a single molecule and at a faster rate. Overall, this work defines new areas of application for FM1000 within downstream processing and presents it as a versatile process aid, where its addition and removal are tunable depending on the needs of each product.

Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R2 = 0.85-0.90) and plasma (SPEED R2 = 0.84; SDC R2 = 0.76, S-Trap R2 = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired.

Innovations in double digest restriction-site associated DNA sequencing (ddRAD-Seq) method for more efficient SNP identification

We present an improved ddRAD-Seq protocol for identifying single nucleotide polymorphisms (SNPs). It utilizes selected restriction enzyme digestion fragments, quick acting ligases that are neutral with the restriction enzyme buffer eliminating buffer exchange steps, and adapters designed to be compatible with Illumina index primers. Library amplification and barcoding are completed in one PCR step, and magnetic beads are used to purify the genomic fragments from the ligation and library generation steps. Our protocol increases the efficiency and decreases the time to complete a ddRAD-Seq experiment. To demonstrate its utility, we compared SNPs from our protocol with those from whole genome resequencing data from Gossypium herbaceum and Gossypium arboreum. Principal component analysis demonstrated that the variability of the combined data was explained by the genotype (PC1) and methodology applied (PC2). Phylogenetic analysis showed that the SNPs from our method clustered with SNPs from the resequencing data of the corresponding genotype. Sequence alignments illustrated that for homozygous loci, more than 90% of the SNPs from the resequencing data were discovered by our method. Our analyses suggest that our ddRAD-Seq method is reliable in identifying SNPs suitable for phylogenetic and association genetic studies while reducing cost and time over known methods.

Development of a gel filtration protocol that eliminates precipitation during refolding of chemically denatured proteins

Purification of recombinant proteins in denaturing conditions has many practical advantages. Chemical denaturants such as urea and guanidine hydrochloride are used to solubilize recombinant proteins from inclusion bodies. Proteins purified in denaturing conditions generally contain fewer contaminants compared to their counterparts purified in native buffers. Denatured proteins can be stored for long and refolded as needed. Refolding is commonly performed by equilibrium dialysis and sometimes on metal affinity or gel filtration columns by diluting out the denaturant and allowing the protein to fold back into its native state. The refolding steps are often tedious and result in significant loss of protein through aggregation. Using a modified gel filtration technique, we successfully refolded a spectrum of proteins that either are highly aggregation‐prone or contain several disulfide bonds or both, back to their native states. Some of the proteins are well known to precipitate significantly during refolding. However, using this technique, precipitation was either minimal or non‐existent. Structural analyses using circular dichroism and fluorescence spectroscopies, gel filtration, and coupled with functional assays confirmed that these proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins, and simultaneously performing buffer exchange and additional purification steps.

Model-based optimization of integrated purification sequences for biopharmaceuticals

Finding the best purification process is a challenging task. Recently, mechanistic models that can accelerate the development of chromatographic unit operations, the most important purification units, became widely available. In previous work, several chromatographic models have been linked together to simulate and optimize integrated processes. However, considering only chromatographic steps may lead to a suboptimal process. Consequently, the aim of this study was to include models for ultra- and diafiltration units into the optimization approach to account for buffer exchange steps before or between chromatography units. This approach was applied to an industrial case, the purification of a monoclonal antibody, where cation exchange, hydrophobic interaction and mixed mode were the possible chromatographic separation modes. It turned out that only the duration of the total filtration step and the duration of the ultrafiltration step were crucial variables for the optimization of the ultra- and diafiltration steps. The ‘best’ in silico purification process was found based on the performance criteria yield and solvent usage. The purity was required to be at least 99.9%.

Hydrophobic-interaction chromatography for purification of influenza A and B virus

Options for hydrophobic-interaction chromatography (HIC) for purification of cell culture-derived influenza A and B virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved.To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 μg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing virus purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived viruses and viral vectors.

An automated platform for the enzyme-mediated assembly of complex oligosaccharides.

