Mixed-mode Resins Research Articles

Capture chromatography with mixed-mode resins: A case study with recombinant human thioredoxin from Escherichia coli

Capture chromatography with mixed-mode resins: A case study with recombinant human thioredoxin from Escherichia coli

High-Throughput Process Development for Recombinant Human Serum Albumin Separation from Pichia pastoris Broth with Mixed-Mode Chromatography

Due to the shortage of blood donation and safe issues of blood-derived pathogens, recombinant human serum albumin (rHSA) expressed by Pichia pastoris is a potential substitute for plasma-derived HSA (pHSA). In this work, four commercialized mixed-mode resins (Capto adhere, Capto MMC, MX-Trp-650M, and Nuvia cPrime) and two ion-exchange resins (Q Sepharose FF and SP Sepharose FF) were used for rHSA separation from Pichia pastoris cell culture broth, and loading and elution conditions were optimized via a high-throughput process using microtiter filter plates. The results indicated that resins with cation-exchange groups had better adsorption performance. The loading conditions at pH 4.0 and dilution rate of 1.0 were suitable for SP Sepharose FF, and no dilution worked for mixed-mode resins. The optimal elution pH was determined to be pH 7.0 for SP Sepharose FF, pH 8.0 for MX-Trp-650M and Nuvia cPrime, and pH 9.0 for Capto MMC. Moreover, rHSA separation from Pichia pastoris broth was performed on column unde...

Advance chromatin extraction improves the performance of electropositive mixed-mode chromatography as a capture step and enables its integration with void-exclusion anion exchange chromatography as a two-column-step purification platform for monoclonal antibody production

Advance chromatin extraction, based on a fatty acid/allantoin/solid phase system, has been reported to significantly improve the purification performance of protein A chromatography, cation exchange chromatography and electronegative mixed-mode chromatography to capture monoclonal antibodies (mAbs). In this study, the beneficial effects of advance chromatin extraction were evaluated using electropositive mixed-mode chromatography and were demonstrated to significantly enhance the dynamic binding capacity and removal efficiency of various impurities. Moreover, retention of immunoglobulin G (IgG) molecules on electropositive mixed-mode resins uniquely followed two phases, which decreased and then increased along with the increase in salt concentration. The eluted IgG was directly loaded and further polished by void-exclusion anion exchange chromatography without adjusting the sample. After these two chromatographic steps, non-histone host cell protein (nh-HCP) content in the end-product was reduced to < 1 ppm, DNA was < 1 ppb, aggregates were < 0.05%, and histone HCP was below the detection limit, with an overall process yield of 89.6%. A simplified and seamless two-column-step purification platform with electropositive mixed-mode chromatography as the primary capture strategy was established to provide a more efficient and economical alternative to current prevailing protein A affinity chromatography-based processes.

Isoform Separation by a Mixed-mode Resin, TOYOPEARL MX-Trp-650M.

TOYOPEARL particles are cross-linked hydroxylated methacrylic polymers available in different pore and particle sizes. They are conjugated with different ligands to generate ion-exchange, hydrophobic interaction and affinity resins. They have excellent physical and chemical properties. A mixed-mode resin, TOYOPEARL MX-Trp-650M, is made of this particle with tryptophan conjugated via N-terminal amino group and hence has both hydrophobic/aromatic side chain and carboxyl group. In this review, I will summarize the properties of the TOYOPEARL particles and MX-Trp-650M resin and application of this resin for purification of proteins and in some detail the separation of disulfide (SS)- scrambled oligomers of insulin-like growth factor-1 (IGF-1). For this particular application, the intact IGF-1 was used to examine binding and elution conditions of TOYOSCREEN MX-Trp-650M column. Strong binding was obtained at pH 4.0, at which arginine, but not NaCl, resulted in elution. Both NaCl and arginine resulted in elution at pH 6.5. In addition, a pH gradient from 4.0 to 8.5 was effective. When applied to SS-scrambled IGF-1 oligomers, both pH and arginine gradient exhibited an efficient separation of the oligomers.

