Five common aggregation mechanisms are described and their. Protein aggregation and bioprocessing springerlink. Five common aggregation mechanisms are described and their relations to. Any mechanism of inhibition of this aggregation should explain the prevention of. Stressinduced antibody aggregates bioprocess international. Alternatively, cell line engineering using a mirna enabled enhancement of specific productivity without negative impact on protein aggregation and glycosylation. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the environment eg, media components and implementing appropriate strategies to minimize the extent of aggregation. Aggregation of therapeutic proteins can occur during bioprocessing, shipping, storage and even delivery to the patient. Why and how protein aggregation has to be studied in vivo. Protein aggregation is one of the most critical stability concerns of protein drug development and manufacturing. Analysis of protein aggregation is therefore an important facet of many areas of protein chemistry and related fields. Scientific and regulatory perspectives on measuring protein. Protein aggregation can be reversible or irreversible and the aggregates formed can be solubleinsoluble, covalentnoncovalent and nativenonnative figure.
Protein aggregation an overview sciencedirect topics. Reduction of mab aggregation during upstream processing by bioprocess optimization could prevent aggregate formation, and thus represent an. Protein aggregation is a common issue encountered during manufacture of biotherapeutics. Principles of protein folding, misfolding and aggregation. Modeling of chemical inhibition from amyloid protein. Nonnative protein aggregation is the typical mechanism encountered in the biopharmaceutical industry and it will be termed protein aggregation in this chapter 23. Amyloid fibrils are found in association with at least two dozen fatal diseases. Not only is mfi more sensitive than traditional techniques, it also lets you discriminate between. Such immune responses can have serious consequences for patients. Aaps open forum on protein aggregation in san francisco, ca on june 5, 2006, and brie. E5729 february 2006 with 634 reads how we measure reads.
Human health can be severely affected by protein misfolding and aggregation resulting from genetic mutations, medical treatment, or prion diseases tauopathies. The structural biology of protein aggregation diseases. A reduction in yield due to protein aggregation can also have a significant impact on development and manufacturing costs. Recently roberts 2007 has shown in more detail the general understanding of aggregation as a multistage process and how most available. Protein aggregation and subvisible particle analysis is a breeze with microflow imaging mfi. Principles, methods, and applications in the pharmaceutical.
Physicochemical principles of protein aggregation benedetta bolognesi, gian gaetano tartaglia centre for genomic regulation, crg and upf, barcelona, spain contents 1. Backgroundaggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Aggregation of therapeutic proteins can result in an incorrect drug dosage, or and undesired and potentially fatal immune response. Experimental kinetics and adequate modelling of chemical inhibition from amyloid aggregation are of practical. Aggregation is a common phenomenon occuring during manufacture and lifetime of biotherapeutics the structure of proteins is very complicated and. The understanding of protein aggregation is a central issue in different fields of protein science, from the heterologous protein production in biotechnology to amyloid aggregation in several neurodegenerative and systemic diseases. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. Principles of protein folding, misfolding and aggregation christopher m. Analyzing protein aggregation in biopharmaceuticals. External factors affecting protein aggregation 119 wei wang, ning li, and stan speaker 4.
Stability of human serum albumin during bioprocessing. The full text of this article is available as a pdf 304k. The kinetics of aggregation is thought to be governed by nucleation. These protein aggregates are often correlated with diseases. Prediction of hot spots of aggregation in polypeptides. Challenges of protein aggregation during purification. Protein aggregation and particle analysis proteinsimple. The presence in cells of misfolded or aggregated proteins triggers a complex biological response. Recently, new analytical methods enabled characterization of mab aggregation during bioprocessing of mammalian cells.
Protein aggregate analysis thermo fisher scientific hk. What are the causes of protein aggregation in biologic formulations. We provided the first direct evidence that amyloid aggregation is indeed governed by saturation behaviour, a prerequisite. A thesis submitted to university college london for the degree of. Although protein aggregation has traditionally been difficult to study, substantial progress in understanding how these diseases arise has been made in the past decade. The importance of sequence diversity in the aggregation and evolution of proteins. Aggregation or reversible selfassociation of protein therapeutics can arise through a number of different mechanisms. Dynamic light scattering, which is a nonperturbing technology, has, for example, been used to monitor inclusion body solubilization, protein refolding, and aggregation near the. Protein aggregation has usually been assumed to involve either unfolded or native states. Identification of process conditions influencing protein aggregation.
