Characterization of aggregation profiles of monoclonal antibodies. Role of water in protein aggregation and amyloid polymorphism d. Tdp43 has been shown to bind to dna and mrna and participate in. Thus, we develop a nonarrhenius model to connect accelerated aggregation experiments at high temperatrue to longterm storage experiments at low temperatrue. Factors that influence protein aggregation protein aggregation is an undesired interaction between protein monomers and can be caused by factors such as ph, ionic strength, mechanical agitation, protein concentration, freezethaw operations, temperature, and packaging materials and agents such as. In all cases, there is an argument for trying to engineer the protein i. 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. The hypothesis that protein aggregation is a causative process in aging is testable now since some models of delayed aging are in hand.
Thus, we develop a non arrhenius model to connect accelerated aggregation experiments at high temperatrue to longterm storage experiments at low temperatrue. Protein aggregation is undesired interaction between protein monomers and can be caused by factors such as ph, ionic strength, mechanical agitation, protein concentration, freezethaw operations, temperature, and packaging materials and agents such as glass and silicone oil. The protein searches for a set of critical contacts unify the description of protein folding and aggregation. It is well established that heating cooling can accelerate slow aggregation by populating depopulating unfolded or partially unfolded monomer states that are key intermediates in aggregation processes.
Helixstrand interaction regulates stability and aggregation. Singleresidue physicochemical characteristics kinetically. Linkage between dynamics and catalysis in a thermophilic. Modeling protein degradation processes and the development of.
The occurrence of protein aggregation during bioprocessing steps such as puri. Apr 25, 20 protein aggregation is a complex, multistep process. It affects not only product quality but also potentially impacts safety, as protein aggregates have been shown to be linked with cytotoxicity and patient immunogenicity. What is protein aggregation and what factors influence protein aggregation. Ma presented mechanistic insights into protein aggregation, and the development of rational approaches and predictive tools for the stabilization of biopharmaceuticals.
Protein aggregation in affected motor neurons is a central hallmark of als, and recent genetic, cellular and histological studies have enlarged our understanding of the molecular composition of these aggregates. A paradigmatic example is provided by a comparison of the already mentioned case of deoxyhbs and that of coliganded hbs cohbs. The aggregation of monoclonal antibodies occurs, with non arrhenius kinetics, via a partially unfolded conformation. Role of water in protein aggregation and amyloid polymorphism. Department of mechanical engineering, the university of texas at dallas, richardson, texas 75080, united states department of chemistry and biochemistry, the university of texas at dallas. Temperaturedependent aggregation kinetics could be divided into two regimes. Preventing protein aggregation detection of aggregates. It is also significant to note that multidomain proteins such as mabs usually aggregate with the nonarrhenius kinetics over a wide temperature. Request pdf nonarrhenius protein aggregation protein aggregation presents one of the key challenges in the development of protein biotherapeutics. Moreover, protein aggregation is an important aspect for regulatory agencies and. Changes in conformational stability, proteinprotein interactions, and aggregation of ns0produced human fc1 were quantified experimentally as a function of ph 4 to 6 and temperature 30 to 77 c, using a combination of differential scanning calorimetry, laser light scattering, sizeexclusion chromatography, and capillary electrophoresis. The physical processes of aggregation, especially in salt solutions, are not well understood. Understanding protein aggregation is important as this process is implicated in many human diseases such as alzheimers, parkinsons, prions and type ii diabetes. The nonarrhenius regime was most pronounced for lower temperatures.
