Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

甘蓝叶制汁工艺研究

对甘蓝叶的预处理、榨汁方式和澄清工艺等进行了研究,结果以经预冻、解冻预处理,以离心式打浆榨汁与真空抽滤相结合的方式制汁,所得出汁率最高,为52.22%.所得甘蓝叶原汁经85℃,5 min;4 000 r/min,15 min 的离心后,再用复合澄清荆(A组份200 r/min,B组份400 mg/kg)进行澄清,可以获得良好澄清效果,其汁液透光率可达69.8%.     

茶树鲜花饮料澄清技术研究

茶树鲜花与茶叶具有相似的营养成分,榨汁后经调配可开发成适口性好、风味独特的鲜花饮料。对茶树鲜花饮料的壳聚糖絮凝、酶法澄清、超滤澄清效果进行了比较,结果表明,采用10万分子截留量的超滤膜处理获得能够较好的澄清效果,且对茶多酚造成的操作损失较小。     

Effect of high pressure homogenization and papain on the preparation of autolysed yeast extract

The effect of high pressure homogenization (600 and 1000 bar) prior to autolysis of a commercial pressed baker's yeast was examined. High pressure homogenization released a maximum of 30% of the solids and 34% of the total nitrogen (TN). After autolysis of the whole homogenized slurry, high yields of solids and TN (up to 81 and 85%, respectively) were obtained. Autolysis of non-homogenized controls yielded much lower yield values (30 and 39%, respectively), whereas autolysis in the presence of papain but without prior disruption gave intermediate values (50 and 61%, respectively). The various treatments led to changes in the extract composition: standard autolysates had the highest total nitrogen and true protein weight contents and the lowest carbohydrate content, whereas this trend was reversed when cells were first disrupted before autolysis. In contrast to controls obtained by standard autolysis without or with papain, centrifuged autolysates from pre-homogenized fractions were not clear. Treatment with a combination of a flocculation and a weighting agent clarified the extracts but resulted in a loss of solids (approximately 20%), including nitrogen and carbohydrates.     

Induction,purification, and characterization of a thermo and pH stable laccase from Abortiporus biennis J2 and its application on the clarification of litchi juice

A fungus J2 producing laccase with high yield was screened in soils and identified as Abortiporus biennis. The production of laccase was induced by 0.1 mM Cu²⁺, 0.1 mM tannic acid, and 0.5 M ethanol. The laccase from Abortiporus biennis J2 was purified to electrophoretic homogeneity by a couple of steps. The N-terminal amino acid sequence of the enzyme was AIGPTADLNISNADI. The properties of the purified laccase were investigated. The result showed the laccase from Abortiporus biennis J2 is a thermo and pH stable enzyme. The laccase activity was inhibited by Hg²⁺, Cd²⁺, Fe²⁺, Ag⁺, Cu²⁺, and Zn²⁺, while promoted by Mg²⁺, Mn²⁺ at 10 mM level. Purified laccase was used to the clarification of litchi juice. After treatment with this laccase, the phenolic content of litchi juice had been found to be greatly reduced along with an increase in the clarity of the juice. The result indicated the potential of this laccase for application in juice procession.     

应用果胶酶澄清甘蔗汁的研究

以甘蔗为原料,采用果胶酶和自然澄清方法进行甘蔗汁澄清比较,结果表明,酶促效果好于自然澄清,100ml甘蔗汁添加200U果胶酶在25℃、12h或45℃、1h条件下,均可使甘蔗汁的澄清度由5%提高到95%,粘度由4.0mPa·s下降到1.1mPa·s。     

Interaction of cell culture with downstream purification: a case study

Separation of product from secreting mammalian cells in the culture broth means the transition from product generation to product isolation. This interface within a biotech production process has to perform a proper solid/liquid phase separation of the cell suspension to make the product containing fluid amenable for further purification. These subsequent steps require fluid with low occurence of contaminants in order to function properly. The goal of this study was to evaluate some economic and fast cell separation methods for the preparation of a product fluid ready for use in further ultrafiltration and chromatographic processes. We have performed experiments to test the usefulness of disc stack centrifuges and tangential flow microfiltration units at large scale. Both systems revealed outstanding prospects with regard to throughput and scale up properties. However, the centrificgation did not lead to a fluid sufficiently free of particles for direct ultrafiltration or chromatography. Thus, an additional filtration step was necessary. On the other hand microfiltration led to an acceptable quality of process fluid directly. By optimisation of process parameters an effective, reproducible and robust cell separation can be obtained. However, our experience has been that such optimal conditions are somewhat specific for a narrow range. Thus, even the equipment functioning well with one type of cell would possibly not perform as well with another cell or even with the same cell under conditions slightly different to the usual situation.     