Preparation of copolymer-grafted mixed-mode resins for immunoglobulin G adsorption

The mixed-mode resins for protein adsorption have been prepared by a novel strategy, copolymer grafting. Specially, the copolymer-grafted resins CG-M-A with two functional groups, 5-amino-benzimidazole (ABI) and methacryloxyethyltrimethyl ammonium chloride (METAC), have been prepared through surface-initiated activator generated by electron transfer for atom transfer radical polymerization of METAC and glycidyl methacrylate (GMA), followed by a ring-open reaction to introduce ABI. The charge and hydrophobicity of CG-MA resins could be controlled by manipulating the addition of METAC and GMA/ABI. Besides, METAC and ABI provided positive effects together in both protein adsorption and elution: dynamic binding capacity of human Immunoglobulin G (hIgG) onto CG-M-A resin with the highest ligand ratio of METAC to ABI is 46.8 mg∙g–1 at pH 9 and the elution recovery of hIgG is 97.0% at pH 5. The separation experiment showed that purity and recovery of monoclonal antibody from cell culture supernatant are 96.0% and 86.5%, respectively, indicating that copolymer-grafted mixed-mode resins could be used for antibody purification.

A parallel demonstration of different resins' antibody aggregate removing capability by a case study

Aggregation is a major concern for therapeutic monoclonal antibody (mAb), as aggregates reduce drug efficacy and safety. In addition to aggregation control, aggregate removal by downstream processing is crucial. Hydrophobic and mixed-mode resins are widely used for aggregate removal in different cases, but they are seldom compared side by side. In this study, the aggregate removing capability of eight resins belonging to different chromatographic types was demonstrated by a case study. This work, by providing multiple options for aggregate removal, allows more flexibility to be gained in downstream processing.

Adsorption behaviors of avian immunoglobulins and purification of immunoglobulin Y from chicken serum with mixed-mode resins

Abstract The importance of immunoglobulin Y (IgY) as a specific antibody equivalent to mammalian immunoglobulin G (IgG) is well recognized. However, production of highly purified IgY is still difficult due to the lack of specific purification methods. In this study, adsorption behaviors of IgY on four mixed-mode resins with functional ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methyli-midazole (MMI), 5-aminobenzi-midazole (ABI) and tryptophan-5-aminobenzi-midazole (W-ABI) were evaluated. The results showed that high adsorption ratio were found at pH 6.0–7.0 with little adsorption under acidic conditions. The resin with ABI ligand was then used to separate IgY from immunized chicken serum. An efficient process with IgY purity of 95% and recovery of 90% was developed after optimization of loading and elution pH and injection volume. The biological activity of the purified IgY was fully maintained. These results indicated that mixed-mode chromatography with specially-designed ligands has great potential for the separation of IgY from crude feedstock.

High-throughput screening and optimization of mixed-mode resins for human serum albumin separation with microtiter filter plate

High-throughput approaches become increasingly important on process development for protein separation. In this work, four mixed-mode resins (Capto adhere, Capto MMC, MX-Trp–650 M and Nuvia cPrime) were investigated for human serum albumin (HSA) separation and compared with two ion-exchangers (Q Sepharose FF and SP Sepharose FF). High-throughput method based on microtiter filter plate was used to determine adsorption properties and optimize separation conditions. For resins with anion-exchange groups alkaline condition would be favorable for HSA adsorption, while acidic condition for resins with cation-exchange groups. Mixed-mode resins showed obviously salt-tolerant properties. Salt addition had little effect on Nuvia cPrime, and could promote HSA adsorption for Capto MMC and MX-Trp–650 M. The elution conditions were also optimized and high recovery could be obtained. In addition, the optimized conditions obtained by high-throughput approaches were verified by lab-scale column chromatography. It was found that Capto MMC and MX-Trp–650 M would be more suitable for HSA separation and DBC10% could reach more than 40 mg/ml under 200 mM NaCl addition. Finally, rHSA separation from P. pastoris culture broth was tested with MX-Trp–650 M, and high purity and recovery could be obtained. This study demonstrated that high-throughput screening method is a powerful tool to optimize the chromatographic separation. Mixed-mode cation-exchange resins showed an efficient practicability for HSA separation with high capacity, suitable salt-tolerance and high elution recovery.