The work highlights i the significance of incorporating unfolding thermodynamics in protein aggregation rate prediction, ii the advantages and challenges associated with the use of the extended lumryeyring ele model for rate prediction and iii the utility of using the arrhenius and the ele models in tandem during product development. Predicting accelerated aggregation rates for monoclonal. Using this framework and monte carlo simulations, we show that reaction rates for protein aggregation are of the arrheniuseyring type and. They are created, modulated, and destroyed according to a temporal plan that meets the survival needs of the cell, organism, and species. Protein aggregation is an extremely complex process, characterized by different steps which routinely consist in the formation of early aggregates, followed by a variety of intermediates, usually evolving into mature fibrils. Protein aggregation an overview sciencedirect topics. Insight into the correlation between lag time and aggregation rate in the kinetics of protein aggregation stefan auer1, and dimo kashchiev2 1centre for molecular nanoscience, university of leeds, leeds, ls2 9jt, uk 2institite of physical chemistry, bulgarian academy of sciences, ul. A method of determining the activation energy e a for degradation of a chemical species includes in sequence the steps of a simultaneously incubating a plurality of samples of the chemical species in a single unitary device at a plurality of constant temperatures t, in each case for an incubation time t selected to result in loss of at most 20 mol % of the amount originally present. Protein aggregation in amyotrophic lateral sclerosis, acta. Any or all of these can be exploited to induce or inhibit aggregation. Aug 29, 2004 transverse nmr relaxation rates r 2 were measured as a function of protein concentration to ensure that there was no contribution to the observed r 2 from protein aggregation at concentrations. Understanding aggregation mechanisms and developing mitigating strategies are imperative to ensure the. Tools to monitor protein aggregation enzo life sciences.
Mechanisms of protein aggregation current pharmaceutical biotechnology, 2009, vol. It affects not only product quality but also potentially impacts safety, as protein aggregates. Connecting hightemperature and lowtemperature protein. Nonarrhenius aggregation behavior may lead to an underestimation of protein aggregation rate at the recommended storage temperature based on accelerated stability data wang and roberts, 20. Transient intermediates in the aggregation pathway appear as non arrhenius behavior profiles at high temperatures only for some mutants and only in low dprs figs.
It could be used at characterization of mab aggregation comparison of hplc method and a quick method developed using the cary 60 uvvis authors arunkumar padmanaban, sreelakshmy menon agilent technologies, inc. Jan 31, 2012 protein folding kinetics is known to be non arrhenius temperature dependent. Nonnative protein aggregation is a common challenge associated with processing, handling, and storage of recombinant proteinsparticularly those intended as drug products. Aging and proteins nick weir second year graduate student, denic lab department of molecular and cellular biology harvard university. Therefore, for designing therapeutics, the exact mechanism of protein aggregation should be clear. Protein aggregation and soluble aggregate formation. Us9632095b2 device and method for determining reaction. Disruption of this delicate balance can expose internal.
Protein aggregation in amyotrophic lateral sclerosis pdf. Top a, roberts cj, kiick kl conformational and aggregation properties of a pegylated alaninerich polypeptide biomacromolecules 12. Gibbs energy wells has been used to analyse the nonarrhenius behaviour of. Protein aggregation in amyotrophic lateral sclerosis. The arrhenius equation in the study of foods 89 if we assume that the deterioration of the foods follows the arrhenius equation and treat the storage data accordingly, we find that approximately straight lines can be drawn. Mechanism of protein kinetic stabilization by engineered. Jul 14, 2015 in such intricate scenario, both experimental and computational studies suggest the existence of general physical principles which govern protein aggregation and supramolecular structure formation 6365. It is also significant to note that multidomain proteins such as mabs usually aggregate with the non arrhenius kinetics over a wide temperature. Weak protein interactions and ph and temperaturedependent.
Straub biophysics program, institute for physical science and. 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. Nonnative aggregation is one of the most common degradation routes that may affect. Dobson2 oxford centre for molecular sciences, new chemistry laboratory. The role of solvent in protein folding and in aggregation. Jun 16, 2007 a mathematical model is presented of the kinetics of nonnative protein aggregation that combines lumry. Blanco ma, sahin e, li y, roberts cj reexamining protein protein and protein solvent interactions from kirkwoodbuff analysis of light scattering in multicomponent solutions. In a small number of cases, native reversible aggregation is a major benefit to product quality, as the native oligomers have higher stability and potentially higher solubility than their monomeric counterparts insulin in the presence of zinc ions is the canonical example of this situation for a protein pharmaceutical product 11,47. In particular, we find that the kinetics can be described by a secondorder model and are non arrhenius. The kinetics and structural transitions of nonnative aggregation of.
