AGGRESCAN and its evolution: A two-decade perspective on protein aggregation prediction

Two Decades of Publishing Excellence in Pharmaceutical Biotechnology

Aggregation is an ancient threat that must be overcome by proteins from all organisms to maintain their native functional states. This is essential for the maintenance of metabolic flux and viability of their cellular machineries. Here, we compare the aggregation-resistance strategies adapted by the thermophilic proteins and their mesophilic homologs using a dataset of 373 protein families. Like their mesophilic homologs, the thermophilic protein sequences also contain potential aggregation prone regions (APRs), capable of forming cross-β motif and amyloid-like fibrils. Tetrapeptide and hexapeptide amyloid-like fibril forming sequence patterns and experimentally proven amyloid-like fibril forming peptide sequences were also detected in the thermophilic proteins. Both the thermophilic and the mesophilic proteins use similar strategies to resist aggregation. However, the thermophilic proteins show superior utilization of these strategies. The thermophilic protein monomers show greater ability to "stow away" the APRs in the hydrophobic cores to protect them from solvent exposure. The thermophilic proteins are also better at gatekeeping the APRs by surrounding them with charged residues (Asp, Glu, Lys, and Arg) and Pro to a greater extent. While thermophilic and mesophilic proteins in our dataset are highly homologous and show strong overall sequence conservation, the APRs are not conserved between the homologs. These findings indicate that evolution is working to avoid amyloidogenic regions in proteins. Our results are also consistent with the observation that thermophilic cells often accumulate small molecule osmolytes capable of stabilizing their proteins and other macromolecules. This study has important implications for rational design and formulation of therapeutic proteins and antibodies.

Arginine (Arg) is a natural amino acid with an acceptable safety profile and a unique chemical structure. Arg and its salts are highly effective in enhancing protein refolding and solubilization, suppressing protein-protein interaction and aggregation and reducing viscosity of high concentration protein formulations. Arg and its salts have been used in research and 20 approved protein injectables. This review summarizes the effects of Arg as an excipient in therapeutic protein formulations with the focus on its physicochemical properties, safety, applications in approved protein products, beneficial and detrimental effects in liquid and lyophilized protein formulations when combined with different counterions and mechanism on protein stabilization and destabilization. The decade literature review indicates that the benefits of Arg overweigh its risks when it is used appropriately. It is recommended to add Arg along with glutamate as a counterion to high concentration protein formulations on top of sugars or polyols to counterbalance the negative effects of Arg hydrochloride. The use of Arg as a viscosity reducer and protein stabilizer in high concentration formulations will be the inevitable future trend of the biopharmaceutical industry for subcutaneous administration.

Disrupted protein folding or decreased protein stability can lead to the accumulation of (partially) un- or misfolded proteins, which ultimately cause the formation of protein aggregates. Much of the interest in protein aggregation is associated with its involvement in a wide range of human diseases and the challenges it poses for large-scale biopharmaceutical manufacturing and formulation of therapeutic proteins and peptides. On the other hand, protein aggregates can also be functional, as observed in nature, which triggered its use in the development of biomaterials or therapeutics as well as for the improvement of food characteristics. Thus, unmasking the various steps involved in protein aggregation is critical to obtain a better understanding of the underlying mechanism of amyloid formation. This knowledge will allow a more tailored development of diagnostic methods and treatments for amyloid-associated diseases, as well as applications in the fields of new (bio)materials, food technology and therapeutics. However, the complex and dynamic nature of the aggregation process makes the study of protein aggregation challenging. To provide guidance on how to analyse protein aggregation, in this review we summarize the most commonly investigated aspects of protein aggregation with some popular corresponding methods.

The ability to control or reverse protein aggregation is vital to the production and formulation of therapeutic proteins and may be the key to the prevention of a number of neurodegenerative diseases. In recent years, laboratory studies of the phenomenon have been accompanied by a growing number of computational treatments aimed at elucidating the molecular mechanisms of aggregation. The present article is a continuation of our simulation studies of coarse-grained model oligopeptides that mimic aggregating proteins. The potential function of a multichain system is expressed in terms of a generalized Go model for a set of sequences with varying contents of secondary-structural motifs akin to α-helices and β-sheets. Conformational evolution is considered by conventional Monte Carlo simulation, and by a variation of the Replica Monte Carlo technique that facilitates barrier-crossing in glasslike aggregated systems. The foldability and aggregation propensity are monitored as functions of the extent of different secondary structures and the length of the chains. Our results indicate that an increased proportion of sheetlike structures facilitates folding of isolated chains, but strongly favors the formation of misfolded aggregates in multichain systems, in agreement with experimental observations. This behavior is interpreted in terms of cooperativity effects associated with the formation of multiple residue–residue bonds involving adjacent monomers in interacting segments, which enhance both intramolecular binding and interprotein association.

