Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates.

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities.

Mechanism of how carbamylation reduces albumin binding to FcRn contributing to increased vascular clearance.

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin's reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library.

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.

Predicting Formulation Conditions During Ultrafiltration and Dilution to Drug Substance Using a Donnan Model with Homology-Model Based Protein Charge.

In the manufacturing of therapeutic monoclonal antibodies (mAbs), the final steps of the purification process are typically ultrafiltration/diafiltration (UF/DF), dilution, and conditioning. These steps are developed such that the final drug substance (DS) is formulated to the desired mAb, buffer, and excipient concentrations. To develop these processes, process and formulation development scientists often perform experiments to account for the Gibbs-Donnan and volume-exclusion effects during UF/DF, which affect the output pH and buffer concentration of the UF/DF process. This work describes the development of an in silico model for predicting the DS pH and buffer concentration after accounting for the Gibbs-Donnan and volume-exclusion effects during the UF/DF operation and the subsequent dilution and conditioning steps. The model was validated using statistical analysis to compare model predictions against experimental results for nine molecules of varying protein concentrations and formulations. In addition, our results showed that the structure-based in silico approach used to calculate the protein charge was more accurate than a sequence-based approach. Finally, we used the model to gain fundamental insights about the Gibbs-Donnan effect by highlighting the role of the protein charge concentration (the protein concentration multiplied with protein charge at the formulation pH) on the Gibbs-Donnan effect. Overall, this work demonstrates that the Gibbs-Donnan and volume-exclusions effects can be predicted using an in silico model, potentially alleviating the need for experiments.

Evolutionary changes in the number of dissociable amino acids on spike proteins and nucleoproteins of SARS-CoV-2 variants.

The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for target recognition, cellular entry, and endosomal escape of the virus. At the same time, it is the part of the virus that exhibits the greatest sequence variation across the many variants which have emerged during its evolution. Recent studies have indicated that with progressive lineage emergence, the positive charge on the spike protein has been increasing, with certain positively charged amino acids (AAs) improving the binding of the spike protein to cell receptors. We have performed a detailed analysis of dissociable AAs of more than 1400 different SARS-CoV-2 lineages, which confirms these observations while suggesting that this progression has reached a plateau with Omicron and its subvariants and that the positive charge is not increasing further. Analysis of the nucleocapsid protein shows no similar increase in positive charge with novel variants, which further indicates that positive charge of the spike protein is being evolutionarily selected for. Furthermore, comparison with the spike proteins of known coronaviruses shows that already the wild-type SARS-CoV-2 spike protein carries an unusually large amount of positively charged AAs when compared to most other betacoronaviruses. Our study sheds light on the evolutionary changes in the number of dissociable AAs on the spike protein of SARS-CoV-2, complementing existing studies and providing a stepping stone towards a better understanding of the relationship between the spike protein charge and viral infectivity and transmissibility.

Theoretical charge plots as a tool for targeted and accelerated ion exchange chromatography method development of NANOBODYⓇ molecules.

NANOBODYⓇ molecules are an innovative class of biotherapeutics based on heavy chain only VHH immunoglobulins. Much like canonical antibodies, they are prone to the formation of charge variants and other post-translational modifications, which can potentially impact their critical quality attributes. Therefore, establishing high-resolution product-specific methods, such as IEX chromatography, is essential for evaluating the purity of these molecules. However, due to the lower surface charge of NANOBODYⓇ molecules, their charge-based elution behavior can differ considerably from that of classical antibodies, resulting in a more extensive method development set-up for these smaller molecules. Using an initial pH screening gradient based on theoretical protein charge plots, we investigated the IEX retention behavior of eight NANOBODYⓇ molecules with a wide range of pI values (pI 5.0 to 10.0). Our findings reveal that the charge-based chromatographic behavior of NANOBODYⓇ molecules cannot be solely attributed to the isoelectric point (pI) of the protein. Rather, a molecule-specific charge threshold was identified as a critical parameter for NANOBODYⓇ molecule retention. Furthermore, the protein charge plot also showed that NANOBODYⓇ molecule elution can be characterized by a charge plateau where the net charge of the protein remains constant over a certain pH range (∼ pH 5.5 to pH 8.0), further challenging the paradigm that elution pH and pI are fixed values. The application of this theoretical approach using protein charge plots to define NANOBODYⓇ molecule charge threshold and charge plateau parameters, can reduce overall IEX method development turnaround time by at least 2-fold.

