Mixed-mode Resins Research Articles

Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs.

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

Optimization of the polishing process by integrating experimental design and high-throughput screening

Complex bispecific antibody formats tend to form more product-related impurities than monoclonal antibodies. The primary constraints are yield and purity in the polishing stage. The purpose of this study was to enhance the understanding of the optimal working window for four mixed-mode resins and to reduce the burden of resin screening and parameter optimization during process development. This study optimized the loading and elution conditions of four different mixed-mode cationic resins to enhance the yield and purity by integrating Design of Experiments with High-Throughput Screening. It was observed that despite being weakly acidic mixed-mode cationic resins, these four resins exhibited significant differences in their adsorption and elution performances and varied tolerances to salt concentrations. Capto MMC demonstrated strong hydrophobicity, while the performance profiles of MX-Trp-650 M and Nuvia cPrime were similar, with Nuvia cPrime showing superior purification effects. Eshmuno CMX, by extending its side chain ligand, achieved higher binding efficiency and capacity. Through the optimization of salt concentrations or the application of dual-gradient elution strategies, the target protein with high yield (77 %) and purity over 99 % was successfully obtained. This research not only provides in-depth insights into the application of mixed-mode chromatography in the biopharmaceutical field but also offers practical optimization strategies for the industrial-scale purification of bispecific antibodies.

Machine learning assisted QSAR analysis to predict protein adsorption capacities on mixed-mode resins

Mixed-mode chromatography is promising for protein separation, but the structural diversities of proteins result in distinct adsorption behaviors. This study used machine learning methods to establish the quantitative structure–activity relationships (QSAR) between the protein adsorption capacity on mixed-mode resins and the molecule properties of target proteins and mixed-mode ligands. Four mixed-mode resins and twenty proteins were tested at different pHs and salt concentrations. Two machine learning models, random forest and gradient boosting, were developed successfully to predict protein adsorption capacities. The determination coefficients (R2) of the training dataset, validation dataset, and test dataset ranged around 0.94–0.97, 0.79–0.82, and 0.90–0.93, respectively. Several key descriptors that have significant impacts on adsorption capacities were identified by a two-step descriptor elimination method. Moreover, the SHapley Additive exPlanations (SHAP) method was used to reveal the mechanism of target protein adsorption on mixed-mode resins. The results provided a valuable guidance for the design and selection mixed-mode resins for the separation and purification of target proteins.

Two-step separation strategy with mixed-mode chromatography to purify recombinant human serum albumin from Pichia pastoris culture broth

Mixed-mode chromatography (MMC) with the specially-designed ligands shows good adsorption selectivity for protein separation. In this study MMC was explored to purify recombinant human serum albumin (rHSA) from Pichia pastoris culture broth, which combined bind-elute and flow-through modes. Four mixed-mode resins were evaluated for their adsorption capacities of HSA. It was found that two home-made MMC resins showed special performances. IC-B-6FF resin with indole-3-acetic acid-cysteine ligand had high adsorption and selectivity towards HSA, and SFA-B-6FF resin with sulfapyridine ligand showed minimal binding to HSA. Therefore, a two-step MMC separation strategy was designed that IC-B-6FF was used to capture rHSA directly from yeast culture broth and then SFA-B-6FF resin was used to polish the remaining impurities by flow-through mode. The results revealed that the purity of rHSA reached 95.1% with a yield of 81.2% after the two-step separation. The removal rate of host cell proteins (HCPs) was 99.8% with final HCP level as low as 50 ppm. The results demonstrate that MMC combining bind-elute and flow-through modes has a promising potential to separate protein with high selectivity and process efficiency.

High throughput screening for rapid and reliable prediction of monovalent antibody binding behavior in flowthrough mode.