Monitoring particles within therapeutic protein formulations is necessary to evaluate drug safety and efficacy. Current regulations require analysis of particles greater than 10 μm, however quantitation of particles 1–10 μm is increasingly expected due to potential immunogenicity. These small particles are difficult to measure with current methods, and new methods must be developed. Amnis Imaging Flow Cytometry (IFC) is an existing technique that is conceptually well-suited for this application because provides: (1) Sensitive detection particles across a wide size range (1–100 μm), (2) Direct particle size/shape measurements from brightfield imagery (3) Particle identification using fluorescent dyes, (4) High sampling efficiency (95%) (5) Low sample volume requirement (20 μL). Here we demonstrate IFC may be used to measure particles common to this application, including protein aggregates, silicone oil droplets, polystyrene bead standards, and newly developed protein surrogate standards. IFC provided sensitive detection of particles as small as 100 nm and could be used to discriminate protein aggregates from silicone oil. Interestingly, IFC analysis of one sample revealed transparent protein aggregates invisible using current gold-standard methods. The results demonstrate that IFC is a useful orthogonal technique to current methods for interrogating small and translucent particles within protein formulations.

Ionic liquids (ILs) have recently emerged as versatile solvents and additives in the field of biotechnology, particularly as stabilizers of proteins and enzymes. Of interest to the biotechnology industry is the formulation of stable biopharmaceuticals, therapeutic proteins, and vaccines which have revolutionized the treatment of many diseases including debilitating conditions such as cancers and auto-immune diseases. The stabilization of therapeutic proteins is typically achieved using additives that prevent unfolding and aggregation of these proteins during manufacture, transport, and long-term storage. To determine if ILs could be used in the formulation of stable therapeutic proteins, a thorough understanding of the effects of ILs on protein stability is needed, as well as understanding the toxicity of ILs on humans, and other considerations for formulation development such as viscosity and osmolality. In this review, we summarize recent developments on the stabilization of proteins and enzymes using ILs, with emphasis on identifying biocompatible ILs that may be suitable for the formulation of stable biopharmaceuticals in the future.

Most protein sequences contain one or several short aggregation prone regions (APR) that can nucleate protein aggregation. Under normal conditions these APRs are protected from aggregation by protein interactions or because they are buried in the hydrophobic core of native protein domains. However, mutation, physiological stress or age-related disregulation of protein homeostasis increases the probability that aggregation-nucleating regions become solvent exposed. Aggregation then results from the self-assembly of APRs into β-structured agglomerates that vary from small soluble oligomeric assemblies to large insoluble inclusions containing thousands of molecules. The functional effects of APR-driven aggregation are diverse and protein-specific leading to distinct disease phenotypes ranging from neurodegeneration to cancer. On a cellular and physiological level both wild type loss-of-function as well as aggregation-dependent gain-of-function effects have been shown to contribute to disease. Several molecular mechanism have been proposed to contribute to gain-of-function activity of protein aggregates including cellular membrane disregulation, saturation of the protein quality control machinery or the ability of aggregates to engage non-native interactions with proteins and nucleic acids. These different mechanisms will all, to some extent, contribute to gain-of-function as in essence they all contribute to the rewiring of the cellular interactome by aggregation-specific interactions, resulting for instance in the pronounced neurotoxicity of TDP43 aggregates by the sequestration of RNA molecules or the promotion of cell proliferation by the entrapment of homologous tumor suppressor proteins in p53 aggregates in cancer. In this review we discuss the mechanism of APR driven aggregation and how APRs contribute to modifying the cellular interactome by recruiting both misfolded as well as active proteins thereby inhibiting or activating specific cellular functions. Finally, we discuss the ubiquity of APRs in protein sequences and how selective pressure shaped protein sequences to minimize APR aggregation.

Intermolecular interactions in highly concentrated formulations of recombinant therapeutic proteins

Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of “biobetter” antibodies.

Protein aggregation is a common problem during the purification and formulation of therapeutic proteins. Here we report that polyphenolic disaccharides are unusually effective at preventing protein aggregation. We find that two polyphenolic glycosides-naringin and rutin-endow diverse proteins with the ability to unfold without aggregating when heated, as well as the ability to refold without aggregating when cooled at low glycoside concentrations (<5 mM). This extreme solubilizing activity is a synergistic combination of the glycone and aglycone moieties, as combinations of polyphenols and sugars fail to suppress aggregation. Moreover, the activity of polyphenolic disaccharides is remarkably specific since their monosaccharide counterparts (as well as other common excipients such as arginine, trehalose, and cyclodextrin) fail to prevent aggregation at similar concentrations (<25 mM). We expect that polyphenolic disaccharides will be valuable additives for enhancing the solubility of proteins in applications plagued by protein aggregation.

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The ReFOLD assay for protein formulation studies and prediction of protein aggregation during long-term storage