Multimodal Resin Research Articles

Clear insight into complex multimodal resins and impurities to overcome recombinant protein purification challenges: A review.

Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed-mode chromatography (MMC) resins for launching selective, orthogonal, non-affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step-by-step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product-related impurities (e.g., aggregates, charge variants, fragments), host-related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.

Micromechanical interlocking-inspired dendritic porous silica-based multimodal resin composites for the tooth restoration

Micromechanical interlocking-inspired dendritic porous silica-based multimodal resin composites for the tooth restoration

Enhanced Downstream Processing for a Cell-Based Avian Influenza (H5N1) Vaccine.

H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high-performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines.

Exploring preferred binding domains of IgG1 mAbs to multimodal adsorbents using a combined biophysics and simulation approach.

In this work, we employ a recently developed biophysical technique that uses diethylpyrocarbonate (DEPC) covalent labeling and mass spectrometry for the identification of mAb binding patches to two multimodal cation exchange resins at different pH. This approach compares the labeling results obtained in the bound and unbound states to identify residues that are sterically shielded and thus located in the mAb binding domains. The results at pH 6 for one mAb (mAb B) indicated that while the complementarity determining region (CDR) had minimal interactions with both resins, the FC domain was actively involved in binding. In contrast, DEPC/MS data with another mAb (mAb C) indicated that both the CDR and FC domains were actively involved in binding. These results corroborated chromatographic retention data with these two mAbs and their fragments and helped to explain the significantly stronger retention of both the intact mAb C and its Fab fragment. In contrast, labeling results with mAb C at pH 7, indicated that only the CDR played a significant role in resin binding, again corroborating chromatographic data. The binding domains identified from the DEPC/MS experiments were also examined using protein surface hydrophobicity maps obtained using a recently developed sparse sampling molecular dynamics (MD) approach in concert with electrostatic potential maps. These results demonstrate that the DEPC covalent labeling/mass spectrometry technique can provide important information about the domain contributions of multidomain proteins such as monoclonal antibodies when interacting with multimodal resins over a range of pH conditions.

Determining buffer conditions for downstream processing of VLP-based recombinant hepatitis B surface antigen using multimodal resins in bind-elute and flow-through purification modes

The difficulties in purification of VLP-based recombinant hepatitis B surface antigen (rHBsAg) are mainly emerged from inefficient semi-purification step plus proteins physicochemical properties and these issues make the downstream processing (DSP) very lengthy and expensive. In this study, optimization of rHBsAg (recombinantly-expressed in Pichia pastoris) DSP was performed using selection of buffering conditions in the semi-purification step. In the semi-purification optimization step, up to 73% of the protein impurities were eliminated and the utmost increase in rHBsAg purity (ca. 3.6-fold) was achieved using 20 mM sodium acetate, pH 4.5. By using rHBsAg binding and nonbinding situations obtained from the response surface plot in design of experiments (DOE), additional bind-elute and flow-through purification mode experiments were conducted and rHBsAg with high purity (near 100%) and recovery (> 83%) was achieved. Following assessment of critical quality attributes (i.e., purity, particle size distribution, host cell DNA, host cell protein, secondary structures, specific activity and relative potency), it was indicated that the characteristics of rHBsAg purified by the new DSP were similar or superior to the ones obtained from conventional DSP. The purification performance of the resin was constantly retained (97–100%) and no significant resin damage took place after 10 adsorption–elution–cleaning cycles. The new DSP developed for production of rHBsAg in this study can substitute the conventional one with granting satisfactory target protein quality, long-lasting resin efficacy, shorter and less expensive process. This process may be also employable for purification of both non-VLP- and VLP- based target proteins expressed in the yeast.