Flowthrough (FT) anion exchange (AEX) chromatography is a widely used polishing step for the purification of monoclonal antibody (mAb) formats. To accelerate downstream process development, high throughput screening (HTS) tools have proven useful. In this study, the binding behavior of six monovalent mAbs (mvAbs) was investigated by HTS in batch binding mode on different AEX and mixed-mode resins at process-relevant pH and NaCl concentrations. The HTS entailed the evaluation of mvAb partition coefficients (Kp) and visualization of results in surface-response models. Interestingly, the HTS data grouped the mvAbs into either a strong-binding group or a weak-binding/FT group independent of theoretical Isoelectric point. Mapping the charged and hydrophobic patches by in silico protein surface property analyses revealed that the distribution of patches play a major role in predicting FT behavior. Importantly, the conditions identified by HTS were successfully verified by 1 mL on-column experiments. Finally, employing the optimal FT conditions (7-9 mS/cm and pH 7.0) at a mini-pilot scale (CV = 259 mL) resulted in 99% yield and a 21-23-fold reduction of host cell protein to <100 ppm, depending on the varying host cell protein (HCP) levels in the load. This work opens the possibility of using HTS in FT mode to accelerate downstream process development for mvAb candidates in early research.

SIMULTANEOUS REMOVAL OF ACIDIC AND NEUTRAL POLLUTANTS FROM WATER USING MIXED-MODE RESINS

Polycyclic aromatic hydrocarbons and endocrine-disrupting phenols are both ubiquitous pollutants in the environment. Human activities through urbanisation and rapid industrialisation are at the forefront of global environmental pollution. Therefore, removing pollutants in miscellaneous characteristics is important to eliminate the stage-by-stage reduction of pollutants, especially from wastewater, for a cost-effective and time-effective treatment. A simple preliminary study was conducted for the concurrent removal of acidic 2, 4, 6-trichlorophenol (2, 4, 6-TCP) and neutral fluorene (FLU) from water by mixed-mode resins that consist of Anion Exchange Chromatography (AEC) and reversed-phase C18 sorbents. The one-variable-at-time approach was applied in the optimisation of the removal process, where sorbent dosage, exposing time, stirring speed, and sample pH were investigated to enhance the removal efficiency. Under the optimal conditions, 20 mg of each AEC and C18 successfully removed 74.8% of 2, 4, 6-TCP and 96.5% of FLU from the water sample spiked with 50 mg/L of 2, 4, 6-TCP and 0.5 mg/L of FLU that was pre-modified to pH 9 and agitated at 600 rpm for 10 min. The AEC and C18 achieved a removal capacity of 37.4 mg/g for 2, 4, 6-TCP and 0.48 mg/g for FLU, respectively, upon treating 20 mL of the water sample. The mixed-mode resins combine the capabilities of two different retention mechanisms and allow for the simultaneous removal of a broad range of compounds from environmental samples.

Antibody sequence-based prediction of pH gradient elution in multimodal chromatography

Multimodal chromatography has emerged as a promising technique for antibody purification, owing to its capacity to selectively capture and separate target molecules. However, the optimization of chromatography parameters remains a challenge due to the intricate nature of protein-ligand interactions. To tackle this issue, efficient predictive tools are essential for the development and optimization of multimodal chromatography processes. In this study, we introduce a methodology that predicts the elution behavior of antibodies in multimodal chromatography based on their amino acid sequences. We analyzed a total of 64 full-length antibodies, including IgG1, IgG4, and IgG-like multispecific formats, which were eluted using linear pH gradients from pH 9.0 to 4.0 on the anionic mixed-mode resin Capto adhere. Homology models were constructed, and 1312 antibody-specific physicochemical descriptors were calculated for each molecule. Our analysis identified six key structural features of the multimodal antibody interaction, which were correlated with the elution behavior, emphasizing the antibody variable region. The results show that our methodology can predict pH gradient elution for a diverse range of antibodies and antibody formats, with a test set R² of 0.898. The developed model can inform process development by predicting initial conditions for multimodal elution, thereby reducing trial and error during process optimization. Furthermore, the model holds the potential to enable an in silico manufacturability assessment by screening target antibodies that adhere to standardized purification conditions. In conclusion, this study highlights the feasibility of using structure-based prediction to enhance antibody purification in the biopharmaceutical industry. This approach can lead to more efficient and cost-effective process development while increasing process understanding.

