What are the different methods of determining protein. Bioprocessing tutorial attaining high transient titers in cho. Glycerol is known to shift the native protein ensemble to more compact states. Addressing protein aggregation the formation of protein aggregates in biopharmaceuticals can be difficult to predict and control. Cambridge healthtech institute peptalk palm springs, ca. Benjamin patrick werner and gerhard winter 11222017. Protein aggregation and particle analysis proteinsimple. Misfolded proteins, in theory, undergo proteolysis in the endosome and the resultant amino acids are recycled in the unfolded protein response cudna and dickson 2003 and the endoplasmic. Microaggregated subvisible particles generated anywhere in a manufacturing process can develop into larger particles over time as a product is stored 62. Dec 27, 2017 protein aggregation of monoclonal antibodies mabs is a common phenomenon associated with the production of these biopharmaceuticals. 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.
Bioprocessing is the use of biological materials organisms, cells, organelles, enzymes to carry out a process for commercial, medical or scientific reasons. Bioprocessing tutorial attaining high transient titers in. Reduction of mab aggregation during upstream processing by bioprocess optimization could prevent aggregate formation, and thus represent an. Understanding of the roles of aggregates in immunogenicity of therapeutic protein products is critical. The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Inducing protein aggregation by extensional flow pnas.
Protein aggregation is a biological phenomenon in which misfolded proteins aggregate i. Scientific and regulatory perspectives on measuring. Unfortunately, cho cells typically express lower levels of protein than hek293 in transient systems, in some instances 50100 times less than the best 293based systems, and only modest titer improvements are obtained through the optimization of individual components of existing. May 25, 2007 protein aggregation problems are not confined to upstream bioprocessing. 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. Heres how we can improve patient safety through primary packaging materials and bedside filtration. Protein aggregation and bioprocessing springerlink. Glycerol also inhibits protein aggregation during the refolding of many proteins. Sheila has implemented quality systems and has managed external analytical and qc activities for multiple biopharmaceutical products. Protein aggregation to explain this di erence, we use as an example the stability of a monoclonal antibody at low ph. Protein misfolding, aggregation and methionine oxidation. Not only is mfi more sensitive than traditional techniques, it also lets you discriminate between protein aggregates and other particulates.
The assay does not require any prior knowledge of protein structure, sequence or ligand binding activity, and is performed on a thermally regulated fluorimeter or rtpcr instrument. Optimizing protein aggregate analysis by size exclusion. In fact, protein aggregates have been implicated in a wide variety of disease known as amyloidoses, including als, alzheimers. Physical and chemical transitions, such as concentration of protein on the adsorbent surface and change in buffer composition, may challenge the structural integrity of proteins, increasing their propensity for aggregation. Protein aggregation is a common issue encountered during manufacture of biotherapeutics. Sep 15, 2006 protein aggregation is a common issue encountered during manufacture of biotherapeutics. The influence of elution from protein a on the aggregation rate of an igg4 during a subsequent low ph hold was investigated.
Overview of upstream and downstream processing of biopharmaceuticals 1. However, for pharmacological applications, protein aggregation represents a major obstacle to the development of protein based pharmaceuticals. Pdf protein aggregation is a common issue encountered during manufacture of biotherapeutics. E5729 february 2006 with 634 reads how we measure reads. Disulfide bond formation resulting from previously unpaired free thiols is a common mechanism for covalent aggregation.
It may be a transient moiety because it can trigger protein aggregation and precipitation, which can obscure isolation of 2ohis as an. Guest editor steve shire protein aggregation and bioprocessing. Physical changes include protein unfolding, binding to surfaces, and aggregation. Second is the problem of understanding potential proteinprotein and proteinexcipient interactions. 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. Clinical studies have shown adverse effects and safety issues caused by protein aggregation 2, 3. A expichos cells at passage 10 or passage 34 postthaw consistently attain. Understanding the influence of adsorptionmediated processes on antibody aggregation in bioprocessing. Protein aggregation has been recognized as one of the major challenges in the development and commercialization of successful protein based drug products because of the inherent and high aggregation tendency during various manufacturing processes, storage, and administration. Protein aggregation is a common issue encountered during. Protein aggregation of monoclonal antibodies mabs is a common phenomenon associated with the production of these biopharmaceuticals.