Purification of a monoclonal antibody using a novel high-capacity multimodal cation exchange nonwoven membrane

A high-capacity, multimodal cation exchange (MMC) chromatographic membrane was developed by conjugating a multimodal ligand – 2-mercaptopyridine-3-carboxylic acid (MPCA) – on a polybutylene terepthalate (PBT) nonwoven fabric. The membrane features an equilibrium binding capacity of ≈ 1000 mg of human polyclonal IgG (IgG) per g of membrane and dynamic binding capacities (DBC10%) ranging from 77.5 to 115.1 mg/mL (residence times of 1 and 5 min, respectively); these values are 2-to-3-fold higher than those of commercial MMC adsorbents. The effects of buffer composition, pH, conductivity on the binding behavior of the MMC-MPCA membrane were investigated in detail. As a moderate cation exchange binder, MPCA enables effective protein elution using buffers with mild pH (8.0–9.0) and conductivity (≈13 mS/cm), thus circumventing the harsh conditions often needed in multimodal chromatography. The MMC-MPCA membrane was evaluated for product capture in bind-and-elute mode on a Chinese hamster ovary (CHO) cell culture harvest containing therapeutic monoclonal antibodies, using commercial multimodal (Capto MMC and MX-Trp-650M) and affinity (AF-rProtein A HC-650F) resins as controls. The MMC-MPCA membrane outperformed the multimodal resins in terms of binding capacity as well as clearance of host cell proteins (HCPs) and aggregates. The membrane was then evaluated by polishing the mAb from a Protein A eluate in bind-and-elute mode. The MMC-MPCA membrane reduced the level of high molecular weight components from 11% to 4% and the HCP content from 1319.7 ppm to 48.7 ppm (LRV of 1.4). Most notably, proteomics analysis of the product demonstrated the clearance of a significant fraction of persistent, high-risk HCPs from the Protein A eluate.

Next generation multimodal chromatography resins via an iterative mapping approach: Chemical diversity, high-throughput screening, and chromatographic modelling

Multimodal chromatography resins are becoming a key tool in the purification of biomolecules. The main objective of this research was the establishment of an iterative framework for the rapid development of new multimodal resins to provide novel selectivity for the future purification challenges. A large chemically diverse virtual library of 100 multimodal Capto™ MMC ligand analogues was created, and a broad array of chemical descriptors were calculated for each ligand in silico. Principal component analysis (PCA) was used to map the chemical diversity and guide selection of ligands for synthesis and coupling to the Capto ImpRes agarose base matrix. Twelve new ligands were prepared in two groups: ‘group one’ consist of L00-L07 and ‘group two’ consist of L08-L12. These ligands are diverse in the influence of varied secondary interactions such as hydrophobic interactions, H-bonding, etc. Additional resin prototypes were also prepared to look at the chromatographic impact of ligand density variation. High-throughput plate-based studies were performed for parallel resin screening for batch-binding of six model proteins at different chromatographic binding pH and sodium chloride concentration conditions. Principal component analysis of the binding data provided a chromatographic diversity map leading to the identification of ligands with improved binding. Further, the new ligands have improved separation resolution between a monoclonal antibody (mAb1) and product related impurities, a Fab fragment and high molecular weight (HMW) aggregates, using linear salt gradient elutions. To quantify the importance of secondary interactions, analysis of the retention factor of mAb1 on the ligands at various isocratic conditions lead to estimations of (a) the total number of water molecules and counter salt ions released during adsorption, and (b) hydrophobic contact area (HCA). The iterative mapping approach of chemical and chromatography diversity maps described in the paper proves to be a promising method for identifying new chromatography ligands for biopharmaceutical purification challenges.

Standardized method for mechanistic modeling of multimodal anion exchange chromatography in flow through operation

Multimodal chromatography offers an increased selectivity compared to unimodal chromatographic methods and is often employed for challenging separation tasks in industrial downstream processing (DSP). Unfortunately, the implementation of multimodal polishing into a generic downstream platform can be hampered by non-robust platform conditions leading to a time and cost intensive process development. Mechanistic modeling can assist experimental process development but readily applicable and easy to calibrate multimodal chromatography models are lacking. In this work, we present a mechanistic modeling aided approach that paves the way for an accelerated development of anionic mixed-mode chromatography (MMC) for biopharmaceutical purification. A modified multimodal isotherm model was calibrated using only three chromatographic experiments and was employed in the retention prediction of four antibody formats including a Fab, a bispecific, as well as an IgG1 and IgG4 antibody subtype at pH 5.0 and 6.0. The chromatographic experiments were conducted using the anionic mixed-mode resin Capto adhere at industrial relevant process conditions to enable flow through purification. An existing multimodal isotherm model was reduced to hydrophobic interactions in the linear range of the adsorption isotherm and successfully employed in the simulation of six chromatographic experiments per molecule in concert with the transport dispersive model (TDM). The model reduction to only three parameters did prevent structural parameter non-identifiability and enabled an analytical isotherm parameter determination that was further refined by incorporation of size exclusion effects of the selected multimodal resin. During the model calibration, three linear salt gradient elution experiments were performed for each molecule followed by an isotherm parameter uncertainty assessment. Lastly, each model was validated with a set of step and isocratic elution experiments. This standardized modeling approach facilitates the implementation of multimodal chromatography as a key unit operation for the biopharmaceutical downstream platform, while increasing the mechanistic insight to the multimodal adsorption behavior of complex biologics.

