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Arginine in a free-state and as part of peptides and proteins shows distinct tendency to form clusters. In free-form, it has been found useful in cryoprotection, as a drug excipient for both solid and liquid formulations, as an aggregation suppressor, and an eluent in protein chromatography. In many cases, the mechanisms by which arginine acts in all these applications is either debatable or at least continues to attract interest. It is quite possible that arginine clusters may be involved in many such applications. Furthermore, it is possible that such clusters are likely to behave as intrinsically disordered polypeptides. These considerations may help in understanding the roles of arginine in diverse applications and may even lead to better strategies for using arginine in different situations.

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A fluid dynamics model has been developed to describe flow behavior in a lab-scale chromatographic system dedicated for protein processing. The case study included a detailed analysis of elution pattern of a protein, which was a monoclonal antibody, glycerol, and their mixtures in aqueous solutions. Glycerol solutions mimicked viscous environment of the concentrated protein solutions. The model accounted for concentration dependences of solution viscosity and density, and dispersion anisotropy in the packed bed. It was implemented into a commercial computational fluid dynamics software using user-defined functions. The prediction efficiency was successfully verified by comparing the model simulations in the form of the concentration profiles and their variances with the corresponding experimental data. The contribution of the individual elements of the chromatographic system to protein band broadening was evaluated for different configurations: for the extra-column volumes in the absence of the chromatographic column, for the zero-length column without the packed bed and for the column containing the packed bed. The influence of the operating variables, including: the mobile phase flowrate, the type of the injection system, i.e., the injection loop capillary or the superloop, the injection volume and the length of the packed bed, on band broadening of the protein was determined under nonadsorbing conditions. For protein solutions having viscosity comparable with the mobile phase, the flow behavior either in the column hardware or in the injection system made major contributions to band broadening, which depended on the type of the injection system. For highly viscous protein solution, the flow behavior in the packed bed exerted a dominant influence on band broadening.

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Multi-size optimization of ion exchangers based on protein characteristics and understanding of underlying mechanism is crucial to achieve maximum separation performance in terms of adsorption capacity and uptake kinetic. Herein, we characterize the effects of three different sizes, macropore size, protein size, and ligand length, on the protein adsorption capacity and uptake kinetic of macroporous cellulose beads, and provide insights into the underlying mechanism. In detail, (1) for smaller bovine serum albumin, macropore size has a negligible effect on the adsorption capacity, while for larger γ-globulin, larger macropores improve the adsorption capacity due to the high accessibility of binding sites; (2) there is a critical pore size (CPZ), at which the adsorption uptake kinetic is minimum. When pore sizes are higher than the CPZ, uptake kinetics are enhanced by pore diffusion. When pore sizes are lower than CPZ, uptake kinetics are enhanced by surface diffusion; (3) increasing ligand length improves the adsorption capacity by three-dimensionally extended polymer chains in pores and enhances uptake kinetic by improved surface diffusion. This study offers an integrated perspective to qualitatively assess the effects of multiple sizes, providing guidance for designing advanced ion exchangers for protein chromatography.

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Multienzyme reactions play an important role in cellular metabolic functions. The assembly of a metabolon is often observed, in which the position and the orientation of composite enzymes are optimized to facilitate the substrate transport. The recent progress of DNA nanotechnology is promising to organize the assembly of bimolecular complexes with precise controlled geometric patterns at nanoscale, such as enzyme cascades assembly, biomimetic substrate channeling, and compartmentalization. Here, we present detailed protocols of using DNA nanoscaffolds to assemble a multienzyme system with control over spatial interactions and arrangements of individual components. The protocols include the preparation and purification of DNA nanostructures, the bioconjugation of DNA with proteins and cofactors, the chromatography purification of DNA-conjugated biomolecules, the characterization of assemblies by routine gel electrophoresis and advanced AFM imaging, as well as the activity evaluation of multienzyme assemblies.

