Mixed-mode Chromatography Research Articles

Application of mixed modal resin for purification of a fibrinolytic enzyme

ABSTRACTRecent advances in purification technologies for therapeutic molecules have stirred the research consortium. Mixed mode chromatography, having multiple interactions with the solute molecule, has drawn significant attention due to its overall advantage over traditional ion-exchange and reverse-phase chromatography. Capto adhere, a mixed mode chromatography resin with strong anion-exchange and reverse-phase interaction with solutes, was explored for purification of fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Static and dynamic resin binding study revealed that 30°C temperature, pH 8, and 0.5 mL/min flow rate were optimum for maximum binding of fibrinolytic enzyme. Maximum static dynamic binding and breakthrough capacities for Capto adhere were 249 and 196 U/mL of resin, respectively. Final purification with Sephadex G 100 gel chromatography resulted in 38-fold purity of fibrinolytic enzyme with 39% enzyme recovery. Purified enzyme was further characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis to homogeneity, and molecular mass was found to be around 55–70 kD. Like most of the serine alkaline proteases, purified fibrinolytic enzyme was stable in a temperature range of 25–40°C and pH range of 7–9. Offshoots of our research findings have revealed a broad application area of mixed mode chromatography.

Imidazolium embedded C8 based stationary phase for simultaneous reversed-phase/hydrophilic interaction mixed-mode chromatography

A new imidazolium embedded C8 based stationary phase (SIL-MPS-VOL) was facilely prepared by two steps and characterized by Fourier transform infrared spectrometry and thermogravimetric analysis. Due to the introduction of quaternary imidazolium group to the traditional C8 stationary phase, the developed SIL-MPS-VOL column demonstrated both reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) retention mechanisms. A series of hydrophobic and hydrophilic test samples, including benzene homologues, anilines, positional isomers, nucleosides and nucleotides, were used to evaluate the developed SIL-MPS-VOL stationary phase. A rapid separation time, high separation efficiency and planar selectivity were achieved, compared with the commercially available C8 column. Moreover, the developed stationary phase was further used to detect and separate of melamine in powdered infant formula and high polar component of secondary metabolites of Trichoderma, and improved separation efficiency was achieved, indicating the potential merits of the developed SIL-MPS-VOL stationary phase for simultaneous separation of complex hydrophobic and hydrophilic samples with high selectivity.

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.

Isolation of ribosomes by chromatography.

Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g., heparin, which is used to inhibit RNases in yeast). A robust protocol for Escherichia coli is given here as an example.

An Eco-Friendly Direct Injection HPLC Method for Methyldopa Determination in Serum by Mixed-Mode Chromatography Using a Single Protein-Coated Column.

A simple, rapid and environment-friendly direct injection HPLC method for the determination of methyldopa (MTD) in human serum has been developed and validated. The method was based on cleanup and separation of MTD from serum by mixed-mode liquid chromatography using a single protein-coated TSK gel ODS-80 TM analytical column (50 × 4.0 mm i.d., 5 µm). The protein-coated column exhibited excellent resolution, selectivity and functioned in two chromatographic modes: size-exclusion chromatography [i.e., solid-phase extraction (SPE) for serum proteins] and reversed-phase chromatography for the final separation of MTD. SPE and HPLC separation were carried out simultaneously with a green mobile phase consisting of acetate buffer (0.1 M, pH 2.4) at a flow rate of 1 mL/min and at room temperature (23 ± 1°C). The eluent was monitored at emission and excitation wavelengths of 320 and 270 nm, respectively. A calibration curve was linear over the range of 0.1-30 µg/mL with a detection limit of 0.027 µg/mL. This online SPE method was successfully applied to real samples obtained from patients receiving MTD therapy.

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.

A new anion-exchange/hydrophobic monolith as stationary phase for nano liquid chromatography of small organic molecules and inorganic anions

