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. Finally, mechanical stresses may cause protein aggregation. 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. It may be a transient moiety because it can trigger protein aggregation and precipitation, which can obscure isolation of 2ohis as an. May 25, 2007 protein aggregation problems are not confined to upstream bioprocessing. Lumry and eyring 1954 polymerization model has been used as a starting point to study protein aggregation. 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. Physical changes include protein unfolding, binding to surfaces, and aggregation. 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.
Optimizing protein aggregate analysis by size exclusion. Analytical strategies for fixeddose coformulated protein. 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. Overview of upstream and downstream processing of biopharmaceuticals 1. 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. Bioprocess development upstream and downstream technologies. Aggregation also affects the mass balance of protein solutions, decreasing the concentration of the target protein. 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. Protein misfolding and aggregation is a fastgrowing frontier in molecular medicine and protein chemistry.
Bioprocessing tutorial attaining high transient titers in cho. 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. E5729 february 2006 with 634 reads how we measure reads. What are the different methods of determining protein. Second is the problem of understanding potential proteinprotein and proteinexcipient interactions. Inducing protein aggregation by extensional flow pnas. Various aggregates hold the potential for adversely impacting production and patients in a variety of ways. Reduction of mab aggregation during upstream processing by bioprocess optimization could prevent aggregate formation, and thus represent an. Disruption of this delicate balance can expose internal. The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Protein aggregation has usually been assumed to involve either unfolded or native states. Protein aggregation is a biological phenomenon in which misfolded proteins aggregate i.
Guest editor steve shire protein aggregation and bioprocessing. Bioprocessing is the use of biological materials organisms, cells, organelles, enzymes to carry out a process for commercial, medical or scientific reasons. Sheila has implemented quality systems and has managed external analytical and qc activities for multiple biopharmaceutical products. Stability of human serum albumin during bioprocessing. 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. However, for pharmacological applications, protein aggregation represents a major obstacle to the development of proteinbased pharmaceuticals. Mechanism, characterization and consequences protein aggregation is recognized by regulatory agencies and the biopharmaceutical industry as a key quality attribute of biotherapeutic products. In the present context, this may include both reversible selfassociation and effectively irreversible aggregation.
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. Aggregation in biopharmaceuticals remains a major concern and threatens the stability of a product. It is possible to influence the amount of aggregate. Simultaneously, protein aggregation can occur during manufacturing and bioprocessing due to pumping, stirring and shaking. Glycerol also inhibits protein aggregation during the refolding of many proteins. Host cell protein per mg of mab in order to ensure product quality, mab concentration monitoring is one of the cqas. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the envi. Proteinsimple 3001 orchard parkway san jose, california, 954 usa. Biopharmaceutical product stability considerations, part. Protein aggregation and subvisible particle analysis is a breeze with microflow imaging mfi. Each of these environmental factors is typically encountered during bioprocessing.
In leather tanning, hides are softened and hair removed using the proteases in faeces. 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. 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. What are the different methods of determining protein aggregation.
Recently, new analytical methods enabled characterization of mab aggregation during bioprocessing of mammalian cells. Not only is mfi more sensitive than traditional techniques, it also lets you discriminate between protein aggregates and other particulates. Microaggregated subvisible particles generated anywhere in a manufacturing process can develop into larger particles over time as a product is stored 62. Bioprocessing tutorial attaining high transient titers in. Understanding the influence of adsorptionmediated processes on antibody aggregation in bioprocessing. Sep 15, 2006 protein aggregation is a common issue encountered during manufacture of biotherapeutics. These observations rationalize the diverse effects of hydrodynamic flow on protein structure and aggregation propensity seen in both nature and in protein manufacture. Glycerol is known to shift the native protein ensemble to more compact states. The kinetics of protein aggregation has been extensively investigated. Protein aggregation is a common issue encountered during manufacture of biotherapeutics. 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. Mechanisms of protein stabilization and prevention of. Mechanisms of protein aggregation current pharmaceutical biotechnology, 2009, vol.
Related disorders include cataracts, arthritis, cystic fibrosis, lateonset diabetes mellitus, and numerous neurodegenerative diseases like alzheimers and parkinsons. Scientific and regulatory perspectives on measuring. Addressing protein aggregation the formation of protein aggregates in biopharmaceuticals can be difficult to predict and control. Protein aggregation during bioprocessing of monoclonal. Continuous bioprocessing at the industrial scale has long been a goal for bioprocessing. Attenuated total reflectance fourier transform infrared spectroscopy atrftir was used to study the conformation of aggregated proteins in vivo and in vitro.