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Antibody fragments and their engineered variants show true potential as next-generation therapeutics as they combine excellent targeting with superior biodistribution and blood clearance. Unlike full antibodies, however, antibody fragments do not yet have a standard platform purification process for large-scale production. Short peptide ligands are viable alternatives to protein ligands in affinity chromatography. In this work, an integrated computational and experimental scheme is described to de novo design 9-mer peptides that bind to Fab fragments. The first cohort of designed sequences was tested experimentally using human polyclonal Fab, and the top performing sequence was selected as a prototype for a subsequent round of ligand refinement in silico. The resulting peptides were conjugated to chromatographic resins and evaluated via equilibrium and dynamic binding studies using human Fab-κ and Fab-λ. The equilibrium studies returned values of binding capacities up to 32 mg of Fab per mL of resin with mild affinity (KD ∼ 10-5 M) that are conducive to high product capture and recovery. Dynamic studies returned values of product yield up to ∼90%. Preliminary purification studies provided purities of 83-93% and yields of 11-89%. These results lay the groundwork for future development of these ligands towards biomanufacturing translation.

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This paper presents a straightforward approach for measuring and quantifying orthogonality directly in complex cell culture fluids (CCFs) without the requirement for tracking the retention behaviors of large sets of proteins. Null-producing CCFs were fractionated using linear salt gradients at constant pH on a set of multimodal resins. Fractions were then analyzed by ultraperformance-reversed phase liquid chromatography and the resulting chromatograms provided host cell protein (HCP) "fingerprints." Using these fingerprints, an inner product vector-based approach was employed to quantify the degree of orthogonality between pairs of resins and operating conditions for these large HCP protein sets. To compare resin orthogonality behavior in different expression systems, the Chinese hamster ovary and Pichia pastoris null-producing CCFs were examined. Orthogonality in multimodal systems was found to strongly depend on the expression system and the HCPs being screened. We also identified several unexpected pairs of multimodal resins within the same family that exhibited significant orthogonality. Furthermore, "self-orthogonality" was evaluated between resins operated at different pHs, and important operating regimes were identified for maximizing orthogonal selectivities. The framework developed in this paper for calculating orthogonality without the need for labor-intensive HCP tracking has important implications for efficient process development and resin/operating condition selection for both monoclonal antibody (mAb) polishing steps and non-mAb processes. In addition, this study provides a tool to unlock the untapped potential of multimodal resins by aiding in their rational selection and incorporation. Finally, the orthogonality framework here can facilitate the development of sets of next-generation multimodal resins specifically designed to provide highly orthogonal and efficient separations tailored for different expression systems.

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A computational fluid dynamics method was used for prediction of flow behavior and band profiles of small- and macro-molecule compounds eluting in extra-column volumes (ECV) of an Äkta chromatographic system. The model compounds were: acetone, bovine serum albumin and an antibody. The construction of ECV was approximated by different types of geometries, starting from the simplest two-dimensional (2D) arrangement consisting of a straight capillary tube, and ending with a three-dimensional system (3D), which accounted for the flow path curvature of individual elements of ECV, including: injection loop capillary, multi-way valve, connecting capillary and detector cell. The accuracy of the model predictions depended on the flow path length and the eluent flowrate. 2D-geometry models reproduced pretty well the shapes of band profiles recorded at the lowest eluent flowrate used, but they failed for increased flowrates. The 3D-geometry model was found to be sufficiently accurate for all conditions investigated. It was exploited to analyze band broadening in the individual ECV elements. The simulation results revealed that the flow behavior in the injection loop capillaries strongly influenced the shape of band profiles, particularly at higher eluent velocities. This was attributed to the formation of Dean vertices triggered by centrifugal forces in curved parts of the eluent flow path.

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The intestinal diplomonadid Giardia lamblia is a causative agent of persistent diarrhea. Current treatments are based on nitro drugs, especially metronidazole. Nitro compounds are activated by reduction, yielding toxic intermediates. The enzymatic systems responsible for this activation are not completely understood. By fractionating cell free crude extracts by size exclusion chromatography followed by mass spectrometry, enzymes with nitroreductase (NR) activities are identified. The protein encoded by ORF 17150 found in two pools with NR activities is overexpressed and characterized. In pools of fractions with main NR activities, previously-known NRs are identified, as well as a previously uncharacterized protein encoded by ORF 17150. Recombinant protein 17150 is a flavoprotein with NADPH-dependent quinone reductase and NR activities. Besides a set of previously identified NRs, we have identified a novel enzyme with NR activity.