Pseudo-affinity capture of K. phaffii host cell proteins in flow-through mode: Purification of protein therapeutics and proteomic study

K. phaffii is a versatile expression system that is increasingly utilized to produce biological therapeutics – including enzymes, engineered antibodies, and gene-editing tools – that feature multiple subunits and complex post-translational modifications. Two major roadblocks limit the adoption of K. phaffii in industrial biomanufacturing: its proteome, while known, has not been linked to downstream process operations and detailed knowledge is missing on problematic host cell proteins (HCPs) that endanger patient safety or product stability. Furthermore, the purification toolbox has not evolved beyond the capture of monospecific antibodies, and few solutions are available for engineered antibody fragments and other protein therapeutics. To unlock the potential of yeast-based biopharmaceutical manufacturing, this study presents the development and performance validation of a novel adsorbent – PichiaGuard – functionalized with peptide ligands that target the whole spectrum of K. phaffii HCPs and designed for protein purification in flow-through mode. The PichiaGuard adsorbent features high HCP binding capacity (∼25 g per liter of resin) and successfully purified a monoclonal antibody and an ScFv fragment from clarified K. phaffii harvests, affording > 300-fold removal of HCPs and high product yields (70–80%). Notably, PichiaGuard outperformed commercial ion exchange and mixed-mode resins without salt gradients or optimization in removing high-risk HCPs – including aspartic proteases, ribosomal subunits, and other peptidases – thus demonstrating its value in modern biopharmaceutical processing.

Separation of bispecific antibody related impurities with mixed-mode chromatography

The complex structure of bispecific antibody molecules and the rapid increase in upstream expression pose a significant challenge for downstream purification. Compared to mAbs, bsAbs typically contain a higher proportion of aggregates and fragments that are difficult to remove. Extensive study and optimization are often required because the properties of impurities are close to the target molecules. In this investigation, ion exchange resins and mixed-mode resins were used to evaluate the effectiveness in removing byproducts. Mixed-mode anion chromatography was found to be effective in polishing step of bsAbs with strong hydrophobicity. Hydroxyapatite chromatography using arginine hydrochloride as an eluent provided excellent separation performance compared to that of cation exchange resins and conventional mixed-mode resins. The separation process was optimized and verified in a 200 L pilot batch. The final products were obtained with excellent purity (99.0 %) and yield (80.70 %). This work provides a new idea for high-yield polishing in downstream processing, which improves the economics of large-scale production.

Adsorption of bovine serum albumin on a mixed-mode resin - influence of salts and the pH value

The separation and purification of proteins is often carried out by chromatography. Mixed-mode adsorbents are interesting as they may combine favorable features of different chromatographic methods, such as ion-exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC). Systematic experimental studies and models of adsorption isotherms of proteins on mixed-mode resins covering a wide range of parameters are still rare, which hampers both the scientific analysis of the complex processes that occur upon the adsorption of proteins on these resins as well as the practical separation design. Therefore, such studies were carried out in the present work for a model system: the adsorption of bovine serum albumin (BSA) on the mixed-mode resin Toyopearl MX-Trp-650M, which combines features of IEC and HIC resins. Adsorption isotherms were measured using sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate at ionic strengths up to 3000 mM and for pH 4.0, 4.7, and 7.0 at 25 ^circC. In the studied pH ranges, BSA exhibits strongly varying net charges and undergoes a conformational change. At pH 4.0 and 4.7, an exponential decay of the BSA adsorption with increasing ionic strength was found at ionic strengths up to approximately 1000 mM, while a rather linear increase was observed at higher ionic strengths for all studied salts; for all salts and ionic strengths, decreasing adsorption with increasing pH value was found. Moreover, a mathematical model was developed, which enables the prediction of equilibrium adsorption isotherms of BSA on Toyopearl MX-Trp-650M for any ionic strength of the studied salts.

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.