An automated platform that can synthesize a wide range of complex carbohydrates will greatly increase their accessibility and should facilitate progress in glycoscience. Here we report a fully automated process for enzyme-mediated oligosaccharide synthesis that can give easy access to different classes of complex glycans including poly-N-acetyllactosamine derivatives, human milk oligosaccharides, gangliosides and N-glycans. Our automated platform uses a catch and release approach in which glycosyltransferase-catalyzed reactions are performed in solution and product purification is accomplished by solid phase extraction. We developed a sulfonate tag that can easily be installed and enables highly efficient solid phase extraction and product release using a single set of washing conditions regardless of the complexity of the glycan. Using this custom-built synthesizer, as many as 15 reaction cycles can be performed in an automated fashion without a need for lyophilization or buffer exchange steps.

Integration of Ion Mobility MSE after Fully Automated, Online, High-Resolution Liquid Extraction Surface Analysis Micro-Liquid Chromatography.

Direct analysis by mass spectrometry (imaging) has become increasingly deployed in preclinical and clinical research due to its rapid and accurate readouts. However, when it comes to biomarker discovery or histopathological diagnostics, more sensitive and in-depth profiling from localized areas is required. We developed a comprehensive, fully automated online platform for high-resolution liquid extraction surface analysis (HR-LESA) followed by micro–liquid chromatography (LC) separation and a data-independent acquisition strategy for untargeted and low abundant analyte identification directly from tissue sections. Applied to tissue sections of rat pituitary, the platform demonstrated improved spatial resolution, allowing sample areas as small as 400 μm to be studied, a major advantage over conventional LESA. The platform integrates an online buffer exchange and washing step for removal of salts and other endogenous contamination that originates from local tissue extraction. Our carry over–free platform showed high reproducibility, with an interextraction variability below 30%. Another strength of the platform is the additional selectivity provided by a postsampling gas-phase ion mobility separation. This allowed distinguishing coeluted isobaric compounds without requiring additional separation time. Furthermore, we identified untargeted and low-abundance analytes, including neuropeptides deriving from the pro-opiomelanocortin precursor protein and localized a specific area of the pituitary gland (i.e., adenohypophysis) known to secrete neuropeptides and other small metabolites related to development, growth, and metabolism. This platform can thus be applied for the in-depth study of small samples of complex tissues with histologic features of ∼400 μm or more, including potential neuropeptide markers involved in many diseases such as neurodegenerative diseases, obesity, bulimia, and anorexia nervosa.

A membrane-based purification process for cell culture-derived influenza A virus

A simple membrane-based purification process for cell culture-derived influenza virus was established that relies on only two chromatographic unit operations to achieve the contamination limits required according to regulatory authorities. After clarification and concentration, a pseudo-affinity membrane adsorber (sulfated cellulose, SCMA) was applied for virus capture. The subsequent polishing step consisted of a salt-tolerant anion exchange membrane adsorber (STMA) to bind residual DNA. For the presented process neither a buffer exchange step nor a nuclease step for further DNA digestion were required. As a starting point, a two-salt strategy (including a polyvalent ion) was employed to screen STMA conditions in a 96-well plate format. After optimization on chromatographic laboratory scale, the virus recovery was up to 97% with a residual DNA level below 0.82%. In addition, the STMA was characterized regarding its dynamic binding capacity and the impact of flow rate on yields and contamination levels. Overall, the total virus yield for influenza virus A/PR/8/34 (H1/N1) of this two-step membrane process was 75%, while the protein and the DNA contamination level could be reduced to 24% and at least 0.5%, respectively. With 19.8μg protein and 1.2ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived vaccines for human use. Overall, the presented downstream process might serve as a generic and economic platform technology for production of cell culture-derived viruses and viral vectors.

An integrated microfluidic chip for immunocapture, preconcentration and separation of β-amyloid peptides.