Continuous manufacturing of monoclonal antibodies: Dynamic control of multiple integrated polishing chromatography steps using BioSMB

We propose a strategy for automation and control of multi-step polishing chromatography in integrated continuous manufacturing of monoclonal antibodies. The strategy is demonstrated for a multi-step polishing process consisting of cation exchange chromatography in bind-and-elute mode followed by mixed-mode chromatography in flowthrough mode. A BioSMB system with a customized Python control layer is used for automation and scheduling of both the chromatography steps. Further, the BioSMB valve manifold is leveraged for in-line conditioning between the two steps, as tight control of pH and conductivity is essential when operating with multimodal resins because even slight fluctuations in load conditions adversely affect the chromatography performance. The pH and conductivity of the load to the multimodal chromatography columns is consistent, despite the elution gradient of the preceding cation exchange chromatography step. Inputs from the BioSMB pH and conductivity sensors are used for real-time control of the 7 pumps and 240 valves to achieve in-line conditioning inside the BioSMB manifold in a fully automated manner. This is confirmed by showcasing different elution strategies in cation exchange chromatography, including linear gradient, step gradient and process deviations like tubing leakage. In all the above cases, the model was able to maintain the pH and conductivity of multimodal chromatography load within the range of 6 ± 0.1 pH and 7 ± 0.3 mS/cm conductivity. The strategy eliminates the need for using multiple BioSMB units or integrating external pumps, valves, mixers, surge tanks, or sensors between the two steps as is currently the standard approach, thus offering a simple and robust structure for integrating multiple polishing chromatography steps in continuous downstream monoclonal antibody purification trains.

Protein adsorption and separation with monomodal and multimodal anion exchange chromatography resins. Part II. Mechanisms of protein aggregation on the chromatographic surface

AbstractBACKGROUNDProteins on chromatographic surfaces can undergo conformational changes, leading to aggregate formation. Such effects have been reported previously for hydrophobic interaction and cation exchange chromatography. In Part I of this work, we examined protein adsorption behavior on monomodal (Nuvia HP‐Q) and multimodal anion exchangers (Nuvia aPrime and Capto Adhere), observing that aggregates are also formed on these multimodal resins. In this work we examine the underlying mechanisms and the conditions that affect aggregate formation.RESULTSIncubating the multimodal anion exchangers with pure bovine serum albumin monomer at low ionic strength results in the formation of dimer and higher‐order oligomers (up to hexamers) to an extent that depends on the incubation time. The percentage of total protein initially bound that is desorbed decreased from >85% with a 5 min incubation to <40% with 24 h incubation at pH 7.0, indicating that additional irreversibly bound species are formed on the resin surface. Load buffers leading to stronger binding (higher pH and/or lower salt) and higher temperatures (32 °C) result in more extensive aggregation. Minimal aggregation occurs, instead, for Nuvia HP‐Q.CONCLUSIONBSA oligomers are formed directly on the surface of the multimodal resins studied and are eluted as discrete species at increasing salt concentrations. Aggregate formation is promoted by the ligand hydrophobicity and affects the separation of monomer–dimer mixtures by frontal chromatography, as the ability of the dimer in the feed to displace the adsorbed protein is impaired by gradual conversion of the monomer to stronger bound species. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Protein adsorption and separation with monomodal and multimodal anion exchange chromatography resins. Part I. Multicomponent adsorption properties and frontal chromatography