Pdf aggregation or reversible selfassociation of protein therapeutics can arise. This presentation will mainly cover the causing factors of and control strategies for protein aggregation during protein formulation development, manufacturing process, and clinical use. Pdf mechanisms of protein aggregation researchgate. To this goal, it became more and more evident the crucial relevance of studying protein aggregation in the complex cellular environment, since it allows to take. A membrane structure and function structure of model membrane, lipid bilayer and membrane protein diffusion, osmosis, ion channels, active transport, membrane pumps, mechanism of sorting and regulation of intracellular transport,electrical properties of membranes. Enter the peptide sequences in fasta formatfasta format. Finally, it was discovered that protein aggregation during bioprocessing occurs at two different levels, in particular at the cellular level and at the level of the bioprocess. To assess the impact of various bioprocessing steps on thestability of freshly precipitated human serum albumin hsa obtainedfrom pooled human plasma. Guest editor steve shire protein aggregation and bioprocessing. Protein aggregation propensity calculations were performed on wild type and mutant sequences of three diverse proteins including superoxide. Aggregation is a general term that encompasses several types of interactions or characteristics. An integrated continuous bioprocessing platform containing a coiled flow inverter reactor for protein precipitation, protein capture using cex, polishing steps using mm chromatography, and a salttolerant aex membrane has been used for three different mabs, and the process continued for 48 h using 1. A membrane structure and function structure of model membrane, lipid bilayer and membrane protein diffusion, osmosis, ion channels, active transport, membrane pumps, mechanism of. The popular protein aggregation and emerging analytical tools conference covers latest trends, challenges and solutions in understanding, characterization and mitigation of problems.
Protein aggregation during bioprocessing of monoclonal. Protein aggregation can increase the immunogenicity of biotherapeutics, and can be a key factor in causing adverse events associated with immunogenicity in the clinic rosenberg, 2006. Predicting and measuring molecular mechanisms of protein. Measuring protein aggregation of biotechnology products ashutosh rao, ph. We hypothesise that the aggregation of these domains occurs from a misfolded dimeric species, leading to kinetic traces that are hyperbolic at long timescales. Protein aggregation is a biological process in which misfolded proteins aggregate. Aggregation stability of a monoclonal antibody during. Not only is mfi more sensitive than traditional techniques, it also lets you discriminate between protein aggregates and other particulates. We provided the first direct evidence that amyloid aggregation is indeed governed by saturation behaviour, a prerequisite for the applicability of the nucleation theory sengupta et al. This presentation will mainly cover the causing factors of and control.
Molecular mechanisms of protein folding and misfolding. The tendency of numerous proteins to convert into amyloidlike fibrils poses fundamental questions for. Aggregates in monoclonal antibody manufacturing processes. The understanding of protein aggregation is a central issue in different fields of protein science, from the heterologous protein production in biotechnology to amyloid.
Time to market is crucial in process development, and many companies developing and manufacturing therapeutic agents have. Methods for measuring protein aggregation bentham science. Little information has been published on the extent and causes of aggregation during bioprocessing for pharmaceutical proteins. Host cell protein analysis in therapeutic protein bioprocessing methods and applications. Department of chemistry, university of cambridge, lens. Protein aggregation and stability in biopharmaceutical products.
Advances in process analytical technology pat provide an opportunity for more effective control of protein aggregation in bioprocessing, according to vedantham. Note that proline is a cyclic imino acid and its structure is shown in its entirety. Protein purification and bioprocessing miniature and parallel. Aggregation of kkq nkk repeat peptides at high and low concentrations. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions.
Tscheliessnig al1, konrath j, bates r, jungbauer a. First, a thorough investigation that compared nine protein aggregation prediction techniques was performed. Consequently, monitoring protein aggregates is important. After initial precipitation of hsa from plasma, the resultantpaste is either a lyophilized or b washed with acetone and thenairdried in order to obtain a dry powder. Time to market is crucial in process development, and many companies developing and manufacturing therapeutic agents have invested in automated protein purification and high throughput bioprocessing. Sep 15, 2006 protein aggregation is a common issue encountered during manufacture of biotherapeutics. Understanding of the roles of aggregates in immunogenicity of therapeutic protein products is critical. We propose a mechanism based on the hydrophobic interactions of. The process of amyloid proteins aggregation causes several human neuropathologies. This is a fast, highthroughput, platebased fluorometric assay format and can detect as low as 0. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the envi.
To assess the impact of various bioprocessing steps on thestability of freshly precipitated human serum albumin hsa obtainedfrom pooled human. Recent developments in bioprocessing of recombinant. Protein aggregation is a biological phenomenon in which misfolded proteins aggregate i. In fact, protein aggregates have been implicated in a wide variety of disease known as amyloidoses, including als, alzheimers. Biovisions antibodyprotein aggregation assay kit is a platform to estimate the extent of aggregation within a proteinantibody sample. Inclusion body formation and other aggregates formed during protein folding have been. Overview of upstream and downstream processing of biopharmaceuticals 1. Inclusion body formation and other aggregates formed during protein folding have been assumed to arise from hydrophobic aggregation of the unfolded or denatured states, whereas amyloid fibrils and other extracellular aggregates have been assumed to arise from nativelike conformations in a process. Aggregation is a common phenomenon occuring during manufacture and lifetime of biotherapeutics the structure of proteins is very complicated and denaturation due to smal pertubations can lead aggregates and particles aggregation is triggered by environmental and engineering factors. Aggregates of proteins may arise from several mechanisms and may be classified in numerous ways, including solubleinsoluble, covalentnoncovalent, reversibleirreversible, and nativedenatured. Cromwell,1 eric hilario,1 and fred jacobson2 1early 2early stage pharmaceutical development, genentech inc, south san francisco, ca stage analytical development, genentech inc, south san francisco, ca. The structure of hsa wasexamined using fourier transform. It is possible to influence the amount of aggregate produced during the cell.