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. 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. A paradigmatic example is provided by a comparison of the already mentioned case of deoxyhbs and that of coliganded hbs cohbs. Using this framework and monte carlo simulations, we show that reaction rates for protein aggregation are of the arrheniuseyring type and. It is also significant to note that multidomain proteins such as mabs usually aggregate with the nonarrhenius kinetics over a wide temperature. Role of protein aggregation in neurodegenerative diseases. Jan 31, 2012 protein folding kinetics is known to be non arrhenius temperature dependent. Nonnative aggregation is one of the most common degradation routes that may affect. 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. Thus, we develop a non arrhenius model to connect accelerated aggregation experiments at high temperatrue to longterm storage experiments at low temperatrue. Singleresidue physicochemical characteristics kinetically. 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.
They are created, modulated, and destroyed according to a temporal plan that meets the survival needs of the cell, organism, and species. Protein aggregation mechanisms, detection, and control. It affects not only product quality but also potentially impacts safety, as protein aggregates. A statistical thermodynamic model of the protein ensemble.
Role of water in protein aggregation and amyloid polymorphism. Nonarrhenius protein aggregation request pdf researchgate. Top a, roberts cj, kiick kl conformational and aggregation properties of a pegylated alaninerich polypeptide biomacromolecules 12. Connecting hightemperature and lowtemperature protein. 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. 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. Role of water in protein aggregation and amyloid polymorphism d. Oct 20, 2017 aggregation kinetics frequently show non arrhenius behaviour even over short temperature ranges. Singleresidue physicochemical characteristics kinetically partition. Modeling protein degradation processes and the development of. Predicting accelerated aggregation rates for monoclonal.
Any or all of these can be exploited to induce or inhibit aggregation. Application note biopharma abstract protein aggregation, where molecules assemble into dimers, trimers, tetramers, or larger multimers, is a. Computational design and biophysical characterization of. Nonnative protein aggregation is a common challenge associated with processing, handling, and storage of recombinant proteinsparticularly those intended as drug products. Effects of macromolecular crowding on protein folding and. However, protein aggregation often exhibits nonarrhenius temperature dependence, even over relatively narrow temperature ranges. 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. What are the causes of protein aggregation in biologic formulations. Nonarrhenius reactiondiffusion kinetics for protein. Helixstrand interaction regulates stability and aggregation. Temperature dependence of protein folding in live cell. A comparison of thermodynamic, kinetic, and spectroscopic data shows that aggregation under nonnativefavoring. Ma presented mechanistic insights into protein aggregation, and the development of rational approaches and predictive tools for the stabilization of biopharmaceuticals. 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.
Understanding aggregation mechanisms and developing mitigating strategies are imperative to ensure the. Evaluation of a nonarrhenius model for therapeutic. 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. Analysis of twostate folding using parabolic approximation iii arxiv. 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. Mechanistic insights into the role of protein interactions on the aggregation behavior of antistreptavidin immunoglobulin gamma1 by gregory v. Protein aggregation why it matters, and how to study it. Together with the weak protein protein repulsions, these highlight challenges that are expected for maintaining longterm stability of biotechnology products. 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. The aggregation of monoclonal antibodies occurs, with non arrhenius kinetics, via a partially unfolded conformation. Nonarrhenius modes in the relaxation of model proteins. Connecting hightemperature and lowtemperature protein stability. Linkage between dynamics and catalysis in a thermophilic. 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. Nonnative protein aggregation is a common challenge associated. Preventing protein aggregation detection of aggregates. In fact, minor protein structuralconformational changes have proven to affect aggregation properties via both the location and shape of the instability region. 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. No, increasing protein aggregation during aging and in disease doesnt give evidence for programmed aging in humans. 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. 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.
Protein aggregation is a longstanding problem for preservation of proteins in both laboratory settings and for commercial biotechnology products. Crispr helps identify new protein potentially involved in. Request pdf nonarrhenius protein aggregation protein aggregation presents one of the key challenges in the development of protein biotherapeutics. Desolvation is a likely origin of robust enthalpic barriers. Protein aggregation is a complex, multistep process. Protein aggregation presents one of the key challenges in the development of protein biotherapeutics.
Significance of unfolding thermodynamics for predicting. Tdp43 has been shown to bind to dna and mrna and participate in. Weak protein interactions and ph and temperaturedependent. Aggregation kinetics for monoclonal antibody products. Dobson2 oxford centre for molecular sciences, new chemistry laboratory.