Purification of hydrophobic complex antibody formats using a moderately hydrophobic mixed mode cation exchange resin

Immunoglobulins of complex formats possess great potential for increased biopharmaceutical efficacy. However, challenges arise during their purification as the removal of numerous product-related impurities typically requires several expensive chromatographic steps. Additionally, many complex antibody formats have a high hydrophobicity which impairs the use of conventional mixed mode chromatography. In the present study, both of these challenges were addressed through the development of an innovative mixed mode resin with 2-amino-4methylpentanoic acid ligands that combines weak cation exchange with moderate hydrophobic interactions. Supported by high throughput partition coefficient screens for identification of preferable pH and salt concentration ranges in bind and elute mode, this mixed mode resin successfully demonstrated efficient impurity separation from an extremely hydrophobic bispecific antibody with a single unit operation. High purity (>97%) was obtained as a result of significant reduction of product-related impurities as well as process-related host cell proteins (>3 log scale), while maintaining satisfactory recovery (70%). This also supports that highly hydrophobic antibody formats can be efficiently purified using a resin with moderate hydrophobic characteristics. Studies involving additional antibodies possessing different formats and a wide range of hydrophobicity confirmed the broad applicability of the new resin. In view of its high selectivity and robust operating ranges, as well as the elimination of the need for an additional column step, the novel resin enables simplified downstream processing and economic manufacturing of complex antibody formats.

Simulation of Adsorption Process of l-Tryptophan on Mixed-Mode Resin HD-1 with Combined Physical Adsorption and Ion Exchange.

The mass-transfer process of l-tryptophan (l-Trp) in the hydrophobic interaction/ion-exchange mixed-mode resin HD-1 particles and fixed bed was studied experimentally and theoretically. The adsorption kinetics of l-Trp in single-component and multicomponent adsorption systems was investigated under different pH conditions. The co-adsorption of sodium ions (Na+) and l-Trp anions was found to be negligible. A modified liquid-film linear driving force model considering the physical adsorption of l-Trp zwitterions and anions as well as ion exchange of l-Trp cations was proposed. The dissociation equilibria of l-Trp molecules and functional groups on the resin were introduced in the model. The model could well fit the kinetic adsorption curves of l-Trp at different pH values. The presence of Na+ and the impurity amino acid l-glutamic acid (l-Glu) did not significantly affect the mass-transfer rate of l-Trp. The dynamic adsorption processes of l-Trp under different pH and concentration conditions were studied. A modified transport-dispersive model considering axial diffusion, liquid-film mass transfer, and a combined physical adsorption and ion-exchange equilibrium was established, which could predict the adsorption breakthrough curves of l-Trp well. During the dynamic adsorption process, the pH of mobile phase in the fixed bed changed with changing the l-Trp concentration in the mobile phase. l-Trp was well separated from Na+ and l-Glu with the purity of l-Trp higher than 99%, the recovery rate higher than 95%, and a concentration of 4.69 × 10–3 mol/L. The elution chromatographic peaks of l-Trp, l-Glu, and Na+ and the pH of the outlet solution were predicted satisfactorily.

Adsorption separation of guanosine 5′-Monophosphate and cytidine 5′-Monophosphate by mixed-mode Resin HD-1: Experimental study and mathematical modeling

The preparative separation of guanosine 5′-monophosphate (GMP) and cytidine 5′-monophosphate (CMP) on mixed-mode resin HD-1 was experimentally and theoretically investigated. The adsorption mechanisms of the two nucleotides were elucidated by analyzing adsorption equilibria and kinetics at different pH values. The adsorption dynamics of GMP and CMP in a fixed bed packed with resin HD-1 were studied. All nucleotide monovalent cations, zwitterions, and monovalent anions were adsorbed by the resin. Further, a general adsorption isotherm model was developed by considering the adsorption of different nucleotide species and the dissociation equilibrium behaviors of resin ligands. The model fit the adsorption isotherm data of GMP and CMP well. A modified liquid-film linear driving force model with the combined physical adsorption of nucleotides in different dissociation states and ion exchange of Na+ was established. The dissociation equilibria of resin ligands and nucleotides were considered. The model satisfactorily predicted the adsorption kinetic data at different pH values. The values of the efficient diffusion coefficients for GMP and CMP were not significantly influenced by the solution pH. A modified transport-diffusion model with pH gradient elution was proposed. The model accurately predicted the elution chromatographic peaks of GMP and CMP, as well as the pH at the outlet of the fixed bed packed with resin HD-1.