AbstractBACKGROUNDMultimodal ion exchangers, containing ligands combining charged and hydrophobic moieties, exhibit enhanced separation compared to monomodal ion exchangers under low loading conditions. Commercial applications often require high protein loads and such conditions have not been investigated extensively, especially for multimodal anion exchangers. This work compares protein adsorption and separation by frontal chromatography for a commercial monomodal (Nuvia HP‐Q) and two multimodal anion exchangers (Nuvia aPrime and Capto Adhere) using bovine serum albumin (BSA) monomer and dimer as a model.RESULTSGreater dimer–monomer selectivity is observed for Nuvia aPrime and Capto Adhere than for Nuvia HP‐Q at low protein loads. However, binary adsorption isotherms at high protein loads show higher binding capacity and greater selectivity for Nuvia HP‐Q, despite having the same ligand density. Higher salt concentrations reduce the binding strength in all cases, but this reduction is less pronounced for the multimodal resins. As a result, selectivity increases for the latter at higher salt concentrations. Confocal laser scanning microscopy shows that, during co‐adsorption, displacement of the monomer by the dimer occurs for each resin. However, displacement becomes very slow for the multimodal resins during sequential adsorption. The multimodal resins give poorer separation by frontal chromatography, accompanied by low recovery and substantial on‐column formation of protein aggregates.CONCLUSIONThe selectivity of multimodal anion exchangers for separation of the model proteins studied varies with salt concentration and protein load. At high protein loads, multimodal resins can result in on‐column aggregate formation negatively impacting the kinetics of competitive binding and separation. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Protein adsorption on core-shell resins for flow-through purifications: Effect of protein molecular size, shape, and salt concentration.

This work addresses the functional properties of the core-shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose-based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.

A framework for calculating orthogonal selectivities in multimodal systems directly from cell culture fluid.

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.

Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography.

In this article, a systematic workflow was formulated and implemented to understand selectivity differences and preferred binding patches for bispecific monoclonal antibodies (mAbs) and their parental mAbs on three multimodal cation exchange resin systems. This workflow incorporates chromatographic screening of the parent mAbs and their fragments at various pH followed by surface property mapping and protein footprinting using covalent labeling followed by liquid chromatography-mass spectrometry analysis. The chromatography screens on multimodal resins with the intact mAbs indicated enhanced selectivity as compared to single-mode interaction systems. While the bispecific antibody (bsAb) eluted between the two parental mAbs on most of the resins, the retention of the bispecific transitioned from co-eluting with one parental mAb to the other parental mAb on Capto MMC. To investigate the contribution of different domains, mAb fragments were evaluated and the results indicated that the interactions were likely dominated by the Fab domain at higher pH. Protein surface property maps were then employed to hypothesize the potential preferred binding patches in the solvent-exposed regions of the parental Fabs. Finally, protein footprinting was carried out with the parental mAbs and the bsAb in the bound and unbound states at pH 7.5 to identify the preferred binding patches. Results with the intact mAb analysis supported the hypothesis that interactions with the resins were primarily driven by the residues in the Fab fragments and not the Fc. Furthermore, peptide mapping data indicated that the light chain may be playing a more important role in the higher binding of Parent A as compared with Parent B in these resin systems. Finally, results with the bsAb indicated that both halves of the molecule contributed to binding with the resins, albeit with subtle differences as compared to the parental mAbs. The workflow presented in this paper lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules which can provide insights into improved biomanufacturability and expedited downstream bioprocess development.

Microfluidics as a high-throughput solution for chromatographic process development – The complexity of multimodal chromatography used as a proof of concept

High-throughput technologies are fundamental to expedite the implementation of novel purification platforms. The possibility of performing process development within short periods of time while saving consumables and biological material are prime features for any high-throughput screening device. In this work, a microfluidic device is evaluated as high-throughput solution for a complete study of chromatographic operation conditions on ten different multimodal resins. The potential of this class of purification solutions is generally hindered by its complexity. Taking this into consideration, the microfluidic platform was herein applied and assessed as a tool for high-throughput applications. The commercially available multimodal ligands were studied for the binding of three antibody-based biomolecules (polyclonal mixture of whole antibodies, Fab and Fc fragments) at different pH and salt conditions, in a total of 450 experiments. The results obtained with the microfluidic device were comparable to a standard 96-well filtering microplate high-throughput tool. Additionally, five of the ten multimodal ligands tested were packed into a bench-scale column to perform a final validation of the microfluidic results obtained. All the data acquired in this work using different screening protocols corroborate each other, showing that microfluidic chromatography is a valuable tool for the fast implementation of a new purification step, particularly, if the goal is to narrow the downstream possibilities by being a first point of decision.

