1,721,027 research outputs found
Advances in Membrane Chromatography for the Capture Step of Monoclonal Antibodies
Full text linkBackground: Monoclonal antibodies are nowadays by far the most important of all biotherapeutics. Unfortunately, they are complex proteins, so that their production is complicated and expensive, which eventually leads to an elevated average cost per treatment per patient. An important research effort is dedicated to the development of a process that may allow a reduction of antibodies production costs.Objective: In particular, the main target is to replace the capture step based on the very expensive use of protein A beads, which is, and has been, the standard for the last 20 years. Among the possible alternatives the use of membrane chromatography for antibody capture will be considered in this work. Despite the development of new convective stationary phases with improved binding capacity, the use of membrane adsorbers for capture chromatography is still limited to niche applications. Conventional packed bead columns are still preferred due to their higher binding capacity even if they suffer from several limitations such as high pressure drop, slow mass transfer through the diffusive pores and strong dependence of the binding capacity on flow rate. An overview of the recent work performed in the field and a critical review of how technology advances could make a breakthrough will be presented here
Dynamic characterization of affinity membranes for monoclonal antibodies purification
No full textDownstream processes for the purification of biological products are often the cost determining production steps. Affinity technology is widely used for the primary capture stage, based on chromatographic beads. In the last decades, significant attention has been devoted to affinity chromatography using microporous membranes as chromatographic supports. Membrane chromatography can overcome the limitation associated to conventional packed-bed columns, such as high pressure drops and slow mass transfer.
This work is focused on the purification of Immunoglobulin G (IgG) via affinity membranes. A new support, Sartoepoxy Protein A membranes (Sartorius, Göettingen, Germany) has been tested in detail in dynamic experiments, using pure solutions of polyclonal IgG as well as the supernatant of a fermentation broth containing monoclonal IgG. All the relevant parameters, namely the dynamic binding capacity, process yield and recovery have been evaluated. The influence of several operating parameters on the adsorption and elution performances has been studied to determine the optimal process conditions.
A mathematical model including convection, diffusion and multi-component adsorption is proposed to simulate the adsorption, washing and elution steps; the model also considers the possible effects of dead end volumes and flow distribution. Results of the simulation have been compared with the experimental data, giving a good description of the global process
Performance of a New Protein A Affinity Membrane for the Primary Recovery of Antibodies
No full textRecovery of antibodies with Protein A affinity chromatography columns has become the standard for the biotechnology industry. Membrane affinity chromatography has not yet experienced
extensive application due to the lower capacity of membrane supports compared to chromatographic beads. In this work, new affinity membranes endowed with an interesting binding capacity
for human IgG are studied in view of their application in the capturing step of a monoclonal antibody production process. The membranes have been extensively tested with pure IgG solutions
and with a cell culture supernatant containing IgG1. The effects of feed flow rate and IgG concentration on the separation performances have been studied in detail, considering in particular
binding capacity, selectivity and recovery. These new high capacity affinity membranes appear good candidates to avoid the throughput limitations and other well-known drawbacks of traditional bead-based chromatographic columns
Modelling and simulation of affinity membrane adsorption
No full textA mathematical model for the adsorption of biomolecules on affinity membranes is presented. The model considers convection, diffusion and adsorption kinetics on the membrane module as well as the influence of dead end volumes and lag times; an analysis of flow distribution on the whole system is also included. The parameters used in the simulations were obtained from equilibrium and dynamic experimental data measured for the adsorption of human IgG on A2P-Sartoepoxy affinity membranes. The identification of a bi-Langmuir kinetic mechanisms for the experimental system investigated was paramount for a correct process description and the simulated breakthrough curves were in good agreement with the experimental data. The proposed model provides a new insight into the phenomena involved in the adsorption on affinity membranes and it is a valuable tool to assess the use of membrane adsorbers in large scale processes
Analysis of different affinity membranes for the primary capture step in antibody manufacturing
No full textIn this work, different new affinity membranes are considered for the capturing step of a monoclonal antibody production process. The membranes with immobilized protein A and synthetic ligands, have been extensively tested with pure IgG solutions and with a cell culture supernatant containing IgG1. The effects of flow rate and IgG concentration in the feed on the separation performances like binding capacity, selectivity and process yield have been studied in detail.
The behaviour and efficiency of the different affinity membranes will be presented and will be also compared with data available for protein A chromatography beads.
A model simulation study of the relevant kinetic and transport phenomena led to the development of a mathematical model capable to describe the separation process based on affinity membrane. Model validation has been successfully performed using the experimental data available for the different membrane tested
Evaluation of affinity membrane adsorbers for antibody purification
No full textRecent development of therapeutics based on monoclonal antibodies has lead biotech industry to look at alternatives to chromatography in view of the increased production needs and affinity membrane adsorbers are among the process considered.
A2P mimetic (Prometic Biosciences, UK) has been coupled to Sartobind epoxy (Sartorius AG, Germany) pre-activated cellulose membranes and characterised with pure polyclonal human IgG and MAbs from a cell culture supernatant.
Membrane capacity and selectivity towards the target proteins have been measured together with all the relevant kinetic parameters, which characterise adsorption and elution. The membranes were also studied with respect to stability of the stationary phase and ligand leakage and compared to affinity chromatography resins.
A2P-Sartobind epoxy affinity membranes were extensively tested in dynamic experiments. The effects of feed concentration and flow rate were investigated in order to find the better conditions for binding and elution steps.
The equilibrium and kinetic parameters measured experimentally were used in a mathematical model developed to describe the behaviour of affinity membrane systems in binding and elution steps. The influence of different adsorption kinetics as well as the effects of dead end volumes, lag times and analysis of flow distribution were used to assess the use of the model for scale-up purposes
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Experimental evaluation and theoretical analysis of convective affinity adsorbers for chromatographic applications
No full textAffinity chromatography represents one of the most important and widely used unit operations in the biotechnology industry. However, traditional packed bead columns suffer from several limitations such as high pressure drop, slow mass transfer through the diffusive pores and strong dependence of the binding capacity on flow rate. One possible alternative to overcome these drawbacks is represented by convective media columns packed with affinity membrane or monoliths.
This work presents the purification of immunoglobulin G in columns packed with convective media, and discusses the enhanced performance versus packed bead columns. The stationary phases studied derive from the functionalization of membranes and monoliths with natural and synthetic affinity ligands that show high specificity towards IgG.
The affinity materials are completely characterized through adsorption, washing and elution cycles. The separation performance of the affinity supports has been determined by feeding both pure IgG solutions and a cell culture supernatant. Relevant process parameters, such as maximum adsorption capacity, affinity equilibrium constant and selectivity, are evaluated and carefully compared among the different affinity supports. The scale-up of the improved affinity materials for industrial applications is also addressed.
The experimental data collected have been used for the validation of a proposed simulation model. Such chromatographic model is based on species mass balance equation over the convective medium, coupled with a suitable kinetic equation which represents the interaction between the IgG target molecule and the ligand immobilized on the porous support. Model simulations are in good agreement with all of the experimental affinity cycles, demonstrating the accuracy of the model to describe the transport phenomena in the column and the adsorption binding mechanism
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