Evaluation of adsorption selectivity of immunoglobulins M, A and G and purification of immunoglobulin M with mixed-mode resins

This study investigated adsorption selectivity of immunoglobulin M (IgM), immunoglobulin A (IgA) and immunoglobulin (IgG) on four mixed-mode resins with the functional ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. IgM purification processes with mixed-mode resins were also proposed. All resins showed typical pH-dependent adsorption, and high adsorption capacity was found at pH 5.0–8.0 with low adsorption capacity under acidic conditions. Meanwhile, high selectivity of IgM/IgA and IgM/IgG was obtained with ABI-4FF and MMI-4FF resins at pH 4.0–5.0, which was used to develop a method for IgM, IgA and IgG separation by controlling loading and elution pH. Capture of monoclonal IgM from cell culture supernatant with ABI-4FF resins was studied and high purity (∼99%) and good recovery (80.8%) were obtained. Moreover, IgM direct separation from human serum with combined two-step chromatography (ABI-4FF and MMI-4FF) was investigated, and IgM purity of 65.2% and a purification factor of 28.3 were obtained after optimization. The antibody activity of IgM was maintained after purification. The results demonstrated that mixed-mode chromatography with specially-designed ligands is a promising way to improve adsorption selectivity and process efficiency of IgM purification from complex feedstock.

Monoclonal antibody fragment removal mediated by mixed mode resins

Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. The mechanism of fragment removal was investigated in two series of experiments. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. The addition of ethylene glycol decreased LHF removal by half. Further decreases in LHF separation were seen upon incubation with urea and arginine. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities are required.

Preparation and evaluation of mixed-mode resins with tryptophan analogues as functional ligands for human serum albumin separation

Abstract Five tryptophan analogues with a hydrophobic indole ring and an amino group on each molecule were used as functional ligands of mixed-mode resins for human serum albumin (HSA) purification. Their adsorption performance was evaluated and the effects of pH and salt addition on HSA adsorption were studied. The resins prepared showed typical pH-dependent adsorption and the highest adsorption capacity and affinity were found at pH 5.0 for all the resins tested. The saturated adsorption capacity was 138.02 mg·g− 1 with the tryptamine-functionalized resin, which significantly decreased at pH below 4.0 due to electrostatic repulsion between ligands and HSA. Moreover, the addition of NaCl or (NH4)2SO4 in media reduced HSA adsorption capacity, although the two salts showed different affecting profiles. The tryptamine-functionalized resin showed the best salt-tolerant performance, and its high adsorption capacity was maintained under high salt concentrations. In addition, the five resins prepared showed good adsorption selectivity for recombinant HSA from Pichia pastoris broth. Molecular docking results between tryptamine and HSA indicated that tryptamine was favorable to bind on Site II (indole-binding site) of HSA.

Selectivity evaluation and separation of human immunoglobulin G, Fab and Fc fragments with mixed-mode resins.

Adsorption selectivity is critical important for mixed-mode chromatography with specially-designed ligands. Human immunoglobulin G (hIgG), Fc and Fab fragments were used in the present work to evaluate adsorption behavior and binding selectivity of four mixed-mode resins with the ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. The resins showed an obvious pH-dependent adsorption behavior. High adsorption capacities were found at neutral pH for hIgG, Fc and Fab, and almost no adsorption happened under acidic conditions. An adsorption selectivity index was proposed to evaluate separation efficiency. High specificity of hIgG/Fc was found at pH 8.9 for MEP resin, and for W-ABI resin at pH 8.0 and 8.9. In addition, isothermal titration calorimetry was used to evaluate ligand-protein interactions. Finally, the separation of hIgG and Fc (1:1) was optimized with mixed-mode resins, and the best separation performance was obtained with W-ABI-based resin. Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4% and recovery of 98.8%, while elution at pH 3.6 provided hIgG with purity of 99.7% and recovery of 86.5%.