Connecting hightemperature and lowtemperature protein stability. Temperature dependence of protein folding in live cell. Oct 20, 2017 aggregation kinetics frequently show non arrhenius behaviour even over short temperature ranges. Protein aggregation is a complex, multistep process.
Due to the complexity of the aggregation process and temperaturedependent conformational stability, temperatureinduced protein aggregation is often nonarrhenius over even relatively small temperature windows relevant for product development, and this makes lowtemperature extrapolation difficult based simply on accelerated stability studies at high temperatures. Computational design and biophysical characterization of. What are the causes of protein aggregation in biologic formulations. Analysis of twostate folding using parabolic approximation iii arxiv. If the development of protein aggregates was an aging independent process, slowing down aging will show no effect on the rate of proteotoxicity over time. A case study on high concentration solutions of a multidomain protein, pharmaceutical research on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips.
Desolvation is a likely origin of robust enthalpic barriers. Palma infm, progetto sud and unita di palermo, at department of physical and astronomical sciences, university of palermo, palermo, italy. This is a common concern, as even a small percentage of aggregated molecules can increase the risk of undesired patient immune responses. Crispr helps identify new protein potentially involved in. What are the different methods of determining protein. Starting at the folded monomer state center of figure 1, proteins can reversibly form small dimers and oligomers that are stable complexes that can be separated from the monomer or are in dynamic equilibrium with the monomer state. Nonnative protein aggregation is a common challenge associated. However, there are many factors that can contribute to protein aggregation including ionic strength, ph, temperature, agitation, freezethaw cycles and even the container you store your protein in. Protein aggregationcrystallization and minor structural. Evaluation of a nonarrhenius model for therapeutic. Nonarrhenius protein aggregation request pdf researchgate. Protein aggregation and its inhibition in biopharmaceutics. Example of the aggregation behavior of a monoclonal antibody solution at low ph the order of a few nanometers, and are thus lying in the colloidal range. Nonlinear or nonarrhenius aggregation rates are most typically observed for proteins that exist primarily in folded states in their monomeric form, although it also occurs when there is a change in ratelimiting step e.
Aggregation kinetics for monoclonal antibody products. A mathematical model of nonnative protein aggregation has also been presented which combines the lumry eyring and nucleated polymerization lenp descriptions that is useful under conditions where detailed kinetics of. With modulated heating laser, we are able to measure the thermodynamics of pgk rapidly across the midpoint of transition of protein unfolding to minimize baseline shifts coming. A statistical thermodynamic model of the protein ensemble. The role of solvent in protein folding and in aggregation s.
A comparison of thermodynamic, kinetic, and spectroscopic data shows that aggregation under nonnativefavoring. Bonchev 11, sofia 11, bulgaria corresponding author. Protein aggregation is a longstanding problem for preservation of proteins in both laboratory settings and for commercial biotechnology products. Desolvation is a likely origin of robust enthalpic barriers to protein folding zhirong liu and hue sun chan protein engineering network of centres of excellence department of biochemistry and department of medical genetics and microbiology faculty of medicine, university of toronto, toronto, ont. Nonlinear or non arrhenius aggregation rates are most typically observed for proteins that exist primarily in folded states in their monomeric form, although it also occurs when there is a change in ratelimiting step e. Conformational stability and aggregation of therapeutic monoclonal.
The lenp model is solved for cases in which aggregation rates are slow compared to folding. Eyring and nucleated polymerization lenp descriptions. Significance of unfolding thermodynamics for predicting. Effects of macromolecular crowding on protein folding and. Protein aggregation presents one of the key challenges in the development of protein biotherapeutics. Together with the weak protein protein repulsions, these highlight challenges that are expected for maintaining longterm stability of biotechnology products. Protein aggregation indian institute of technology bombay. Nonarrhenius modes in the relaxation of model proteins. Jan 31, 2005 read protein aggregation and its inhibition in biopharmaceutics, international journal of pharmaceutics on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. However, mechanisms of protein aggregation can be exceedingly complex, involving conformational rearrangement, nucleation and growth steps, as well as processes of aggregate fragmentation, aggregate coalescence to yield larger aggregates and phase separation of insoluble aggregates. Application note biopharma abstract protein aggregation, where molecules assemble into dimers, trimers, tetramers, or larger multimers, is a.