We present an integrated microfluidic chip for detection of β-amyloid (Aβ) peptides. Aβ peptides are major biomarkers for the diagnosis of Alzheimer's disease (AD) in its early stages. This microfluidic device consists of three main parts: (1) An immunocapture microcolumn based on self-assembled magnetic beads coated with antibodies specific to Aβ peptides, (2) a nano-porous membrane made of photopolymerized hydrogel for preconcentration, and (3) a microchip electrophoresis (MCE) channel with fluorescent detection. Sub-milliliter sample volume is either mixed off-chip with antibody coated magnetic beads and injected into the device or is injected into an already self-assembled column of magnetic beads in the microchannel. The captured peptides on the beads are then electrokinetically eluted and re-concentrated onto the nano-membrane in a few nano-liters. By integrating the nano-membrane, total assay time was reduced and also off-chip re-concentration or buffer exchange steps were not needed. Finally, the concentrated peptides in the chip are separated by electrophoresis in a polymer-based matrix. The device was applied to the capture and MCE analysis of differently truncated peptides Aβ (1-37, 1-39, 1-40, and 1-42) and was able to detect as low as 25 ng of synthetic Aβ peptides spiked in undiluted cerebrospinal fluid (CSF). The device was also tested with CSF samples from healthy donors. CSF samples were fluorescently labelled and pre-mixed with the magnetic beads and injected into the device. The results indicated that Aβ1-40, an important biomarker for distinguishing patients with frontotemporal lobe dementia from controls and AD patients, was detectable. Although the sensitivity of this device is not yet enough to detect all Aβ subtypes in CSF, this is the first report on an integrated or semi-integrated device for capturing and analyzing of differently truncated Aβ peptides. The method is less demanding and faster than the conventional Western blotting method currently used for research.

On-bead antibody-small molecule conjugation using high-capacity magnetic beads

Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Workflow for large-scale analysis of melanoma tissue samples

The aim of the present study was to create an optimal workflow for analysing a large cohort of malignant melanoma tissue samples. Samples were lysed with urea and enzymatically digested with trypsin or trypsin/Lys C. Buffer exchange or dilution was used to reduce urea concentration prior to digestion. The tissue digests were analysed directly or following strong cation exchange (SCX) fractionation by nano LC–MS/MS. The approach which resulted in the largest number of protein IDs involved a buffer exchange step before enzymatic digestion with trypsin and chromatographic separation in 120min gradient followed by SCX–RP separation of peptides.

Use of gradient gel filtration to simultaneously exchange buffer, purify and refold denatured proteins

Purification of recombinant proteins in denaturing conditions, 8 M urea or 6 M guanidine hydrochloride (GuHCl), has many practical advantages. Recombinant proteins can be solubilized from inclusion bodies in the presence of urea or GuHCl in buffers. Proteins purified in denaturing buffers generally contain fewer contaminants compared to their counterparts purified in native conditions; thus reducing the number of steps in the purification scheme. Denatured proteins can be stored for long time in buffers containing denaturants and then refolded as required. However, one major disadvantage of denaturing protein purification is the laborious refolding step, where the denaturant is carefully diluted out to allow the denatured protein to fold back into its native state. This step is not only time-consuming but can also result in significant loss of the protein through aggregation. Using a spectrum of proteins with measurable functional activities, we report the use of a modified gel filtration technique, gradient gel filtration, to successfully refold these aggregation-prone proteins back to their native states during denaturing purification. Some of the proteins chosen are well-known to precipitate significantly during refolding. However, using this technique, precipitation was either so minimal or non-existent. Circular dichroism, enzymatic assays and dynamic molecular mass determination confirmed that the proteins were correctly refolded and biologically active. This study provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification step.

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.

Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor.

We successfully transferred a two-stage batch precipitation-based antibody capture step to continuous mode using continuous tubular reactors. The precipitation process solely employs a cheap mineral salt (CaCl2 ) and an organic solvent (ethanol) and could replace the costly protein A capture step in the purification of recombinant antibodies from cell culture supernatant. The time from startup untill attaining steady state conditions was reached in less than 15 minutes and both reactors were operated for several hours at steady state without manual intervention, delivering antibody at a constant yield and purity. An overall yield of > 90 percent, with a host cell protein reduction from 42 777 to 9000 ppm and a DNA reduction from 359 ppm to 7 ppm, could be achieved for the antibody investigated. The precipitated antibody can be dissolved at very high concentrations (> 40 g/L) in numerous buffer systems of various pH and high and low ionic strength, thereby rendering a subsequent concentration or buffer exchange step redundant. This system enables cell culture supernatants with low or high antibody titer to be processed with constant reactor size and without changing any parameters or increasing precipitant consumption. Aggregate levels were below 1% under all conditions tested. Purification by precipitation did not affect binding to CD16a or the isoform distribution of the antibody.