Thermodynamic modeling of protein retention in mixed-mode chromatography: An extended model for isocratic and dual gradient elution chromatography

An extended model is developed to describe protein retention in mixed-mode chromatography based on thermodynamic principles. Special features are the incorporation of pH dependence of the ionic interaction on a mixed-mode resin and the addition of a water term into the model which enables one to describe the total number of water molecules released at the hydrophobic interfaces upon protein-ligand binding. Examples are presented on how to determine the model parameters using isocratic elution chromatography. Four mixed-mode anion-exchanger prototype resins with different surface chemistries and ligand densities were tested using isocratic elution of two monoclonal antibodies at different pH values (7–10) and encompassed a wide range of NaCl concentrations (0–5M). U-shape mixed-mode retention curves were observed for all four resins. By taking into account of the deprotonation and protonation of the weak cationic functional groups in these mixed-mode anion-exchanger prototype resins, conditions which favor protein-ligand binding via mixed-mode strong cationic ligands as well as conditions which favor protein-ligand binding via both mixed-mode strong cationic ligands and non-hydrophobic weak cationic ligands were identified. The changes in the retention curves with pH, salt, protein, and ligand can be described very well by the extended model using meaningful thermodynamic parameters like Gibbs energy, number of ionic and hydrophobic interactions, total number of released water molecules as well as modulator interaction constant. Furthermore, the fitted model parameters based on isocratic elution data can also be used to predict protein retention in dual salt-pH gradient elution chromatography.

Static and dynamic binding behavior of an IgG2 monoclonal antibody with several new mixed mode affinity adsorbents

The binding behavior of several mixed-mode chromatographic adsorbents, derived from different terpyridine-based ligands immobilized onto Sepharose FF™, has been investigated with a humanized IgG2 monoclonal antibody. Static adsorption isotherms were determined and the derived parameters used to guide the choice of ligand structure and adsorption conditions to achieve favorable IgG2 mAb binding under dynamic loading conditions. The binding and elution behavior of selected adsorbents in packed chromatographic columns were studied with the purified IgG2 mAb and crude cell culture broths containing the same IgG2 mAb. Clearance of host cell proteins was found to be strongly influenced by the structure of the ligand used to generate these mixed mode resins. One ligand candidate in particular, ES-(Cl.S.Cl)terpy, was found to possess selectivity on a par with the traditionally employed Protein A affinity adsorbents for the purification of monoclonal IgG2s with an excellent level of clearance of host cell proteins. Moreover, high binding capacities, e.g. between 34 and 70 mg IgG2 mAb/mL resin, were achieved with these new adsorbents.

Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases.

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L−1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.

Modeling of dual gradient elution in ion exchange and mixed-mode chromatography

Protein retention using dual gradient elution in ion exchange- and mixed-mode chromatography can be modeled using the combination of a modified Yamamoto's LGE model and a conversion term to correlate the elution salt concentration and pH at any given gradient slope. Incorporation of the pH dependence of the binding charges into the model also provides some insights on the dual effects of salt and pH in protein–ligand interaction. The fitted thermodynamic parameters (ΔGP0/RT, ΔGS0/RT, number of charged amino acids involved in binding) of the dual gradient elution data using lysozyme and mAbs on SP Sepharose® FF, Eshmuno® HCX, and Capto® MMC ImpRes were consistent to the results of mono gradient data. This gives rise to an approach to perform thermodynamic modeling of protein retention in ion exchange- and mixed-mode chromatography by combining both salt and pH gradient into a single run of dual gradient elution which will increase time and cost efficiency. The dual gradients used in this study encompassed a wide range of pH (4–8) and NaCl concentrations (0–1M). Curve fits showed that ΔGP0/RT is protein type and ligand dependent. ΔGS0/RT is strongly dependent on the stationary phase but not the protein. For mAb04 on mixed-mode resin Capto® MMC, ΔGS0/RT is 5–6 times higher than the result reported for the same protein on cation exchanger Fractogel® EMD SO3− (S).

