新型亲和技术在下游过程中的应用

基于生物分子之间特异性的亲和作用而发展起来的新的分离纯化与分析技术－膜亲和过滤、亲和萃取、亲和沉淀和亲和电泳在生物技术产品的分离纯化中显示巨大的开发应用前景。本文综述有关的研究结果。     

Advances in the production and downstream processing of antibodies

Sales of monoclonal antibody (mAbs) therapies exceeded $ 40 billion in 2010 and are expected to reach $ 70 billion by 2015. The majority of the approved antibodies are targeting cancer and autoimmune diseases with the top 5 grossing antibodies populating these two areas. In addition over 100 monoclonal antibodies are in Phase II and III of clinical development and numerous others are in various pre-clinical and safety studies. Commercial production of monoclonal antibodies is one of the few biotechnology manufacturing areas that has undergone significant improvements and standardization over the last ten years. Platform technologies have been established based on the structural similarities of these molecules and the regulatory requirements. These improvements include better cell lines, advent of high-performing media free of animal-derived components, and advances in bioreactor and purification processes. In this chapter we will examine the progress made in antibody production as well as discuss the future of manufacturing for these molecules, including the emergence of single use technologies.     

Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase

This work presents a comprehensive study between the polymer/salt aqueous two-phase systems (ATPS) and chromatography process for downstream processing of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH). First, the partitioning behavior of recombinant PheDH in polyethylene glycol (PEG)/K₂HPO₄ ATPS was examined. For comparative purpose, a classical chromatographic protocol was performed as well. Investigation of chromatography and ATPS procedures revealed that the ATPS comprising of 9% (w/w) PEG-6000, 16% (w/w) K₂HPO₄ and 16% (w/w) KCl with pH of 8.0, volume ratio (V _R ) of 0.25, temperature of 25 °C and 40% (w/w) cell lysate ensured the most favorable approach for PheDH downstream process. A specific activity of 4,231.4 U/mg, a yield of 96.7% and a recovery of 162.0% were obtained. Furthermore, the shorter process time (4 vs. 48 h) and the lower total cost (4 vs. 20 €) were additionally features that confirmed the suitability of proposed technique.     

Emergence of ideal membrane cascades for downstream processing

An algorithm is developed for describing ideal membrane cascades for fractionation of binary and pseudo-binary mixtures. It is shown that solvent management plays a key role in determining both purification and yield. Development of efficient diafilters is needed if membrane cascades are to achieve their full potential in competing with both chromatography and simulated moving bed operations in downstream processing of proteins. Such a replacement will also be important for fractionation of higher titers and larger substrates, such as plasmids, viruses, and even whole cells.     

Aqueous two-phase extraction for downstream processing of amyloglucosidase

A polymer/salt aqueous two-phase system has been successfully employed for separation and purification of amyloglucosidase. The effects of system pH, molecular weight of polymer and composition of the two-phase system on amyloglucosidase partition behaviour in polyethylene glycol (PEG 4000, 6000)/disodium hydrogen phosphate were investigated. Experimental data are explained based on Kim's theoretical model for the prediction of biomolecule partitioning in a PEG/salt system.     

Optimal synthesis of chromatographic trains for downstream protein processing

Downstream bioprocessing and especially chromatographic steps, commonly used for the purification of multicomponent systems, are significant cost drivers in the production of therapeutic proteins. There has been an increased interest in the development of systematic methods for the design of such processes, and the appropriate selection of a series of chromatographic steps is still a major challenge to be addressed. Several models have been developed previously but have assumed that 100% recovery of the desired product is obtained at each chromatographic step. In this work, a mathematical framework is proposed, based on mixed integer optimisation techniques, that removes this assumption and allows full flexibility on the position of retention time cut-points, between which the desired product fraction is collected. The proposed model is demonstrated on three example protein mixtures, each containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The proposed model results in a reduction of one to three chromatographic steps over solutions that no losses are allowed.     

Comparison of downstream processing methods in purification of highly active laccase

Bioprocess and Biosystems Engineering - Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study...     