In this study, an anion-exchange/hydrophobic polymethacrylate-based stationary phase was prepared for nano-liquid chromatography of small organic molecules and inorganic anions. The stationary phase was synthesized by in situ polymerization of 3-chloro-2-hydroxypropylmethacrylate and ethylene dimethacrylate inside silanized 100μm i.d. fused silica capillary. The porogen mixture consisted of toluene and dodecanol. The pore size distrubution profiles of the resulting monolith were determined by mercury intrusion porosimetry and the morphology of the prepared monolith was investigated by scanning electron microscope. Good permeability, stability and column efficiency were observed on the monolithic column with nano flow. The produced monolithic column, which contains reactive chloro groups, was then modified by reaction with N,N-dimethyl-N-dodecylamine to obtain an anion-exchange/hydrophobic monolithic stationary phase. The functionalized monolith contained ionizable amine groups and hydrophobic groups that are useful of anion-exchange/hydrophobic mixed-mode chromatography. The final monolithic column performance with respect to anion-exchange and hydrophobic interactions was assesed by the separation of alkylbenzene derivatives, phenolic compounds and inorganic anions, respectively. Theoretical plate numbers up to 23,000 plates/m were successfully achieved in the separation of inorganic anions.

Development and scale-up of the recovery and purification of a domain antibody Fc fusion protein-comparison of a two and three-step approach.

A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc-fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale-up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two-step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB-Fc fusion protein. A two-step process was compared to a three-step process. The two-step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three-step process. The three-step process was implemented for manufacture of clinical material to mitigate risk.

Application of HPLC mixed-mode chromatography in determining radiochemical purity of [(14) C] labeled metformin hydrochloride.

Metformin is currently prescribed worldwide to treat type 2 diabetes, and therefore, radiolabeled [(14) C] metformin is often prepared for clinical comparisons of new drug candidates. Prior to using the radiolabeled metformin, the purity needs to be determined to ensure the quality of the material. While typical reversed-phase LC methods are often the first choice for purity analysis, they are not suitable for this determination because the compound is poorly retained under these conditions. Mixed-mode chromatography has been demonstrated to overcome these retention issues, and therefore, this methodology was utilized for the purity determination of radiolabeled metformin.

Purification of rabbit polyclonal immunoglobulin G with ammonium sulphate precipitation and mixed-mode chromatography

Immunoglobulins G (IgG) against hepatitis B core antigen (HBcAg) was successfully purified using a purification scheme comprising ammonium sulphate precipitation and SepFast™ MM AH-1 column chromatography. Ammonium sulphate precipitation performed at 40% saturation was optimum in terms of the recovered polyclonal IgG concentration (7.8mg/ml) and the removal of albumin (72%). The yield, purity and purification factor achieved from this simple purification method were 99%, 94% and 7.8, respectively. The IgG recovered from ammonium sulphate precipitation was subjected to SepFast™ MM AH-1 column chromatography and the purity of IgG was further increased to 98%, corresponding to a purification factor of 8.1. Protein aggregation was also reduced significantly in the purified IgG sample. Furthermore, the salt content in the purified sample was reduced by 75% and therefore the need of desalting final product was eliminated. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of anti-HBcAg IgG obtained after these purification processes was maintained.

Preparation of a weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography stationary phase for protein separation using click chemistry.

In this study, 3-diethylamino-1-propyne was covalently bonded to the azide-silica by a click reaction to obtain a novel dual-function mixed-mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high-salt-concentration mobile phase and weak anion exchange character in a low-salt-concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed-mode chromatography stationary phase, a new off-line two-dimensional liquid chromatography technology using only a single dual-function mixed-mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.

Purification of monoclonal antibodies by chemical affinity mixed mode chromatography

The application of several pyridine-based compounds as immobilised ligands has been investigated for the purification of monoclonal antibodies via mixed mode chromatography. The ligands employed were 4′-terpyridinysulfanylethylamine (4′-TerPSEA), 5-bromo-2-pyridinylsulfanylethylamine (5-Br-2-PSEA), 2-quinolinylsulfanylethylamine (2-QSEA) and 4-pyridinylsulfanylethylamine (4-PSEA). The performance attributes of adsorbents, derived from the immobilisation of these different ligands onto Sepharose 6 Fast Flow™, was evaluated from batch adsorption studies and from chromatographic experiments with humanised IgG1, IgG2 and IgG4 monoclonal antibodies produced by stable CHO cell lines cultured in chemical defined media. These results demonstrated that monoclonal antibodies of different subclasses can be efficiently purified from crude CHO cell culture supernatants using these new chemical affinity chromatographic systems. Moreover, the majority of the CHO host proteins could be eliminated during the chromatographic purification step with these resins, as monitored by a specific ELISA assay.