An integrated and continuous downstream process for microbial virus-like particle vaccine biomanufacture.

In this study, we present the first integrated and continuous downstream process for the production of microbial virus‐like particle vaccines. Modular murine polyomavirus major capsid VP1 with integrated J8 antigen was used as a model virus‐like particle vaccine. The integrated continuous downstream process starts with crude cell lysate and consists of a flow‐through chromatography step followed by periodic counter‐current chromatography (PCC) (bind‐elute) using salt‐tolerant mixed‐mode resin and subsequent in‐line assembly. The automated process showed a robust behavior over different inlet feed concentrations ranging from 1.0 to 3.2 mg ml−1 with only minimal adjustments needed, and produced continuously high‐quality virus‐like particles, free of nucleic acids, with constant purity over extended periods of time. The average size remained constant between 44.8 ± 2.3 and 47.2 ± 2.9 nm comparable to literature. The process had an overall product recovery of 88.6% and a process productivity up to 2.56 mg h−1 mlresin −1 in the PCC step, depending on the inlet concentration. Integrating a flow through step with a subsequent PCC step allowed streamlined processing, showing a possible continuous pathway for a wide range of products of interest.

Adsorption Equilibria, Kinetics, and Column Dynamics of L-Tryptophan on Mixed-Mode Resin HD-1.

The adsorption amount and selectivity of L-tryptophan (l-Trp) on the hydrophobic interaction and ion exchange mixed-mode chromatography medium HD-1 were studied as well as the salt resistance of the resin via adsorption equilibrium experiments. The adsorption mechanisms of l-Trp were illuminated by combining adsorption equilibria and a kinetics analysis. The separation effect was studied by dynamic separation experiments in a fixed-bed. The results indicate that an increase of the concentration proportion of l-Trp zwitterion benefits the adsorption of l-Trp. The resin shows a high adsorption selectivity for l-Trp at different pH values. The adsorption amount of l-Trp is not affected significantly by NaCl. Various groups play a role in the adsorption of l-Trp. An adsorption energy lower than 8 kJ/mol indicates that the adsorption of l-Trp is mainly based on non-electrostatic interactions, with an electrostatic interaction as a supplement. The adsorption equilibrium model considering the dissociation equilibrium of the resin and l-Trp proposed in this work can simulate the adsorption equilibrium data of l-Trp at different pH values as well. The mass transfer rate of l-Trp is controlled by intraparticle and liquid film diffusion simultaneously. The fixed-bed packed with resin HD-1 can separate l-Trp with the purity of l-Trp higher than 99%, recovery rate higher than 95%, and concentration of 4.69 × 10–3 mol/L.

Purification of N-acetylgalactosamine-modified-oligonucleotides using orthogonal anion-exchange and mixed-mode chromatography approaches