Analytical strategies for fixeddose coformulated protein. Mechanisms of protein stabilization and prevention of. Review the effects of shear flow on protein structure and. In fact, protein aggregates have been implicated in a wide variety of disease known as amyloidoses, including als, alzheimers, parkinsons and prion disease after synthesis. However, for pharmacological applications, protein aggregation represents a major obstacle to the development of proteinbased pharmaceuticals. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. Clearly when mechanism 3 is operative improving the chemical stability will also reduce aggregation, and conversely attempts to improve the conformational stability of the monomer may not reduce aggregation. Overview of upstream and downstream processing of biopharmaceuticals 1 ian marison professor of bioprocess engineering and head of school of biotechnology, dublin city university, glasnevin, dublin 9, ireland email. Stresses to the protein such as freezing, exposure to air, or interactions with metal surfaces may result in surface denaturation, which then leads to the formation of aggregates. Expression yields were too low, culture media too primitive, and the tech.
Finally, mechanical stresses may cause protein aggregation. What are the different methods of determining protein aggregation. Various aggregates hold the potential for adversely impacting production and patients in a variety of ways. Continuous bioprocessing at the industrial scale has long been a goal for bioprocessing. In contrast to glycosylation, the other ptms of protein therapeutics are less well studied and understood chirino and miresluis 2004. Aggregates in monoclonal antibody manufacturing processes. Bioprocess development upstream and downstream technologies monica chuong, phd school of pharmacyboston 1. Protein aggregation and subvisible particle analysis is a breeze with microflow imaging mfi. It is possible to influence the amount of aggregate. Understanding the influence of adsorptionmediated processes. Attaining high transient titers in cho cells bioprocessing tutorial october 1, 2015 volume 35, number 17.
Mechanisms of protein aggregation current pharmaceutical biotechnology, 2009, vol. Dunstan1 1 department of chemical and biomolecular engineering, university of melbourne, parkville, vic 3010, australia 2 research and development department, csl biotherapies, 189209 camp rd, broadmeadows, vic 3047, australia. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the envi. Optimizing protein aggregate analysis by size exclusion chromatography authors rowan moore,1 amy farrell,2 jonathan bones,2 and ken cook1 1thermo fisher scienti. These aggregates can lead to adverse side effects in patients upon administration, thus expensive downstream processing steps to remove the higher molecular weight species are inevitable. Review the effects of shear flow on protein structure and function innocent b. The structure of hsa wasexamined using fourier transform. Bioprocess development upstream and downstream technologies.
Protein crystallization screening poster pegs 2019 pdf download insulin homologs and biosimilars poster peptalk 2019 pdf download biologic developability poster bioprocessing summit 2018 pdf. Protein aggregation can occur during all stages of the lifetime of a protein therapeutic, including expression, refolding, purification, sterilization, shipping, storage, and delivery processes 1. Percent aggregation by sizeexclusion hplc figure 3. The full text of this article is available as a pdf 304k. Bekard,1 peter asimakis,1 joseph bertolini,2 dave e. What are the causes of protein aggregation in biologic formulations. 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. Lumry and eyring 1954 polymerization model has been used as a starting point to study protein aggregation. Performance characteristics of the expicho transient expression system. In the present context, this may include both reversible selfassociation and effectively irreversible aggregation. Attenuated total reflectance fourier transform infrared spectroscopy atrftir was used to study the conformation of aggregated proteins in vivo and in vitro. Protein aggregation, along with other physical andor chemical instabilities of proteins, remains to be one of the major road barriers hindering rapid commercialization of potential protein drug. Protein aggregation is favored in the food industry since it imparts desirable texture and flavor. Recently, new analytical methods enabled characterization of mab aggregation during bioprocessing of mammalian cells.