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The strategy of using polymer-grafted media is effective to create protein chromatography of high capacity and uptake rate, giving rise to an excellent performance in high-throughput protein separation due to its high dynamic binding capacity. Taking the scientific development and technological innovation of protein chromatography as the objective, this review is devoted to an overview of polymer-grafted media reported in the last five years, including their fabrication routes, protein adsorption and chromatography, mechanisms behind the adsorption behaviors, limitations of polymer-grafted media and chromatographic operation strategies. Particular emphasis is placed on the elaboration and discussion on the behaviors of ion-exchange chromatography (IEC) with polymer-grafted media because IEC is the most suitable chromatographic mode for this kind of media. Recent advances in both the theoretical and experimental investigations on polymer-grafted media are discussed by focusing on their implications to the rational design of novel chromatographic media and mobile phase conditions for the development of high-performance protein chromatography. It is concluded that polymer-grafted media are suitable for development of IEC and mixed-mode chromatography with charged and low hydrophobic ligands, but not for hydrophobic interaction chromatography with high hydrophobic ligands and affinity chromatography with ligands that have single binding site on the protein.

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Recent studies have shown that by combining orthogonal, non-affinity chromatography steps, it is possible to rapidly develop efficient purification processes for molecules of interest. Here, we build upon previous work to develop a flexible framework for identifying resins that remove optimally orthogonal sets of impurities for a wide variety of products. Our approach involves screening a library of proteins with diverse properties (pI ranging from 5.0-11.4 and varying hydrophobicity measured by retention in a HIC gradient) on a library of resins and quantifying each resin's ability to separate every protein pair in the library. Orthogonality is then defined as the degree to which two resins separate mutually exclusive sets of protein pairs. We applied this approach to a library of model proteins and a series of strong, salt tolerant, and multimodal ion exchangers and evaluated which resin combinations performed well and which performed poorly. In particular, we found that strong cation and strong anion exchangers were orthogonal, while strong and salt tolerant anion exchangers were not orthogonal. Interestingly, salt tolerant and multimodal cation exchangers were found to be orthogonal and the best resin combination included a multimodal cation exchange resin and a tentacular anion exchange resin. This approach for quantifying orthogonality is valuable in that it can be used both as a criteria for resin design as well as process design. We envision that, using this framework, it will be possible to design a set of next generation chromatography ligands that are explicitly engineered to optimize separability and orthogonality.

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The green-light absorbing proteorhodopsin (GPR) is the prototype of bacterial light-driven proton pumps. It has been the focus of continuous research since its discovery 20 years ago and has sparked the development and application of various biophysical techniques. However, a certain controversy and ambiguity about the oligomeric assembly of GPR still remains. We present here the first tag-free purification of pentameric GPR. The combination of ion exchange and size exclusion chromatography yields homogeneous and highly pure untagged pentamers from GPR overexpressing Escherichia coli. The presented purification procedure provides native-like protein and excludes the need for affinity purification tags. Importantly, three-dimensional protein crystals of GPR were successfully grown and analyzed by X-ray crystallography. These results together with data from single particle cryo-electron microscopy provide direct evidence for the pentameric stoichiometry of purified GPR.

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Molecular techniques allow for the rapid discovery of CCM-associated protein targets, crucial to understanding CCM pathogenesis. Here, we describe optimized protein extraction methods that allow for extraction from whole cell, and/or cellular sub-compartments, including nuclear, mitochondria, cytoplasmic, and membrane-bound proteins, from lysates. This allows for the analysis of in vitro co-immunoprecipitation (Co-IP), label-free measurement of protein-protein interactions, multiplex protein-lipid binding assays, and western blots. Together, all these methods allow for a global analysis of the molecular mechanisms underlying CCM pathogenesis.