N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing in vivo; however, their structural complexity requires development of new purification methods to address high purity and recovery requirements. The current study evaluates complementary purification approaches using a mixed-mode Scherzo SS-C18 and anion-exchange (AEX) TSK-gel SuperQ-5PW for a range of single-stranded triantennary GalNAc-oligonucleotides. Initially, the semi-preparative mixed-mode support (10 × 250 mm, 3 µm) was compared against the preparative AEX analogue (21.5 × 300 mm, 13 µm), with the former affording double the recovery and higher purity of 95% over its AEX counterpart displaying 91% for a selected siRNA conjugate. An assortment of GalNAc-modified oligonucleotides was later purified using the mixed-mode resin revealing good recoveries (∼30–60%) and high purities of 90–94% ranging from straightforward to more challenging purifications. High sample loading in the 20 mg range was achieved, which was comparable with the larger preparative TSKgel SuperQ-5PW support. The Scherzo-SS-C18 resin also afforded some degree of resolution between diastereomers containing phosphorothioate functionalities. The TSKgel SuperQ-5PW support was later investigated to provide orthogonal separation selectivity to the Scherzo-SS-C18 column enabling purification of a selected, GalNAc-siRNA conjugate. The developed pH (8.5–11) and salt (0.3–0.7 M) gradients method provided enhanced separation selectivity between the free and conjugated siRNA, while minimizing formation of secondary structures and highlighting a complementary approach to deal with challenging purifications of oligonucleotide-GalNAc conjugates. Together, the use of AEX and mixed-mode columns provide much needed orthogonality to deal with complex GalNAc-modified oligonucleotides and potentially other upcoming modalities.

Influence of pH value and salts on the adsorption of lysozyme in mixed-mode chromatography.

Mixed‐mode chromatography (MMC) is an interesting technique for challenging protein separation processes which typically combines adsorption mechanisms of ion exchange (IEC) and hydrophobic interaction chromatography (HIC). Adsorption equilibria in MMC depend on multiple parameters but systematic studies on their influence are scarce. In the present work, the influence of the pH value and ionic strengths up to 3000 mM of four technically relevant salts (sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate) on the lysozyme adsorption on the mixed‐mode resin Toyopearl MX‐Trp‐650M was studied systematically at 25℃. Equilibrium adsorption isotherms at pH 5.0 and 6.0 were measured and compared to experimental data at pH 7.0 from previous work. For all pH values, an exponential decay of the lysozyme loading with increasing ionic strength was observed. The influence of the pH value was found to depend significantly on the ionic strength with the strongest influence at low ionic strengths where increasing pH values lead to decreasing lysozyme loadings. Furthermore, a mathematical model that describes the influence of salts and the pH value on the adsorption of lysozyme in MMC is presented. The model enables predicting adsorption isotherms of lysozyme on Toyopearl MX‐Trp‐650M for a broad range of technically relevant conditions.

Evaluating a new mixed-mode resin Diamond MMC Mustang using Capto MMC ImpRes as a benchmark

Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. In this work, we evaluated this resin using Cytiva's Capto MMC ImpRes, a well-established mixed-mode resin with similar properties, as a benchmark. The data suggest that in comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable binding capacity and resolution. In addition, the resin under evaluation shows good lot-to-lot consistency. The information provided in this study allows users to have additional options when selecting mixed-mode resin for intermediate purification or final polishing, which is favourable especially at the present time when the supply chains of many manufacturers are negatively impacted by the coronavirus pandemic.

Influence of Salts on the Adsorption of Lysozyme on a Mixed-Mode Resin

Mixed-mode chromatography (MMC), which combines features of ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC), is an interesting method for protein separation and purification. The design of MMC processes is challenging as adsorption equilibria are influenced by many parameters, including ionic strength and the presence of different salts in solution. Systematic studies on the influence of those parameters in MMC are rare. Therefore, in the present work, the influence of four salts, namely, sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate, on the adsorption of lysozyme on the mixed-mode resin Toyopearl MX-Trp-650M at pH 7.0 and 25°C was studied systematically in equilibrium adsorption experiments for ionic strengths between 0 mM and 3000 mM. For all salts, a noticeable adsorption strength was observed over the entire range of studied ionic strengths. An exponential decay of the loading of the resin with increasing ionic strength was found until approx. 1000 mM. For higher ionic strengths, the loading was found to be practically independent of the ionic strength. At constant ionic strength, the highest lysozyme loadings were observed for ammonium sulfate, the lowest for sodium chloride. A mathematical model was developed that correctly describes the influence of the ionic strength as well as the influence of the studied salts. The model is the first that enables the prediction of adsorption isotherms of proteins on mixed-mode resins in a wide range of technically interesting conditions, accounting for the influence of the ionic strength and four salts of practical relevance.