Plasmid processing for mRNA, gene therapy and other vector applications

<h3>Watch the video or read the poster to learn:</h3> <ul><li>Novel three-step purification process for plasmid (p)DNA </li><li>A new high productivity multimodal resin designed specifically for pDNA purification</li><li>Comparison between legacy and novel purification process in terms of productivity </li><li>How a novel fibro format could offer even greater productivity gains in the future</li><div class="authors authors-lg focus-visible" data-focus-visible-added=""><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Headshopt copy.png" data-image="1"></div><div class="author-det"> <b>Henrik Ihre</b> has his roots in biopharma, leadership and product development for the biopharma downstream industry in specific. He is motivated by bringing new solutions and manufacturing of new pharmaceuticals for patients developed by partners of Cytiva. He has been the Director of Strategic Technologies since March 2020 with specific knowledge and background in the downstream purification of biopharmaceuticals for over 20 years.&nbsp; </div></div></div></ul>

The use of multimodal resins for purification of therapeutic monoclonal antibodies

Obtaining protein samples with high yield, high purity, and high activity is the foundation for most modern biological studies. Besides, highly purified protein preparations, such as monoclonal antibodies, have been widely used in the treatment of cancer, genetic and orphan diseases.

Abstract 2851: A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format

Abstract A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format. Removal of unreacted or residual compounds in the sample-preparation workflow is a key success factor for downstream analysis and processing of biomolecules. For example, during sample preparation of biomolecules such as proteins or nucleic acids, there is often a need to label the biomolecules with dyes, affinity tags, radioactive labels, and mass tags. In other instances, there is a need to chemically modify biomolecules such as to reduce, oxidize, methylate, etc. During these treatments, some amount of the labeling or chemical agent remains in the sample as an unreacted label/chemical. These unreacted small molecules can cause several issues during downstream analysis or use of the biomolecule. For example, free unreacted fluorescent dyes after bioconjugation of protein cause non-specific binding of free-dye resulting in high background issues during fluorescent imaging. Commonly used clean-up methods following sample preparation, such as dialysis, are tedious and lengthy with multiple buffer exchanges sometime resulting in loss of protein due to aggregation. Alternatively, size-exclusion chromatography requires expensive instrumentation in addition to lengthy set up time. We have developed the Thermo Scientific™ PierceTM Dye and Biotin Removal Spin Columns, a novel resin that is packed in an easy to use “Spin and Go” format of varying spin column sizes (0.5 mL, 2mL, 5 mL and 10 mL) to accommodate a range of sample volumes (100 uL- 4 mLs). The resin is highly specialized to produce exceptional protein recovery and can be used effectively to remove 4 different class of small molecules: non-conjugated fluorescent dyes, biotinylation reagents, reducing agents, and crosslinkers. It is a multimodal resin with size exclusion properties and proprietary surface chemistry that allows removal of the above-mentioned small molecules, while the size exclusion property allows for the biomolecule to be efficiently recovered. Our results indicate efficient dye removal and protein recovery from fluorescent conjugated antibodies. The results also indicate removal of reducing agents such as TCEP at a concentration as high as 25 mM from protein samples, while recovering &amp;gt;90% of the added protein. Results also indicate &amp;gt;90% removal of biotinylated molecules and crosslinkers from Protein samples with &amp;gt;90% protein recovery. Our results show that the efficiency and binding capacity for removal of small molecules using this resin is better than any resin in the market. This is exemplified by immunofluorescent analysis of ZO-1 monoclonal antibody conjugated Alexa Fluor™ 488 which was purified using PierceTM Dye and Biotin Removal Spin columns and shows high intensity and lesser background in Caco-2 cells than the non-purified conjugate. Citation Format: Deven Kent Etnyre, Ramesh Ganapathy, Marie Nlend, Chris Etienne, Atul M. Deshpande. A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2851.