State of the art of mixed-mode stationary phase

With the development of science and technology, more and more complex samples like peptides and proteins need to be separated. It is difficult to separate them by single mode chromatography. Due to the unique separation character of mixed-mode chromatography, the same separation performance can be obtained in mixed-mode chromatography in one separation as multidimensional chromatography. And some disadvantages of multidimensional chromatography can be avoided in mixed-mode chromatography, such as the complexity of the system, the poor compatibility of mobile phases and the long analytical time. More and more attention is devoted to the mixed-mode chromatography in recent years. The focus on the mixed-mode chromatography is to design new structures of mixed-mode stationary phase. At present, mixed-mode stationary phases included reversed-phase/ion-exchange, reversed-phase/hydrophilic, hydrophilic/ion-exchange, zwitterionic and trimode mixed-mode stationary phases. The kinds of mixed-mode stationary phases are summarized and their features and applications in different fields are discussed in this review.

Application of mixed-mode ultra high performance liquid chromatography to forensic drug analysis

Methodology is presented for the rapid analysis of drugs of abuse using a single UHPLC column with orthogonal separation methods using different combinations of the same solutions in the solvent reservoir.

Preparation of a novel weak cation exchange/hydrophobic interaction chromatography dual-function polymer-based stationary phase for protein separation using "thiol-ene click chemistry".

A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) microspheres was synthesized by thiol-ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96% can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2%, 92.4%, and 2.8 × 10(7) IU/mg, respectively, in IEC mode, and 83.4%, 95.2%, and 4.3 × 10(7) IU/mg, respectively, in HIC mode. The results indicate that the dual-function mixed-mode stationary phase prepared in this study may aid in the development of new two-dimensional liquid chromatography technology with a single column for intensive proteomic studies, and it may also find applications in recombinant protein drug production since it can save column costs and simplify the biotechnological processes.

Poly(4-vinylpyridine): a polymeric ligand for mixed-mode protein chromatography

Poly(4-vinylpyridine) (P4VP) was proposed for use as a polymeric ligand of mixed-mode chromatography (MMC) of proteins. P4VP has linear hydrophobic chains with ionizable pyridyl groups in its backbone. The polymer was coupled onto Sepharose FF gel at a pyridyl group density of 190μmol/mL (FF-P4VP-190) by the substitution reaction of pyridyl amines with brominated Sepharose gel. Thereby the immobilized ligand possesses the intrinsic hydrophobic nature as well as the newly obtained electrostatic interaction properties endowed from the substituted positively charged pyridyl amines. The pore size distribution was measured by inverse size exclusion chromatography, and the results revealed that P4VP formed a three-dimensional layer on the matrix surface with a maximum layer depth of 4.2nm. The adsorption isotherms of γ-globulin and bovine serum albumin (BSA) to FF-P4VP-190 were determined under varying pH values and salt concentrations to provide insights into the adsorption properties of the medium. It was found that the adsorption capacity of γ-globulin and BSA both presented an increase with pH increasing from 8.0 to 9.0. Moreover, FF-P4VP-190 exhibited stronger adsorption for BSA than γ-globulin. The higher affinity for BSA might be attributed to its more net negative charges. Protein adsorption capacities to FF-P4VP-190 decreased with increasing NaCl concentration, but still manifested moderate levels at high salt concentration such as 75mg/mL for γ-globulin and 14mg/mL for BSA at 0.5mol/L NaCl and pH 9.0. The capacity decreases with increasing ionic strength, indicating the dominant role of electrostatic interactions, while the moderate capacity values at 0.5mol/L NaCl confirmed the presence of salt-tolerant feature of FF-P4VP-190, making it function as an MMC material. Column chromatography was conducted to investigate protein elution behavior. Efficient protein recovery was achieved at mild elution conditions such as pH 4.0 for γ-globulin and pH 4.5 for BSA. The results indicate that the P4VP-based adsorbent would provide new possibilities for protein purification by MMC.

Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli

Prokaryotic production systems have been widely used to manufacture recombinant therapeutic proteins. Economically, the prokaryotic production – especially of small therapeutic molecules – is advantageous compared to eukaryotic production strategies. However, due to the potential endotoxin and host cell protein contamination, the requirements for the purification process are disproportionately higher and therefore more expensive and elaborate to circumvent. For this reason, the goal of this work was to develop and establish a rapid, simple, inexpensive and ‘up-scalable’ production and purification process, using the therapeutic relevant protein anti-EGFR scFv hu225 as model molecule. Configuring high cell density cultivation of Escherichia coli – using the rha-BAD expression system as production platform – a specific product concentration up to 20mgscFv/gCDW was obtained. By combining freeze-and-thaw, osmotic shock and pH induced host cell protein precipitation, almost 70% of the product was extracted from the biomass. In a novel approach a mixed mode chromatography was implemented as a capturing and desalting step, which allowed the direct application of further ion exchange chromatography steps for purification up to pharmaceutical grade. Thereby, 50% of the produced scFv could be purified within 10h while maintaining the biological activity.

Application of 4′-terpyridinylsulfanylethylamine resins for the purification of monoclonal antibodies by mixed-mode chromatography

In this study, a pyridine-based compound, 4′-terpyridinylsulfanylethylamine (4′-TerPSEA), has been employed as a ligand to purify via mixed-mode chromatographic procedures a humanised monoclonal antibody of the IgG1 sub-class directly from crude supernatants derived from cultured CHO cells. The antibody binding capacity, selectivity and reusability of the adsorbent, derived from the immobilisation of this ligand onto Sepharose FF™, were compared to a Protein A affinity resin. The chromatographic performance of this mixed mode adsorbent was similar to that shown by the Protein A-based adsorbent with this IgG1 mAb. In addition, the IgG1 mAb was able to bind to the immobilised 4′-TerPSEA under reducing conditions. Through the use of papain-digested IgG1 mAb, fractionated with both the 4′-TerPSEA and Protein A adsorbents, it was found that this IgG1 mAb preferentially bound to the immobilised 4′-TerPSEA Sepharose FF™ resin through its Fc region.

Protein adsorption behavior and immunoglobulin separation with a mixed‐mode resin based on p‐aminohippuric acid

p-Aminohippuric acid is a newly developed ligand for mixed-mode chromatography with a commercial resin name of Nuvia cPrime. In this study, bovine immunoglobulin G and bovine serum albumin were used as two model proteins, and the adsorption isotherms with Nuvia cPrime were investigated under different pH and salt concentrations. The results showed that pH had a strong but different influence on the adsorption of these two proteins. The adsorption capacity for bovine immunoglobulin G and BSA was 170.4 and 28.1 mg/g at pH 6.0, respectively. Different salts also showed varying effects on the protein adsorption. Moreover, the adsorption and elution behaviors of the two proteins in a column were determined under varying pH and salt concentrations. An optimized process showed that feedstock loaded under pH 6.0 with 0.8 M (NH4)2SO4 and eluted under pH 8.0 with 1.0 M NaCl could effectively purify bovine immunoglobulin G from feedstock containing BSA. The purity of bovine immunoglobulin G could reach 99.8% and the recovery was 92.7%. The results demonstrated that the control of pH and salt addition during the loading and elution processes were two key factors in improving separation efficiency with Nuvia cPrime resin.

A novel surface-confined glucaminium-based ionic liquid stationary phase for hydrophilic interaction/anion-exchange mixed-mode chromatography

Glucaminium-based ionic liquids are a new class of recently developed ionic liquids and prepared by functionalizing the amine group of N-methyl-d-glucamine, which renders them good hydrophilicity due to the presence of the glucose structure and charged quaternary ammonium group. In the present study, a glucaminium-based ionic liquid N,N-diallyl-N-methyl-d-glucaminium bromide was synthesized and subsequently bonded to the surface of 3-mercaptopropyl modified silica materials through “thiol-ene” click chemistry. The obtained stationary phase was characterized by elemental analysis and infrared spectroscopy, and then packed as a HPLC column. A mixture of five nucleosides was used to characterize the separation performance of the obtained column under HILIC mode and the column efficiency was determined with cytidine as the test solute, reaching 80,000plates/m. Then, the retention behavior was evaluated by investigating the effect of various chromatographic factors on retention of different types of solutes, and the results revealed that the developed surface-confined glucaminium-based ionic liquid stationary phase exhibited a hydrophilic interaction/anion-exchange mixed-mode retention mechanism. Finally, two mixtures of nucleotides and flavonoids were separated on the glucaminium-based ionic liquid column, respectively under hydrophilic interaction and hydrophilic interaction/anion-exchange mixed-mode chromatography. In conclusion, the multimodal retention capabilities of the glucaminium-based ionic liquid column could offer a wider range of retention behavior and flexible selectivity toward polar and hydrophilic compounds.