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It has been known that anion exchangers prepared by grafting poly(ethyleneimine) (PEI) onto Sepharose FF (PEI-Sepharose) at ionic capacities (IC) over 600 mmol/L show both high protein adsorption capacity and uptake kinetics, and charge reversal of PEI-Sepharose by modification with succinic anhydride can produce protein cation exchangers of high capacity and uptake rate. Previously, a Charge Reversal-then-Reduction procedure (route A) was studied for preparation of cation exchangers of different IC values from PEI-Sepharose. In this work, we proposed a new route, that is, Charge Reduction-then-Reversal route (route B), to develop cation exchangers of different IC values from PEI-Sepharose FF with an IC of 700 mmol/L (FF-PEI-L700) as the starting resin. The two kinds of cation exchangers (route A, PEI-L700-CRn; route B, PEI-Rm-Cn) are compared for lysozyme (Lys) adsorption and chromatography. The two modification routes result in the difference in the ligand structures that significantly affect protein adsorption equilibrium and kinetics. Route A introduces long electroneutral groups that hinder protein adsorption and reduce equilibrium capacity. Moreover, charge reversal by reaction with succinic anhydride could cause diamide formation, which reduces remaining carboxyl groups or the IC. In the charge-reduced FF-PEI-Rm resins of the lowest IC (394 mmol/L) prepared in route B, the diamide formation was little due to the lack of primary and secondary amine groups, so its charge reversal makes a higher-IC cation exchanger. This makes PEI-Rm-Cn show a higher IC (589 mmol/L) than PEI-L700-CRn (463 mmol/L) in which De/D0 jumps about four times. The differences in the adsorption equilibrium and kinetics make the two kinds of resins behave distinctly in dynamic adsorption and chromatography. Namely, PEI-Rm-Cn resins display obviously higher dynamic binding capacities than PEI-L700-CRn resins in the IC range studied. For instance, the DBC (at 10% breakthrough) of PEI-R590-C680 (192 mg/mL) is 33% higher than that of PEI-L700-CR680 (144 mg/mL). This is proved by the purification of Lys from chicken egg white solution, in which the PEI-R590-C680 column purified Lys with a recovery yield 14% higher than the PEI-L700-CR680 column. This research thus demonstrated that Charge Reduction-then-Reversal route is superior over Charge Reversal-then-Reduction route in fabricating a high-capacity cation exchanger from PEI-Sepharose.

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Polyclonal immunoglobulin therapeutics comprising dosed IgG and IgM combinations are powerful tools in fighting cancer and severe infections. The inability of protein ligands to produce polyclonal IgG- and IgM-enriched formulations and recover monoclonal IgM calls for novel ligands with superior biorecognition activity. In this study, a peptoid ligand discovered by our group, and integrated into affinity adsorbents LigaTrap Technologies' "Human IgG" and "Human IgM", were utilized to purify IgG and IgM from complex fluids. IgG purification from human serum using LigaTrap IgG afforded 94.6% purity and 62.9% yield, on par with Protein A/G resins. When challenged with CHO and HEK cell culture harvests with low IgG titer (<1 mg/mL), LigaTrap IgG returned values of yield and purity well above 60% and 90%. LigaTrap IgM was evaluated for purifying IgM in comparison with commercial adsorbents, and afforded a product purity of 93% from a CHO harvest (IgM titer of 1 mg/mL) and 75.1% yield from a HEK harvest (0.5 mg/mL). LigaTrap-M provided IgM enrichment up to 11-fold higher than HiTrap resin. The peptoid adsorbents separated IgG-depleted human serum into IgM- and IgA-enriched fractions. These results demonstrate the potential of the peptoid ligand for manufacturing polyclonal Ig formulations and monoclonal IgM therapeutics.

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Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed.

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Experimental and theoretical analysis of deformation of band profiles in extra-column volumes (ECV) was performed, and its influence on the retention pattern of proteins in a small chromatographic column was quantified. Several macromolecule and small-molecule compounds, and their mixtures were eluted from a chromatographic system in the absence and presence of the column. The peak deformation in ECV was attributed to non-uniform velocity distribution in the radial direction in connecting capillaries. The phenomenon enhanced with increasing molecular weight of the model compound, when radial diffusion dominated the mechanism of band spreading. The band shape was also affected by the geometry of the injection system used, i.e., an injection loop capillary or a superloop. The phenomenon vanished for a small molecule compound, for which plug flow conditions could be established. The difference in flow behaviour of the macromolecule and small-molecule compounds caused them to migrate with different velocities in ECV, which resulted in partial separation of their bands. The ECV effect influenced the retention behaviour of macromolecules in a small column; it caused tailing of peaks and asymmetry of breakthrough curves. To describe the elution profiles in ECV and in the column, a mathematical model was used which accounted for non-ideality of the flow pattern. The model reproduced accurately band profiles of macromolecules within a range of relatively low velocities, typical however for protein chromatography.