The applications of reactive dyes in enzyme and protein downstream processing

Downstream processing of proteins is often a key factor in the overall process of satisfying product specifications and meeting current commercial demands. In this context, affinity chromatography and other techniques based on the affinity concept have revolutionized protein purification technology, although they have failed to demonstrate their broader applicability at the process scale. On the other hand, reactive dyes offer many advantages as pseudoaffinity media and in many occasions have successfully circumvented problems associated with conventional affinity ligands. The main features of reactive dyes include their broad spectrum of interaction with proteins, low cost, ready availability, high reactivity, ease of immobilization, and both biological and chemical stability. Consequently, dye-ligand media now find application in both analytical and process-scale purification of proteins by techniques such as low- and high-pressure performance affinity chromatography, affinity partitioning, and affinity precipitation.     

Surface charge engineering of PQQ glucose dehydrogenase for downstream processing

The ion-exchange chromatography behavior of recombinant glucose dehydrogenase harboring pyrroloquinoline quinone (PQQGDH) was modified to greatly simplify its purification. The surface charge of PQQGDH was engineered by either fusing a three-arginine tail to the C-terminus of PQQGDH (PQQGDH+Arg3) or by substituting three residues exposed on the surface of the enzyme to Arg by site-directed mutagenesis (3RPQQGDH). During cation exchange chromatography, both surface charge-engineered enzymes eluted at much higher salt concentrations than the wild-type enzyme. After the chromatography purification step, both PQQGDH+Arg3 and 3RPQQGDH appeared as single bands on SDS-PAGE, while extra bands appeared with the wild-type protein sample. Although all tested kinetic parameters of both engineered enzymes are similar to those of wild type, both modifications resulted in enzymes with increased thermal stability. Our achievements have resulted in the greater production of an improved quality PQQGDH by a simplified process.     

Enzyme stability in downstream processing. Part 2: quantification of inactivation

In biotechnological recovery processes the instability of the product can lead to large losses in the sequence of recovery processes needed to purify the product. As the cost of the final active product is strongly dependent on the recovery yield, this will lead to an increase in product cost. Therefore knowledge of factors that influence stability is important. This Part 2 provides the basic principles for design and operation of processes in which inactivation takes place. Simple kinetics and reactor modelling are discussed. These are applied to a number of unit operations: cell disruption, membrane filtration, drying and reversed micellar extraction. It is thus shown that the basic tools for modeling of biochemical processes provide us with the data needed for optimal process design and operation.     

Evaluation of recent advances in butanol fermentation, upstream, and downstream processing

Four different processes for butanol production from corn, namely, batch fermentation and distillative recovery (BFDR), batch fermentation and pervaporative recovery (BFPR), fed-batch fermentation and pervaporative recovery (FBFPR), and immobilized cell continuous fermentation and pervaporative recovery (ICCFPR) were evaluated. Pervaporative recovery significantly reduces the cost of butanol production. Depending upon the byproduct credit, which is approximately 3.7 times that of the amount of butanol produced, BFDR, BFPR, FBFPR, and ICCFPR result in a butanol price of 0.55,0.55, 0.14-0.39, 0.12-0.37, and0.12-0.37, and 0.11-0.362kg^-1, respectively. The price of butanol was recently reported at $1.212kg^-1 by Chemical Marketing Reporter. It should be noted that all three components (acetone, butanol, and ethanol: ABE) diffuse through the pervaporation membrane. Further separation and purification of the solvents would require distillation, which has been considered in this exercise. This article also details the impact of byproduct credit, rate of return, and tax on butanol price.     

Differential response in downstream processing of CHO cells grown under mild hypothermic conditions

The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L^?1. However, host cell proteins, measured by ELISA, increased by ～50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI‐TOF MS and 2D‐PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:688–696, 2013     

Fluidized bed design parameters affecting novel lactic Acid downstream processing.

Lactic acid purification was directly done from fermentation utilizing a fluidized bed column refilled with a strong anionic exchange resin. The purpose of this work was to study the influence of two important design parameters, bed-diameter (D) and bed-height (H), in the lactic acid binding and elution capacity of the matrix. By changing the settled bed height from 2.5 to 5 cm for each diameter of column analyzed it was possible to obtain an 50% increase in the binding capacity of the resin in all experiments. This fact was attributed to a higher contact time between the culture broth and the anionic resin produced by the increase of back mixing and lactic acid residence time.