RESUMO
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.
Assuntos
Anticorpos Monoclonais , Proteína Estafilocócica A , Cromatografia de Afinidade , Modelos Lineares , Projetos de PesquisaRESUMO
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 650â¯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.
Assuntos
Cromatografia , Polímeros , Adsorção , Ânions , Calorimetria , TermodinâmicaRESUMO
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.
Assuntos
Anticorpos Monoclonais , Cromatografia de Afinidade , Simulação por Computador , Modelos Químicos , Proteína Estafilocócica A/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificaçãoRESUMO
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.
Assuntos
Cromatografia por Troca Iônica/métodos , Oligonucleotídeos/isolamento & purificação , Ânions , Sítios de Ligação , Difusão , Oligonucleotídeos/química , PolímerosRESUMO
Previously, a predictive model was developed to identify optimal blends of expensive high-quality and cheaper low-quality feedstocks for a given geographical location that can deliver high sugar yields. In this study, the optimal process conditions were tested for application at commercially-relevant higher biomass loadings. We observed lower sugar yields but 100% conversion to ethanol from a blend that contained only 20% high-quality feedstock. The impact of applying this predictive model simultaneously with least cost formulation model for a biorefinery location outside of the US Corn Belt in Lee County, Florida was investigated. A blend ratio of 0.30 EC, 0.45 SG, and 0.25 CS in Lee County was necessary to produce sugars at high yields and ethanol at a capacity of 50 MMGY. This work demonstrates utility in applying predictive model and LCF to reduce feedstock costs and supply chain risks while optimizing for product yields.
Assuntos
Zea mays , Biomassa , Metabolismo dos Carboidratos , Carboidratos , Custos e Análise de Custo , Etanol/economia , Etanol/metabolismo , Fermentação , FloridaRESUMO
BACKGROUND: Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains. RESULTS: Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. CONCLUSIONS: Xylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.