A thermodynamic evaluation of antibody-surface interactions in multimodal cation exchange chromatography.

In this study, the thermodynamics of binding of two industrial mAbs to multimodal cation exchange systems was investigated over a range of buffer and salt conditions via a van't Hoff analysis of retention data. Isocratic chromatography was first employed over a range of temperature and salt conditions on three multimodal resins and the retention data were analyzed in both the low and high salt regimes. While mAb retention decreased with salt for all resins at low salts, retention increased at high salts for two of the resins, suggesting a shift from electrostatic to more hydrophobic driven interactions. The retention data at various temperatures were then employed to generate non-linear van't Hoff plots which were fit to the quadratic form of the van't Hoff equation. At low salts, retention of both mAbs decreased with increasing temperature and the van't Hoff plots were concave downward on Capto MMC and Nuvia cPrime, while being concave upward on Capto MMC ImpRes. Different trends were observed on some of the resins with respect to both the concavity of the van't Hoff plots as well as the impact of temperature on the favorable enthalpies in the low salt regime. Interestingly, while increasingly favorable enthalpy with temperature was observed with Capto MMC and Nuvia cPrime at low salt, favorable enthalpy decreased with temperature for Capto MMC ImpRes. At high salts, binding of both mAbs on the two Capto resins were consistently entropically driven, consistent with desolvation. While the negative heat capacity data at low salts indicated that desolvation of polar/charged groups were important in Capto MMC and Nuvia cPrime, the positive data suggested that desolvation of non-polar groups were more important with Capto MMC ImpRes. Finally, the data at high salts indicated that desolvation of non-polar groups was the major driver for binding of both mAbs to the Capto resins under these conditions.

A rapid and an efficient method to remove unreacted small‐molecules using a versatile resin packed in a convenient Spin‐and‐Go format.

Removal of unreacted or residual compounds in the sample‐preparation workflow is a key success factor for downstream analysis and processing of biomolecules. For example, during sample preparation of biomolecules such as proteins or nucleic acids, there is often a need to label the biomolecules with dyes, affinity tags, radioactive labels, and mass tags. In other instances, there is a need to chemically modify biomolecules such as to reduce, oxidize, methylate, etc. During these treatments, some amount of the labeling or chemical agent remains in the sample as an unreacted label/chemical. These unreacted small molecules can cause several issues during downstream analysis or use of the biomolecule. For example, free unreacted fluorescent dyes after bioconjugation of protein cause non‐specific binding of free‐dye resulting in high background issues during fluorescent imaging. Commonly used clean‐up methods following sample preparation, such as dialysis, are tedious and lengthy with multiple buffer exchanges sometime resulting in loss of protein due to aggregation. Alternatively, size‐exclusion chromatography requires expensive instrumentation in addition to lengthy set up time.We have developed the Thermo Scientific™ Pierce™ Dye and Biotin Removal Spin Columns, a novel resin that is packed in an easy to use “Spin and Go” format of varying spin column sizes (0.5 mL, 2mL, 5 mL and 10 mL) to accommodate a range of sample volumes (100 uL–4 mLs). The resin is highly specialized to produce exceptional protein recovery and can be used effectively to remove 4 different class of small molecules: non‐conjugated fluorescent dyes, biotinylation reagents, reducing agents, and crosslinkers. It is a multimodal resin with size exclusion properties and proprietary surface chemistry that allows removal of the above‐mentioned small molecules, while the size exclusion property allows for the biomolecule to be efficiently recovered.Our results indicate efficient dye removal and protein recovery from fluorescent conjugated antibodies. The results also indicate removal of reducing agents such as TCEP at a concentration as high as 25 mM from protein samples, while recovering &gt;90% of the added protein. Results also indicate &gt;90% removal of biotinylated molecules and crosslinkers from Protein samples with &gt;90% protein recovery. Our results show that the efficiency and binding capacity for removal of small molecules using this resin is better than any resin in the market. This is exemplified by immunofluorescent analysis of ZO‐1 monoclonal antibody conjugated DyLight™ 488 which was purified using Pierce™ Dye and Biotin Removal Spin columns and shows high intensity and lesser background in Caco‐2 cells than the non‐purified conjugate.