Two Agendas for Bioethics: Critique and Integration

Many bioethicists view the primary task of bioethics as ‘value clarification’. In this article, I argue that the field must embrace two more ambitious agendas that go beyond mere clarification. The first agenda, critique, involves unmasking, interrogating, and challenging the presuppositions that underlie bioethical discourse. These largely unarticulated premises establish the boundaries within which problems can be conceptualized and solutions can be imagined. The function of critique, then, is not merely to clarify these premises but to challenge them and the boundaries they define. The second agenda, integration, involves honoring and unifying what is right in competing values. Integration is the morally ideal response to value conflict, offering the potential for transcending win/lose outcomes. The function of integration, then, is to envision actions or policies that not only resolve conflicts, but that do so by jointly realizing many genuine values in deep and compelling ways. My argument proceeds in stages. After critically examining the role and dominant status of value clarification in bioethical discourse, I describe the nature and value of the two agendas, identify concrete examples of where each has been and could be successful, and explain why a critical integrative bioethics – one that appreciates the joint necessity and symbiotic potential of the two agendas – is crucial to the future of the field. The ultimate goal of all of this is to offer a more compelling vision for how bioethics might conduct itself within the larger intellectual and social world it seeks to understand and serve.     

Cell clarification and size separation using continuous countercurrent magnetophoresis

Nonmagnetic microparticles (e.g., cells, polymer beads) immersed in a magnetic fluid (ferrofluid) under a nonuniform magnetic field experience a magnetophoretic force in the direction of decreasing magnetic field strength. This phenomenon was exploited in the development of a continuous magnetophoretic countercurrent separation for the removal and concentration of micron-sized particles from aqueous suspensions, and in particular as a viable approach for cell clarification of raw fermentation broth. A magnetic fluid is added to the cell suspension, the mixture is introduced to the magnetic separator, which consists of an open flow tube passing between pairs of magnets that move in a direction counter to the flow of the suspension. The cells are pushed ahead of the magnet pairs owing to the magnetophoretic forces acting on them, collected in a tube upstream of the feed injection point, and removed as a concentrated suspension for further treatment.     

Clarification of recombinant proteins from high cell density mammalian cell culture systems using new improved depth filters

Increasingly high cell density, high product titer cell cultures containing mammalian cells are being used for the production of recombinant proteins. These high productivity cultures are placing a larger burden on traditional downstream clarification and purification operations due to higher product and impurity levels. Controlled flocculation and precipitation of mammalian cell culture suspensions by acidification or using polymeric flocculants have been employed to enhance clarification throughput and downstream filtration operations. While flocculation is quite effective in agglomerating cell debris and process related impurities such as (host cell) proteins and DNA, the resulting suspension is generally not easily separable solely using conventional depth filtration techniques. As a result, centrifugation is often used for clarification of cells and cell debris before filtration, which can limit process configurations and flexibility due to the investment and fixed nature of a centrifuge. To address this challenge, novel depth filter designs were designed which results in improved primary and secondary direct depth filtration of flocculated high cell density mammalian cell cultures systems feeds, thereby providing single‐use clarification solution. A framework is presented here for optimizing the particle size distribution of the mammalian cell culture systems with the pore size distribution of the gradient depth filter using various pre‐treatment conditions resulting in increased depth filter media utilization and improved clarification capacity. Feed conditions were optimized either by acidification or by polymer flocculation which resulted in the increased average feed particle‐size and improvements in throughput with improved depth filters for several mammalian systems. Biotechnol. Bioeng. 2013; 110: 1964–1972. © 2013 Wiley Periodicals, Inc.