Isoform separation of proteins by mixed-mode chromatography

Mixed-mode chromatography uses a multimodal functional resin, mainly composed of electrostatic and aromatic/hydrophobic groups. Here we have tested 2 mixed-mode resins, anion-exchange Capto adhere and cation-exchange Capto MMC, using 2 model proteins, i.e., an Fc-fusion etanercept, and bovine serum albumin (BSA). When etanercept was produced in Chinese hamster ovary cells, a large amount of misfolded species was generated. A novel technology to achieve effective separation of the misfolded or aggregated species has been developed in this study using these mixed-mode columns and elution conditions that combine pH change and NaCl or arginine at different concentrations. Etanercept, which has been purified by Protein-A chromatography, was bound to the Capto MMC or Capto adhere columns under various conditions and eluted by modulating the pH and salt or arginine concentration. The misfolded species occurred in the fractions at higher salt or arginine concentrations, most likely reflecting stronger electrostatic and hydrophobic interactions of the misfolded species with these mixed-mode resins.Another model protein, BSA, containing several oligomeric species, was also subjected to Capto adhere or Capto MMC chromatography using either NaCl or arginine gradient elution, with a greater recovery by arginine gradient. The oligomers were effectively separated on these mixed-mode columns using either gradient elution, eluting in later fractions similar to etanercept misfolded species.

Protein adsorption and chromatography on novel mixed-mode resins fabricated from butyl-modified poly(ethylenimine)-grafted Sepharose

We have previously studied benzoyl-modified poly(ethylenimine) (PEI)-grafted Sepharose FF resins for mixed-mode chromatography (MMC), and found PEI-grafted resin with an ionic capacity (IC) of about 300mmol/L was suitable for fabricating MMC resins to achieve favorable MMC performance. To extend the application of PEI-grafted resins in MMC, we have herein modified a PEI-grafted resin with an initial IC of 315mmol/L (FF-PEI-L315) with hydrophobic butyl groups, and two MMC resins with residual ICs of 280 and 252mmol/L were obtained (denoted as Bu30-PEI315 and Bu60-PEI315). Bovine serum albumin (BSA) and γ-globulin adsorption at pH 8.0 and elution at pH 3.0 were first explored on the two MMC resins. It was found that with increasing butyl density, protein binding capacity increased but the elution percentage decreased, indicating that high butyl density was beneficial in protein adsorption but not favorable in elution. It was considered that the butyl density of Bu60-PEI315 was so high that the electrostatic repulsion at pH 3.0 was insufficient to make the protein dissociate from the resin surface. Then, the eluent was switched to 3-[(3-cholamido-propyl)-dimethylammonio]-1-propanesulfonate (CHAPS) solution, and complete BSA recovery was obtained on Bu60-PEI315. Complete γ-globulin recovery was obtained on Bu60-PEI315 at a lower CHAPS concentration than that for BSA. This suggests the possibility of selective elution of the two proteins by manipulating eluent concentration. The results indicate that CHAPS was a suitable eluent for protein recovery and separation with the MMC resin, particularly for the purification of antibody from feedstock containing serum albumin. Finally, adsorption equilibria and uptake kinetics onto Bu30-PEI315 and Bu60-PEI315 were investigated. At 0mol/L NaCl, BSA binding on the MMC resins was only by electrostatic interaction while γ-globulin showed a bi-modal binding behavior, including electrostatic and hydrophobic interactions. At high NaCl concentrations, both BSA and γ-globulin were bound by hydrophobic interaction. Favorable binding properties for different proteins in a wide range of NaCl concentrations were observed on Bu60-PEI315, indicating its usefulness as an MMC adsorbent.

Purification process of recombinant monoclonal antibodies with mixed mode chromatography

An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs.