Linear flow-velocity gradient chromatography-An efficient method for increasing the process efficiency of batch and continuous capture chromatography of proteins.

A new method was proposed for increasing the capture chromatography process efficiency, linear flow-velocity gradient (LFG). The method uses a linear decreasing flow-velocity gradient with time during the sample loading. The initial flow velocity, the final flow velocity and the gradient time are the parameters to be tuned. We have developed a method for determining these parameters by using the total column capacity and the total loaded amount as a function of time. The capacity can be calculated by using the relationships between dynamic binding capacity (DBC) and residence time. By leveraging the capacity, loading amount, and the required conditions, the optimum LFG can be designed. The method was verified by ion-exchange and protein A chromatography of monoclonal antibodies (mAbs). A two-fold increase in the productivity during the sample loading was possible by LFG compared with the constant flow-velocity (CF) operation. LFG was also applied to a 4-column continuous process. The simulation showed that the cost of resin per unit amount of processed mAbs can be reduced by 13% while 1.4 times enhancement in productivity was preserved after optimization by LFG compared to CF. The process efficiency improvement is more pronounced when the isotherm is highly favorable and the loading volume is large.

Microcalorimetric Analysis of the Adsorption of Lysozyme and Cytochrome c onto Cation-Exchange Chromatography Resins: Influence of Temperature on Retention.

We studied the adsorption mechanism of two basic proteins, equine cytochrome c (Cyt) and chicken egg-white lysozyme (Lys), adsorbing onto negatively charged chromatography surfaces. In liquid chromatography, the retention volume of Lys was larger than that of Cyt on negatively charged ion-exchange resins. When the temperature increased, the retention volume of Cyt increased, whereas that of Lys clearly decreased. Both Lys and Cyt share similar physical characteristics, so the opposite behavior with increasing temperatures was surprising, indicating a more complex mechanism of adsorption may be involved. We analyzed the adsorption of these proteins by using isothermal titration calorimetry (ITC). The change in adsorption enthalpy determined by ITC allowed the understanding of the reason for and underlying driving forces of protein adsorption that resulted in this opposite behavior. Large exothermic enthalpies of adsorption were observed for Lys (-43.95 kJ/mol), and Lys adsorption was found to be enthalpically driven. On the other hand, endothermic enthalpies were dominant for Cyt adsorption (32.41 kJ/mol), which was entropically driven. These results indicate that dehydration and release of counterions play a more important role in Cyt adsorption and ionic interaction and hydrogen bridges are more significant in Lys adsorption. Understanding of the adsorption mechanism of proteins onto chromatography resins is essential for modeling and developing new, efficient chromatographic processes.

Effect of pore size on performance of monolithic tube chromatography of large biomolecules.

Effect of pore size on the performance of ion-exchange monolith tube chromatography of large biomolecules was investigated. Radial flow 1 mL polymer based monolith tubes of different pore sizes (1.5, 2, and 6 µm) were tested with model samples such as 20 mer poly T-DNA, basic proteins, and acidic proteins (molecular weight 14 000-670 000). Pressure drop, pH transient, the number of binding site, dynamic binding capacity, and peak width were examined. Pressure drop-flow rate curves and dynamic binding capacity values were well correlated with the nominal pore size. While duration of the pH transient curves depends on the pore size, it was found that pH duration normalized on estimated surface area was constant, indicating that the ligand density is the same. This was also confirmed by the constant number of binding site values being independent of pore size. The peak width values were similar to those for axial flow monolith chromatography. These results showed that it is easy to scale up axial flow monolith chromatography to radial flow monolith tube chromatography by choosing the right pore size in terms of the pressure drop and capacity.

A simple method for calculating the productivity of polyphenol separations by polymer-based chromatography.

A simple method for calculating the productivity of chromatography processes was proposed based on the iso-resolution curve concept. The model separation system was polyphenol separations by polystyrene divinylbenzene resins with the ethanol-water mixture mobile phase. The distribution coefficient K was determined as a function of ethanol concentration I by linear gradient elution experiments. The HETP-mobile phase velocity u curves were determined as a function of I. Using K and HETP, the iso-resolution curve was calculated, from which the productivity was determined as a function of I. It was found that there is an optimum I, where the highest productivity with the minimum amount of mobile phase consumption is obtained.

Liposome-Papain Conjugates for Catalytic Digestion of Antibody Producing Fab Fragments.

Papain is useful for the enzymatic digestion of various proteins to produce functional peptides or protein fragments. Immobilized papain being reactive toward proteins and easily removable from a reaction mixture is worth developed. In the present work, liposomes were applied as colloidal carriers of papain for the catalytic digestion of polyclonal immunoglobulin G (IgG). Papain was covalently conjugated at pH = 7.0 via tris-succinimidyl aminotriacetate (TSAT) to liposomes incorporated with 5 mol % poly(ethylene glycol)-tethered lipid with a reactive amino group. The papain-conjugated liposome (liposome-papain) catalyzed the hydrolysis of Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride (BAPNA) at pH = 5.0-7.0. The activity of liposome-papain significantly increased with increasing temperature from 25 to 50 °C. The Michaelis constant Km was determined with respect to the liposome-papain- and free papain-catalyzed reactions with BAPNA at 37 °C as Km = 1.11 ± 0.13 and 11.6 ± 2.9 mM, respectively. Liposome-papain was applied to the catalytic digestion of 10 mg·mL-1 IgG at 37 °C for 24 h at pH = 5.0-7.0. The reaction mixture could be analyzed without pretreatment by using the affinity columns immobilized with the protein A or protein L ligand because colloidal liposome-papain quickly flowed through the chromatographic stationary phase, exhibiting little proteolytic effect on the proteinaceous ligands. The analysis clearly demonstrated the catalytic production of antigen-binding fragments (Fab) from IgG in an enzyme concentration- and pH-dependent manner. Liposome-papain with 15 or 50 mol % anionic lipids also catalyzed the formation of Fab from IgG. The above results demonstrated that liposome-papain was useful to digest IgG and to purify Fab formed with the affinity chromatography.

Measurement of High Protein Concentrations by Optical Rotation: A Case Study for Monitoring of Monoclonal Antibody Drug Downstream Processes.

BACKGROUND: Recent advancements in cell engineering and bioreactor engineering have enabled high monoclonal antibody (mAb) concentrations in harvested solutions for the downstream process (DSP).

Methods: As many unit operations such as capture chromatography, polish chromatography, membrane filtration, virus inactivation, virus filtration, and concentration by ultrafiltration are involved in DSP, it is crucial to monitor the process carefully in order to perform reliable and stable DSP operations. One of the most important signals (process parameter) to be monitored is the protein concentration CP. Although various methods are available, most of them are not suited for measuring high CP. In this paper, we have developed a method for measuring very high CP by optical rotation (OR).

Result: Linear correlations were confirmed between OR and Cp in the range CP = 0 to 80 g/L for mAbs with high repeatability and small variation coefficients. This method was applied to the monitoring of CP in the opaque (colored) solution during the cell culture. The CP by OR was in good agreement with those by the standard Protein A HPLC method.

Conclusion: Monitoring of high CP by OR is expected to be an efficient process analytical tool (PAT) for DSP.

A regressive approach to the design of continuous capture process with multi-column chromatography for monoclonal antibodies.

Although empirical methods have been introduced in the process development of continuous chromatography, the common approach to optimize a multi-column continuous capture chromatography (periodic counter-current chromatography, PCCC) process heavily relies on numerical model simulations and the number of experiments. In addition, different multi-column settings in PCCC add more design variables in process development. In this study, we have developed a rational method for designing PCCC processes based on iterative calculations by mechanistic model-based simulations. Breakthrough curves of a monoclonal antibody were measured at different residence times for three protein A resins of different particle sizes and capacities to obtain the parameters needed for the simulation. Numerical calculations were performed for the protein sample concentration in the range of 1.5 to 4 g/L. Regression curves were developed to describe the relative process performances compared with batch operation, including the resin capacity utilization and the buffer consumption. Another linear correlation was established between breakthrough cut-off (BT%) and a modified group composed of residence time, mass transfer coefficient, and particle size. By normalizing BT% with binding capacity and switching time, the linear regression curves were established for the three protein A resins, which are useful for the design and optimization of PCCC to reduce the process development time.

Rapid Purification of Immunoglobulin G Using a Protein A-​immobilized Monolithic Spin Column with Hydrophilic Polymers.

A rapid purification method was developed for antibody production in Chinese hamster ovary (CHO) cells using a Protein A-immobilized monolithic silica spin column with hydrophilic polymers. Monolithic silica modified with copolymers of 2-hydroxyethylmethacrylate (HEMA) and glycidyl methacrylate (GMA) showed lower non-specific protein absorption than that modified with a silane reagent. The epoxy group of GMA was converted to an amino group, and Protein A was modified by the coupling reagent. The amount of immobilized Protein A was controlled by changing the ratio of GMA to HEMA and the mesopore size of monolith. A modified monolith disk was fixed to a spin column for rapid antibody purification. The linear curves (for the antibody concentrations over 10 - 300 µg/mL) had a correlation coefficient of >0.999. Our column had various analytical advantages over previously reported columns, including a shorter preparation time (<10 min) and smaller sample volumes for purification with Protein A-immobilized agarose.

Correlation between protein desorption behavior and its adsorption enthalpy change in polymer grafted anion exchange chromatography.

Thermodynamic studies on protein adsorption onto chromatographic surfaces mainly focus on the molecular level interaction between proteins and ligands. Yet, not much attention is given to the study of polymer grafted ligand architecture effect on thermodynamic parameters, nor to the relation between chromatographic parameters and the directly obtained thermodynamic parameters. These relations are needed in order to confer meaning and to ease future data interpretation of thermodynamic studies of protein adsorption. In this study, the adsorption of bovine serum albumin monomer (BSAm) onto chromatographic surfaces with grafted ligands was studied from a thermodynamic point of view together with chromatographic data. Isothermal titration calorimetry (ITC) results showed that BSAm adsorption is exothermic (ΔH¯ads < 0) when adsorbs onto Toyopearl GigaCapQ 650 M, Toyopearl Q600AR, and Q Sepharose XL, but endothermic (ΔH¯ads > 0) when adsorbs onto Toyopearl SuperQ and a conventional resin (Q Sepharose Fast Flow), showing clear differences in the driving forces of adsorption caused by different ligand architectures. In addition, we found a new relation between the salt required for protein elution and the change in adsorption enthalpy (ΔH¯ads) directly measured with ITC, intrinsically connecting both adsorption and desorption mechanisms.

Retention and diffusion characteristics of oligonucleotides in a solid phase with polymer grafted anion-exchanger.

In the chromatographic separation process of oligonucleotides (ONs), mechanistic understanding of their binding and diffusion processes is of significant importance to determine operating conditions in a fast and robust way. In this work, we determined the number of binding sites and the diffusivities of ONs in a polymer grafted anion exchange chromatography through linear gradient experiments (LGE) being carried out at selected four to five gradient slopes. Synthetic poly (T)s with length ranging from 3 to 90-mer were employed as a model of an antisense oligonucleotide with typical lengths of 10 - 30 bases. Comparison of the retention was also conducted between the grafted anion exchanger with a conventional ligand and an anion monolith disk. For the ONs up to 50 bases, the number of binding sites determined can be correlated with the length of ONs, and the grafted resin showed a better diffusion and narrower peak width compared to the nongrafted one. The retention behavior became similar for porous media when the longer ONs (> 50mer) were applied. The results obtained suggest that antisense ONs can be separated with grafted ligands without sacrificing mass transfer properties.

Retention Mechanism of Proteins in Hydroxyapatite Chromatography - Multimodal Interaction Based Protein Separations: A Model Study.

BACKGROUND: Retention mechanism of proteins in hydroxyapatite chromatography (HAC) was investigated by linear gradient elution experiments (LGE). MATERIALS AND METHODS: Several mobile phase (buffer) solution strategies and solutes were evaluated in order to probe the relative contributions of two adsorption sites of hydroxyapatite (HA) particles, C-site due to Ca (metal affinity) and P-site due to PO4 (cation-exchange). When P-site was blocked, two basic proteins, lysozyme (Lys) and ribonuclease A(RNase), were not retained whereas cytochrome C(Cyt C) and lactoferrin (LF) were retained and also retention of acidic proteins became stronger as the repulsion due to P-site was eliminated. The number of the binding site B values determined from LGE also increased, which also showed reduction of repulsion forces. CONCLUSION: The selectivity (retention) of four basic proteins (RNase, Lys, Cyt C, LF) in HAC was different from that in ion-exchange chromatography. Moreover, it was possible to tune the selectivity by using NaCl gradient.

Thermodynamic analysis of polyphenols retention in polymer resin chromatography by van't Hoff plot and isothermal titration calorimetry.

Temperature effect on the adsorption enthalpy of polyphenols was analyzed with van't Hoff plots using the distribution coefficient, K, determined with isocratic and gradient elution chromatography. Catechin and epigallocatechin gallate (EGCG) were used as model polyphenols. The stationary phase was polystyrene-divinylbenzene (PS-DVB) resin particles and the mobile phase was an ethanol-water mixture. The values of adsorption enthalpy determined by chromatography and isothermal titration calorimetry were obtained in the temperature range of 283 and 318 K. The results obtained by van't Hoff plots were consistent with the ones obtained with the isothermal titration calorimetry (ITC). The interaction between PS-DVB particles and the polyphenols was found to be exothermic with negative values of enthalpy, -30.1 and -37.7 kJ/mol for catechin and EGCG, respectively.

Reaction-Mediated Desorption of Macromolecules: Novel Phenomenon Enabling Simultaneous Reaction and Separation.

Combining chemical reaction with separation offers several advantages. In this work possibility to induce spontaneous desorption of adsorbed macromolecules, once being PEGylated, through adjustment of the reagent composition is investigated. Bovine serum albumin (BSA) and activated oligonucleotide, 9T, are used as the test molecules and 20 kDa linear activated PEG is used for their PEGylation. BSA solid-phase PEGylation is performed on Q Sepharose HP. Distribution coefficient of BSA and PEG-BSA as a function of NaCl is determined using linear gradient elution (LGE) experiments and Yamamoto model. According to the distribution coefficient the selectivity between BSA and PEG - BSA of around 15 is adjusted by using NaCl. Spontaneous desorption of PEG - BSA is detected with no presence of BSA. However, due to a rather low selectivity, also desorption of BSA occurred at high elution volume. A similar procedure is applied for activated 9T oligonucleotide, this time using monolithic CIM QA disk monolithic column for adsorption. Selectivity of over 2000 is obtained by proper adjustment of PEG reagent composition. High selectivity enables spontaneous desorption of PEG-9T without any desorption of activated 9T. Both experiments demonstrates that reaction-mediated desorption of macromolecules is possible when the reaction conditions are properly tuned.

Effects of protein conformational changes on separation performance in electrostatic interaction chromatography: unfolded proteins and PEGylated proteins.

As it is important to understand how protein conformational changes affect the separation performance in ion exchange chromatography (IEC), we investigated two model systems, unfolded proteins (lysozyme and bovine serum albumin) with urea and dithiothreitol, and PEGylated proteins (lysozyme attached with polyethyleneglycol molecular weight 5000). Linear gradient elution IEC experiments were carried out and the data were analysed by our model previously presented in order to obtain the binding site value B and the peak salt concentration I(R). Unfolded proteins (bovine serum albumin and lysozyme) with urea and dithiothreitol showed weaker retention and larger binding site values compared with the values for native proteins. Multiple PEGylated lysozyme peaks were separated, and eluted earlier than the native peak appeared. There is a good correlation between B and I(R) for PEGylated lysozymes.

Choosing the right protein A affinity chromatography media can remove aggregates efficiently.

Protein A chromatography (PAC) is commonly used as an efficient capture step in monoclonal antibody (mAb) separation processes. Usually dynamic binding capacity is used for choosing the right PAC. However, if aggregates can be efficiently removed during elution, it can make the following polishing steps easier. In this study a method for choosing the right PAC media in terms of mAb aggregate removal is proposed. Linear pH gradient elution experiments of two different mAbs on various PAC columns are carried out, where the elution behavior of aggregates as well as the monomer is measured. Aggregates of one mAb are more strongly retained compared with the mAb monomer. Another mAb showed different elution behavior, where the aggregates are eluted as both the weakly and strongly retained peaks. In order to remove the two types of aggregates by stepwise elution two protocols are tested. The first protocol A consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. No detectable peaks are observed during the wash stage of protocol A whereas significant peaks are monitored during the arginine wash of protocol B. One of the PAC columns showed a smaller peak during the arginine wash. In addition, both aggregate removal and monomer yield are higher with protocol B compared with the other PAC columns. This method is found to be useful for choosing the right PAC column.

Monolith disk chromatography separates PEGylated protein positional isoforms within minutes at low pressure.

Although PEGylation makes proteins drugs more effective, the PEGylation reaction must be controlled carefully in order to obtain a desired PEGylated protein form since various different PEGylated forms may be produced during the reaction. For monitoring the PEGylation reaction, a method with monolith disk ion exchange chromatography, which can separate positional isomers as well as PEGmers, has been developed as a process analytical tool (PAT). The method was optimized for separation of randomly PEGylated protein (lysozyme) isoforms based on the number of resolved peaks, peak resolution, analysis time and pressure drop. In order to increase the retention of mono- and di-PEGylated protein isomers the mobile phase was decreased to pH 4.5, where a large number of mono- and di-PEGylated isomers were resolved within a few minutes. Based on the linear gradient elution optimization model, the following values were determined: gradient slope 0.016 M/mL, disk thickness 3 mm (single disk) and flow rate 10 mL/min. Under these optimal conditions, the analysis was completed within ca. 4 min while the pressure drop was below 1 MPa. As the method was successfully applied to monitoring mono and di-PEGylated positional isoforms in the reaction mixture of random PEGylation of lysozyme, it is expected to be an efficient PAT tool.

Liposome-assisted activity of superoxide dismutase under oxidative stress.

A biological membrane is the front line of defense for cells against various environmental stresses such as heat and reactive oxygen species (ROS) and is expected to play an important role in the antioxidant system with antioxidant enzymes, similarly to its chaperone-like function in cooperation with heat shock proteins. The oxidative stress response of superoxide dismutase (SOD), which is known to catalyze the dismutation of O(2)(-) to H(2)O(2), was investigated in the presence of artificial membranes, liposomes, in order to obtain fundamental information on the biological ROS scavenging system. SOD lost its activity in the presence of H(2)O(2) and was found to have two loops including one which contains an alpha-helix which presents the substrate O(2)(-) to the activity center of SOD (Cu(II)). From circular dichroism analysis of SOD in the presence of H(2)O(2), the contents of the alpha-helix in SOD were found to decrease in correspondence with the inactivation and conformational change of SOD, suggesting that the conformation of the alpha-helix loops affects SOD activity. In the presence of liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), SOD was not inactivated in the presence of H(2)O(2) although the contents of its alpha-helix structure were decreased. The oxidized SOD was found to interact with the liposome surface under oxidative stress using dielectric dispersion analysis. Based on these results, a possible mechanism of SOD protection against ROS on liposomes was presented.

Oxidation of cholesterol catalyzed by amyloid beta-peptide (Abeta)-Cu complex on lipid membrane.

A catalytic reaction of H2O2 production by an amyloid beta-peptide (Abeta)-Cu complex with cholesterol incorporated in a liposome was kinetically analyzed. The Michaelis-Menten model was applied to the H2O2 production reaction using cholesterol as the substrate catalyzed by the Abeta-Cu complex. The Km value for the Abeta-Cu complex catalytic reaction with cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes (Km=0.436 microM for Abeta(1-40); Km=0.641 microM for Abeta(1-42)) was found to be smaller than that with cholesterol-containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes (Km=0.585 microM for Abeta(1-40), Km=0.890 microM for Abeta(1-42)). The results imply that membrane properties could play an important role in the interactions of the Abeta-Cu complex with cholesterol in these liposomes. Considering the physical states of the cholesterol/POPC (liquid disordered phase) and cholesterol/DPPC (liquid ordered phase) liposomes in the present reaction conditions, the data obtained suggests that the H2O2-generating activity of the Abeta-Cu complex, accompanied by oxidation of membrane-incorporated cholesterol, could be effected by the phase of the liposome membranes.

Salt tolerant chromatography provides salt tolerance and a better selectivity for protein monomer separations.

Salt tolerant chromatography (STC) is an attractive method as buffer exchange during protein purification processes can be skipped; however, the retention and separation mechanism of such STC are still not fully understood. We carried out linear gradient elution (LGE) experiments of bovine serum albumin (BSA) including its dimer form by using poly-amine ligand STC. The peak salt concentration IR was measured as a function of normalized gradient slope GH, and the number of binding sites B was determined. The separation performance of monomer and dimer was much higher for STC. The IR values of BSA monomer and dimer for STC were much higher (IR > 0.5M) than those for conventional IEC. The IR values of arginine-Cl gradient decreased markedly compared to those of NaCl gradient whereas they did not change for conventional IEC. This might be due to combined effects of electrostatic and hydrophobic interaction to the retention of proteins in STC. Adding polyethylene glycol (PEG) into the mobile phase of IEC also increased the retention (salt tolerance) and the resolution of BSA monomer and dimer. Higher viscosity and low solubility of proteins due to PEG were disadvantages of this method. STC with poly-amine ligand might be also suited for the continuous flow-through separation of monomer.

Relationship between the mobility of phosphocholine headgroup and the protein-liposome interaction: a dielectric spectroscopic study.

Proteins could affect the headgroup mobility of phospholipid within liposome membranes through the protein-liposome interaction. The variation of headgroup mobility of phospholipid was then investigated by using the dielectric dispersion analysis. The eight proteins (Mw = 4.2-28.7 kDa) were used to investigate the protein-liposome interaction. It has been revealed that the strength of the protein-liposome interaction at 25 °C was linearly correlated with the stability of intramolecular hydrogen bondings of proteins, better than with their hydrophobicity and the surface charge density. Overall, liposomes composed of binary lipid system, appeared to strongly interact with proteins, in contrast to liposomes composed of single, ternary, and quaternary lipid systems. This is probably because liposomes composed of binary lipid system favored to form the microscopic environment where proteins could interact. The present result suggested the heterogeneous phase state of lipid membranes was one of dominant factors for the interaction between proteins and lipid membranes.