Patterns of gene expression in microarrays and expressed sequence tags from normal and cataractous lenses. Nonarrhenius modes in the relaxation of model proteins maksim skorobogatiy, hong guo, and martin zuckermann department of physics and centre for the physics of materials, mcgill university, montre. A consequence of this is that it is challenging to extrapolate the results of aggregation kinetics commonly measured at higher temperatures to lower temperatures. Modeling protein degradation processes and the development of rational approaches to stabilization a new strategy of molecular qbd naresh chennamsetty1, vladimir voynov1, veysel kayser, veysel kayser1, curt schneider1, diwakar shulka1, bernhard helk2 and bernhardt l. Nonarrhenius reactiondiffusion kinetics for protein. Factors affecting the physical stability aggregation of. Protein aggregation is a problem in amyloid and other diseases, and it is a challenge when formulating solutions of biological drugs, such as monoclonal antibodies. Conformational stability and aggregation of therapeutic. In accordance with the eukaryotic systems, it has been documented that protein aggregation also reduces fitness of bacterial cells, but although our understanding of the cellular protein quality control systems is perhaps most detailed in bacteria, the use of bacterial proteostasis as a.
No, increasing protein aggregation during aging and in disease doesnt give evidence for programmed aging in humans. Because of the complex nature of hv3 aggregation, we did not study this deviation in. Mechanistic insights into the role of protein interactions on the aggregation behavior of antistreptavidin immunoglobulin gamma1 by gregory v. Role of protein aggregation in neurodegenerative diseases. Analyzing protein aggregation in biopharmaceuticals. Protein aggregation and its impact on product quality. Blanco ma, sahin e, li y, roberts cj reexamining proteinprotein and proteinsolvent interactions from kirkwoodbuff analysis of light scattering in multicomponent solutions. Jan 08, 2014 read significance of unfolding thermodynamics for predicting aggregation kinetics. Jun 10, 2011 overall, the results demonstrate a novel strategy to quantitatively determine aggregation rates at time scales of industrial interest, based on k obs values from tsml, which are sample.
Such a non arrhenius behavior has been observed for other proteins such as lysozyme matagne et al. Multiple detector approaches to protein aggregation by sec author andrew coffey agilent technologies, inc. Protein aggregation mechanisms, detection, and control. However, protein aggregation often exhibits nonarrhenius temperature dependence, even over relatively narrow temperature ranges. Barnett a dissertation submitted to the faculty of the university of delaware in partial ful llment of the requirements for the degree of doctor of philosophy in chemical engineering fall 2015. In fact, minor protein structuralconformational changes have proven to affect aggregation properties via both the location and shape of the instability region. Multiple detector approaches to protein aggregation by sec. Evaluation of a nonarrhenius model for therapeutic monoclonal antibody aggregation article in journal of pharmaceutical sciences 1007. In particular, we find that the kinetics can be described by a secondorder model and are nonarrhenius.
Preventing protein aggregation protein aggregation cause artifacts in most biophysical techniques, leads to uncertainty in the active concentration of protein in solution and loss of biological activity. The results provide a basis for what to expect for aggregation mediated by partial unfolding of the fc1 when dealing with a range of different fcbased platforms, as well as for igg proteins that have aggregation resistant fab designs. The occurrence of protein aggregation during bioprocessing steps such as purification, formulation and fillfinish, impacts yield and production costs, and must be controlled throughout the. Protein aggregation why it matters, and how to study it. If the protein remains folded upon aggregation and hence is likely undergoing reversible aggregation, then major concerns with aggregation are typically one or more of the following. Singleresidue physicochemical characteristics kinetically partition. Key di erences between proteins and polymer colloids figure 9. The global analysis of e act suggests that similar to stability, the e act is also affected by trp substitution. We use fast relaxation imaging frei to measure stability and folding kinetics of fretlabeled destabilized phosphoglycerate kinase pgk. Unfortunately, it is a common problem for many protein constructs when removed from their biological context e.
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