The physical processes of aggregation, especially in salt solutions, are not well understood. Straub biophysics program, institute for physical science and. Because of the complex nature of hv3 aggregation, we did not study this deviation in. It is also significant to note that multidomain proteins such as mabs usually aggregate with the non arrhenius kinetics over a wide temperature. Protein aggregation in amyotrophic lateral sclerosis pdf. 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.
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. The kinetics and structural transitions of nonnative aggregation of. Temperaturedependent aggregation kinetics could be divided into two regimes. Unfortunately, it is a common problem for many protein constructs when removed from their biological context e. 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. 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. 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. Blanco ma, sahin e, li y, roberts cj reexamining protein protein and protein solvent interactions from kirkwoodbuff analysis of light scattering in multicomponent solutions. What are the different methods of determining protein. 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. Such a non arrhenius behavior has been observed for other proteins such as lysozyme matagne et al. This is a common concern, as even a small percentage of aggregated molecules can increase the risk of undesired patient immune responses. The protein searches for a set of critical contacts unify the description of protein folding and aggregation. Mechanisms of protein aggregation current pharmaceutical biotechnology, 2009, vol.
Patterns of gene expression in microarrays and expressed sequence tags from normal and cataractous lenses. Characterization of aggregation profiles of monoclonal antibodies. Protein aggregation and soluble aggregate formation. Conformational stability and aggregation of therapeutic. The occurrence of protein aggregation during bioprocessing steps such as puri. 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.
A consequence of this is that it is challenging to extrapolate the results of aggregation kinetics commonly measured at higher temperatures to lower temperatures. What is protein aggregation and what factors influence protein aggregation. Jun 16, 2007 a mathematical model is presented of the kinetics of nonnative protein aggregation that combines lumry. In all cases, there is an argument for trying to engineer the protein i. 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. The role of solvent in protein folding and in aggregation. 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.
Multiple detector approaches to protein aggregation by sec. Moreover, protein aggregation is an important aspect for regulatory agencies and. 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. Multiple detector approaches to protein aggregation by sec author andrew coffey agilent technologies, inc. Aging and proteins nick weir second year graduate student, denic lab department of molecular and cellular biology harvard university. The role of solvent in protein folding and in aggregation s. The nonarrhenius regime was most pronounced for lower temperatures. Protein aggregation indian institute of technology bombay. 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. Evaluation of a nonarrhenius model for therapeutic monoclonal antibody aggregation article in journal of pharmaceutical sciences 1007. 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 lenp model is solved for cases in which aggregation rates are slow compared to folding. 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. 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. Protein aggregation an overview sciencedirect topics. Protein aggregation in amyotrophic lateral sclerosis.
Gibbs energy wells has been used to analyse the nonarrhenius behaviour of. 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. Understanding protein aggregation is important as this process is implicated in many human diseases such as alzheimers, parkinsons, prions and type ii diabetes. Mechanism of protein kinetic stabilization by engineered. The hypothesis that protein aggregation is a causative process in aging is testable now since some models of delayed aging are in hand. Jan 08, 2014 read significance of unfolding thermodynamics for predicting aggregation kinetics. Therefore, for designing therapeutics, the exact mechanism of protein aggregation should be clear. 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. 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. Eyring and nucleated polymerization lenp descriptions. We use fast relaxation imaging frei to measure stability and folding kinetics of fretlabeled destabilized phosphoglycerate kinase pgk. 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.
Protein aggregation and its inhibition in biopharmaceutics. 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. 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. 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. 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. The global analysis of e act suggests that similar to stability, the e act is also affected by trp substitution. 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. 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. Bonchev 11, sofia 11, bulgaria corresponding author. 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. Key di erences between proteins and polymer colloids figure 9. Disruption of this delicate balance can expose internal.
Thus, we develop a nonarrhenius model to connect accelerated aggregation experiments at high temperatrue to longterm storage experiments at low temperatrue. In particular, we find that the kinetics can be described by a secondorder model and are non arrhenius. 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. Conformational stability and aggregation of therapeutic monoclonal.
Protein aggregation in amyotrophic lateral sclerosis, acta. Protein aggregationcrystallization and minor structural. Factors affecting the physical stability aggregation of. Us9632095b2 device and method for determining reaction. 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. 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. Palma infm, progetto sud and unita di palermo, at department of physical and astronomical sciences, university of palermo, palermo, italy. 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. Apr 25, 20 protein aggregation is a complex, multistep process.
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