Identification of process conditions influencing protein. However, for pharmacological applications, protein aggregation represents a major obstacle to the development of protein based pharmaceuticals. Understanding of the roles of aggregates in immunogenicity of therapeutic protein products is critical. The effects of shear flow on protein structure and function innocent b. Cambridge healthtech institute peptalk palm springs, ca. Clinical studies have shown adverse effects and safety issues caused by protein aggregation 2, 3. Host cell protein per mg of mab in order to ensure product quality, mab. Analyzing protein aggregation in biopharmaceuticals. Aaps open forum on protein aggregation in san francisco, ca on june 5, 2006, and brie. Benjamin patrick werner and gerhard winter 11222017. Nanotechnology and nanoimaging technology, including cryoelectron microscopy of beta2microglobulin, studying amyloidogensis. Many types of aggregates are known and their size can differ from nanometers to the visible range 46.
The structure of hsa wasexamined using fourier transform. Pdf protein aggregation and bioprocessing researchgate. 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. Those challenges require screening a vast array of conditions. 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.
Mechanisms of protein stabilization and prevention of protein. Identification of process conditions influencing protein aggregation. Protein misfolding, aggregation and methionine oxidation. Igg4 was incubated in elution buffer after protein a chromatography and the monomer concentration in neutralised samples was quantified by size exclusion. 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. 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. Alternatively, cell line engineering using a mirna enabled enhancement of specific productivity without negative impact on protein aggregation and glycosylation. What are the causes of protein aggregation in biologic formulations.
Process analytical technology pat for continuous bioprocessing. 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. Optimizing protein aggregate analysis by size exclusion chromatography authors rowan moore,1 amy farrell,2 jonathan bones,2 and ken cook1 1thermo fisher scienti. 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. Attaining high transient titers in cho cells bioprocessing tutorial october 1, 2015 volume 35, number 17. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. Pdf protein aggregation is a common issue encountered during manufacture of biotherapeutics.
Protein aggregation and particle analysis proteinsimple. Aggregates in monoclonal antibody manufacturing processes. In fact, protein aggregates have been implicated in a wide variety of disease known as amyloidoses, including als, alzheimers. The full text of this article is available as a pdf 304k. Little information has been published on the extent and causes of aggregation during bioprocessing for pharmaceutical proteins. In disease states, protein aggregation can occur as amyloid fibrillogenesis. Aggregation of therapeutic proteins can occur during bioprocessing, shipping, storage and even delivery to the patient. 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.
Protein aggregation mechanisms, detection, and control. 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. Percent aggregation by sizeexclusion hplc figure 3. Disulfide bond formation resulting from previously unpaired free thiols is a common mechanism for covalent aggregation.
Protein aggregation and bioprocessing springerlink. Expression yields were too low, culture media too primitive, and the tech. Measuring protein aggregation of biotechnology products ashutosh rao, ph. 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. Vice president and senior consultant, bptc susan is a seasoned biotechnology.
To assess the impact of various bioprocessing steps on thestability of freshly precipitated human serum albumin hsa obtainedfrom pooled human plasma. Protein aggregation is a common issue encountered during. Review the effects of shear flow on protein structure and. Protein aggregation of monoclonal antibodies mabs is a common phenomenon associated with the production of these biopharmaceuticals. Protein crystallization screening poster pegs 2019 pdf download insulin homologs and biosimilars poster peptalk 2019 pdf download biologic developability poster bioprocessing summit 2018 pdf. Review the effects of shear flow on protein structure and function innocent b. Bekard,1 peter asimakis,1 joseph bertolini,2 dave e. In the type of situation described by a lumry eyring modelsa. 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. The influence of elution from protein a on the aggregation rate of an igg4 during a subsequent low ph hold was investigated. Performance characteristics of the expicho transient expression system. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and ef. Nov 22, 2017 addressing protein aggregation the formation of protein aggregates in biopharmaceuticals can be difficult to predict and control.
A expichos cells at passage 10 or passage 34 postthaw consistently attain. Understanding the influence of adsorptionmediated processes. Some industries have a long tradition of enzyme use. Dec 27, 2017 protein aggregation of monoclonal antibodies mabs is a common phenomenon associated with the production of these biopharmaceuticals. 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. These protein aggregates are often correlated with diseases. Protein aggregation to explain this di erence, we use as an example the stability of a monoclonal antibody at low ph. Heres how we can improve patient safety through primary packaging materials and bedside filtration. Bioprocess development upstream and downstream technologies monica chuong, phd school of pharmacyboston 1. 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. In contrast to glycosylation, the other ptms of protein therapeutics are less well studied and understood chirino and miresluis 2004. Protein aggregation is favored in the food industry since it imparts desirable texture and flavor. Solid state fermentation microorganisms grow on a moistened solid surface without free flowing water, but have free access to air.
379 874 889 1183 446 16 110 1332 1513 471 1430 1239 134 755 988 963 897 372 108 1090 465 535 469 632 1108 490 822 730 1329 854 1284 531 253 144 512 1330 520 1084 1144 172