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An efficient mathematical tool for the design and scaling up of protein chromatography is suggested, in which the model parameters can be determined quickly over a wide operating space without large material investments. The design method is based on mathematical modelling of column dynamics and moment analysis. The accuracy of the dynamic models that are most frequently used for simulations of chromatographic processes is analyzed, and possible errors that can be generated using the moment analysis are indicated. The so-called transport dispersive model was eventually employed for the process simulations. The model was modified to account for the protein dispersion in void volumes of chromatographic systems. The manner of the model calibration was suggested, which was based on a few chromatographic runs and verified over a wide space of the operating parameters, including composition and flow rate of the mobile phase, column dimensions, residence time, and mass loading. The model system for the study was ion-exchange chromatography. The analysis was performed based on the elution profiles of basic fibroblast growth factor 2 and lysozyme, on two different IEX media.

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In this study, we describe a new approach for the characterization of process-related impurities along with an in silico tool to generate orthogonal, integrated downstream purification processes for biological products. A one-time characterization of process-related impurities from product expression in Pichia pastoris was first carried out using linear salt and pH gradients on a library of multimodal, salt-tolerant, and hydrophobic charge induction chromatographic resins. The Reversed-phase ultra-performance liquid chromatography (UPLC) analysis of the fractions from these gradients was then used to generate large data sets of impurity profiles. A retention database of the biological product was also generated using the same linear salt and pH gradients on these resins, without fraction collection. The resulting two data sets were then analyzed using an in silico tool, which incorporated integrated manufacturing constraints to generate and rank potential three-step purification sequences based on their predicted purification performance as well as whole-process "orthogonality" for impurity removal. Highly ranked sequences were further examined to identify templates for process development. The efficacy of this approach was successfully demonstrated for the rapid development of robust integrated processes for human growth hormone and granulocyte-colony stimulating factor.

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Metallothioneins (MTs) are a class of small cysteine-rich proteins essential for Zn and Cu homeostasis, heavy metal detoxification, and cellular redox chemistry. Herein, we describe the separation and characterization of MTs differentially modified with N-ethylmaleimide (NEM) by liquid chromatography-mass spectrometry (LC-MS). The full-length recombinant MT isoform 1a as well as is isolated domain fragments were first alkylated, then separated on column with subsequent detection by ultra-high resolution ESI-MS. Different behavior was observed for the three peptides with the full-length protein and the isolated α-domain exhibiting similar separation characteristics. For the isolated ß-domain, the smallest peptide with 9 cysteines in the sequence, each alkylated species was well separated, indicating large changes in protein conformation. For the full-length (20 cysteines in the sequence) and α-domain (11 cysteiens in the sequence) peptides, the apo- and lightly alkylated species co-eluted, indicating similar structural properties. However, the more extensively alkylated species were well separated from each other, indicating the sequential unfolding of the apo-MT peptides and providing evidence for the mechanistic explanation for the cooperative alkylation reaction observed for NEM and other bulky and hydrophobic alkylation reagents. We show for the first time clear separation of highly similar MTs, differing by only +125 Da, and can infer structural properties from the LC-MS data, analogous to more complicated and less ubiquitous ion-mobility experiments. The data suggest a compact globular structure for each of the apo-MTs, but where the ß-domain is more easily unfolded. This differential folding stability may have biological implications in terms of domain-specific participation of MT in cellular redox chemistry and resulting metal release.

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In protein chromatography, process variations, such as aging of column or process errors, can result in deviations of the product and impurity levels. Consequently, the process performance described by purity, yield, or production rate may decrease. Based on visual inspection of the UV signal, it is hard to identify the source of the error and almost unfeasible to determine the quantity of deviation. The problem becomes even more pronounced, if multiple root causes of the deviation are interconnected and lead to an observable deviation. In the presented work, a novel method based on the combination of mechanistic chromatography models and the artificial neural networks is suggested to solve this problem. In a case study using a model protein mixture, the determination of deviations in column capacity and elution gradient length was shown. Maximal errors of 1.5% and 4.90% for the prediction of deviation in column capacity and elution gradient length respectively demonstrated the capability of this method for root cause investigation.

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