Aaps open forum on protein aggregation in san francisco, ca on june 5, 2006, and brie. Protein aggregation during bioprocessing of monoclonal. Some industries have a long tradition of enzyme use. In the type of situation described by a lumry eyring modelsa. Nov 22, 2017 addressing protein aggregation the formation of protein aggregates in biopharmaceuticals can be difficult to predict and control. Analyzing protein aggregation in biopharmaceuticals. Identification of process conditions influencing protein. Host cell protein per mg of mab in order to ensure product quality, mab concentration monitoring is one of the cqas. Several different protein aggregates, including amyloid fibrils from several peptides and polypeptides, inclusion bodies, folding aggregates, soluble oligomers, and protein extracts from stressed cells, were examined in this study. Host cell protein per mg of mab in order to ensure product quality, mab. Protein crystallization screening poster pegs 2019 pdf download insulin homologs and biosimilars poster peptalk 2019 pdf download biologic developability poster bioprocessing summit 2018 pdf download. The kinetics of protein aggregation has been extensively investigated.
Aggregation in biopharmaceuticals remains a major concern and threatens the stability of a product. Simultaneously, protein aggregation can occur during manufacturing and bioprocessing due to pumping, stirring and shaking. To assess the impact of various bioprocessing steps on thestability of freshly precipitated human serum albumin hsa obtainedfrom pooled human plasma. Aggregation of therapeutic proteins can occur during bioprocessing, shipping, storage and even delivery to the patient. Proteinsimple 3001 orchard parkway san jose, california, 954 usa. Alternatively, cell line engineering using a mirna enabled enhancement of specific productivity without negative impact on protein aggregation and glycosylation. Related disorders include cataracts, arthritis, cystic fibrosis, lateonset diabetes mellitus, and numerous neurodegenerative diseases like alzheimers and parkinsons. In disease states, protein aggregation can occur as amyloid fibrillogenesis. Nanotechnology and nanoimaging technology, including cryoelectron microscopy of beta2microglobulin, studying amyloidogensis. Elution from protein a was found to increase aggregation rates by half an order of magnitude, while other aspects of the aggregation kinetics appeared unaffected. Solid state fermentation microorganisms grow on a moistened solid surface without free flowing water, but have free access to air. Disruption of this delicate balance can expose internal. Little information has been published on the extent and causes of aggregation during bioprocessing for pharmaceutical proteins. In leather tanning, hides are softened and hair removed using the proteases in faeces.
Protein misfolding and aggregation is a fastgrowing frontier in molecular medicine and protein chemistry. Dunstan1 1 department of chemical and biomolecular engineering, university of melbourne, parkville, vic 3010, australia 2 research and development department, csl biotherapies, 189209 camp rd, broadmeadows, vic 3047. 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. Jul 09, 20 attenuated total reflectance fourier transform infrared spectroscopy atrftir was used to study the conformation of aggregated proteins in vivo and in vitro. These observations rationalize the diverse effects of hydrodynamic flow on protein structure and aggregation propensity seen in both nature and in protein manufacture.
Protein aggregation has been recognized as one of the major challenges in the development and commercialization of successful protein or proteinbased drug products ross and wolfe, 2016, singh et al. Many types of aggregates are known and their size can differ from nanometers to the visible range 46. Process analytical technology pat for continuous bioprocessing. These protein aggregates are often correlated with diseases. Mechanism, characterization and consequences protein aggregation is recognized by regulatory agencies and the biopharmaceutical industry as a key quality attribute of biotherapeutic products.
Protein aggregation has been recognized as one of the major challenges in the development and commercialization of successful protein or protein based drug products ross and wolfe, 2016, singh et al. The effects of shear flow on protein structure and function innocent b. Mechanisms of protein stabilization and prevention of protein. Each of these environmental factors is typically encountered during bioprocessing. Aggregation also affects the mass balance of protein solutions, decreasing the concentration of the target protein. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and ef. Identification of process conditions influencing protein aggregation. Low capital investment, water utilization, waste water, and energy requirement no agitation no foam formation simple fermentation media less space less control techniques ease in controlling bacterial contamination. Measuring protein aggregation of biotechnology products ashutosh rao, ph. Vice president and senior consultant, bptc susan is a seasoned biotechnology.
Such immune responses can have serious consequences for patients. Stability of human serum albumin during bioprocessing. Igg4 was incubated in elution buffer after protein a chromatography and the monomer concentration in neutralised samples was quantified by size exclusion. Protein aggregation has usually been assumed to involve either unfolded or native states.
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