Characterization of the aggregation propensity of charge variants of recombinant human growth hormone.

Biopharmaceutical products are subject to in depth analysis to ensure and improve their safety and efficacy. As part of this effort the stability and aggregation mechanisms of the therapeutic protein is characterized over the whole life cycle. The stability and aggregation behavior of single charge variants present in biopharmaceuticals were hardly investigated. In this study we applied a previously established methodology to assess the charge variants of the drug substance (DS) of human growth hormone (hGH). We assessed the stability and aggregation propensity of an acidic variant which forms in DS at a larger extent during short time storage at elevated temperatures. We developed a semi-preparative method to separate and analyze the charge species. Thermal and colloidal stability of this variant was analyzed by light scattering methods and a stability testing in different buffer formulations. The acidic variant showed slightly attractive self-interaction at lower pH. Thermal stress did not result in increased aggregation propensity or decreased stability compared to the DS. Thus, the methodology enabled to assess the risk of a single protein variant within the DS of hGH. The approach can also be utilized for other protein drugs as previously shown for a monoclonal antibody.

High-throughput multiplex analysis of mAb aggregates and charge variants by automated two-dimensional size exclusion-cation exchange chromatography coupled to mass spectrometry.

Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension (1D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension (2D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis.

Storage stability of soy protein isolate powders containing soluble protein aggregates formed at varying pH.

Soy protein is wildly used in food industry due to its high nutritional value and good functionalities. However, the poor storage stability of commercial soy protein products has puzzled both the producers and the users for a long time. The current study assessed the changes in protein solubility, aggregation, oxidation, and conformation of soy protein isolate (SPI) with various soluble aggregates formed at different pH values (pH 5-8) during storage. During storage, SPI samples showed a reduced protein solubility (p < .05), an increased protein oxidation (p < .05), and an attenuated conformational enthalpy (∆H). SPI with a higher pH produced more disulfide-mediated aggregates at the expense of sulfhydryl groups and experienced greater losses of protein tertiary structure and a faster reduction in solubility. Yet, all samples nearly shared similar rising trend during 8-week storage, which indicated the production of protein carbonyls was insensitive to pH. Soluble aggregates present in fresh SPI samples appeared to induce instability of SPI during storage. These findings suggested SPI prepared at pH 6 was in favor of its storage stability, and soluble aggregates presented in fresh samples should be paid more attention for further study of storage stability kinetics.

Exploring the Potential of Dendritic Oligoglycerol Detergents for Protein Mass Spectrometry.

The ability to design detergents that are suitable for protein analysis by mass spectrometry (MS) represents an on-going challenge in the field of native MS. Desirable detergent characteristics include charge-reducing properties and low gas-phase stabilities of complexes formed with proteins. In this work, the gas-phase properties of oligoglycerol detergents (OGDs) are optimized by fine tuning of their molecular structure. Furthermore, a tandem mass spectrometry (MS/MS) approach is presented that estimates the gas-phase properties of detergents simply by studying the dissociation behaviour of protein-detergent complexes (PDCs) formed with the soluble protein ß-lactoglobulin (BLG). Graphical Abstract ᅟ.

IgG Charge: Practical and Biological Implications.

Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab')2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)'2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to -13; (2) the F(ab')2 fragments are nearly neutral for IgG1s and IgG2s, and about -5 for some of the IgG4s; (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; (4) the charge on the intact IgGs does not equal the sum of the F(ab')2 and Fc charge. In no case is the calculated charge, based solely on H+ binding, remotely close to the measured charge. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. A thermodynamically rigorous, concentration-dependent protein-protein interaction parameter is introduced. Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. Example curves are provided.

Comparative study of analytical techniques for determining protein charge.

As interest in high-concentration protein formulations has increased, it has become apparent that routine, accurate protein charge measurements are necessary. There are several techniques for charge measurement, and a comparison of the methods is needed. The electrophoretic mobility, effective charge, and Debye-Hückel-Henry charge have been determined for bovine serum albumin, and human serum albumin. Three different electrophoretic methods were used to measure the electrophoretic mobility: capillary electrophoresis, electrophoretic light scattering, and membrane confined electrophoresis. In addition, the effective charge was measured directly using steady-state electrophoresis. Measurements made at different NaCl concentrations, pH, and temperatures allow comparison with previous charge estimates based on electrophoresis, Donnan equilibrium, and pH titration. Similar charge estimates are obtained by all of the methods. The strengths and limitations of each technique are discussed, as are some general considerations about protein charge and charge determination.