Rapid Intact mass based multi-attribute method in support of mAb upstream process development.

N-linked glycosylation is a critical quality attribute for monoclonal antibodies (mAbs) as it affects product stability, immunogenicity, receptor binding and antibody effector function, clearance and half-life. It has been widely accepted that the glycosylation process is greatly impacted by several factors during bioreactor operations. Therefore, the timely acquisition of N-linked glycosylation information during cell culture process development is critical for the success of the endeavor. In this paper we describe a simple, rapid, and robust Multi-Attribute Method (MAM) based on intact mass analysis. This method, developed for an open access instrument, has been optimized for the analysis of light and heavy chains generated from dithiothreitol (DTT) reduction of intact mAbs sampled directly out of bioreactors. The N-linked glycosylation profile, identity confirmation of light chain and heavy chain, light chain glycation and non-glycosylated heavy chain (NGHC) can all be monitored by this method. Our results confirm that the N-linked glycosylation profile determined using Intact mass based MAM is comparable with a release glycan assay and LC-MS/MS peptide based MAM assay for the most abundant glycoforms. Furthermore, the results for the NGHC attribute determined with our method are comparable to results from capillary gel electrophoresis (CGE) and LC-MS/MS peptide based MAM.

A Multicompany Assessment of Submicron Particle Levels by NTA and RMM in a Wide Range of Late-Phase Clinical and Commercial Biotechnology-Derived Protein Products.

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (<2 µm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurement-based techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products.

Saccharomyces cerevisiae morphological changes and cytokinesis arrest elicited by hypoxia during scale-up for production of therapeutic recombinant proteins.

BACKGROUND: Scaling up of bioprocesses represents a crucial step in the industrial production of biologicals. However, our knowledge about the impact of scale-up on the organism's physiology and function is still incomplete. Our previous studies have suggested the existence of morphological changes during the scale-up of a yeast (Saccharomyces cerevisiae) fermentation process as inferred from the volume fraction occupied by yeast cells and exometabolomics analyses. In the current study, we noticed cell morphology changes during scale-up of a yeast fermentation process from bench (10 L) to industrial scale (10,000 L). We hypothesized that hypoxia observed during scale-up partially impaired the availability of N-acetyl-glucosamine, a precursor of chitin synthesis, a key polysaccharide component of yeast mother-daughter neck formation. RESULTS: Using a combination of flow cytometry with two high throughput cell imaging technologies, Vi-CELL and Flow Imaging, we found changes in the distribution of cell size and morphology as a function of process duration at the industrial scale of the production process. At the end of run, concomitantly with lowest levels of dissolved oxygen (DO), we detected an increase in cell subpopulations exhibiting low aspect ratio corresponding to morphologies exhibited by large-single-budded and multi-budded cells, reflecting incomplete cytokinesis at the M phase of the yeast mitotic cycle. Metabolomics from the intracellular milieu pointed to an impaired supply of precursors for chitin biosynthesis likely affecting the septum formation between mother and daughter and cytokinesis. Inducing hypoxia at the 10 L bench scale by varying DO levels, confirmed the existence and impact of hypoxic conditions on yeast cell size and morphology observed at the industrial scale. CONCLUSIONS: We conclude that the observed increments in wet cell weight at the industrial scale correspond to morphological changes characterized by the large diameter and low aspect ratio exhibited by cell subpopulations comprising large single-budded and multi-budded cells. These changes are consistent with impairment of cytokinesis triggered by hypoxia as indicated by experiments mimicking this condition at DO 5% and 10 L scale. Mechanistically, hypoxia impairs N-acetyl-glucosamine availability, a key precursor of chitin synthesis.

Energetics of charge-charge interactions between residues adjacent in sequence.

Statistical analysis of the residue separation between a pair of ionizable side chains within 4 Å of each other was performed on a set of 1560 non-homologous PDB structures. We found that the frequency of pairs of like charges (i.e., pairs consisting of acidic residues Asp and Glu or pairs consisting of basic residues Arg and Lys) is two orders of magnitude lower than the pairs of oppositely charged residues (salt-bridges). We also found that for pairs of like charges the distribution is skewed dramatically towards short residue separation (<3). On the basis of these observations, we hypothesize that at short residue separation the repulsion between charges does not contribute much to the protein stability and the effects are largely dominated by the long range charge-charge interactions with other ionizable groups in the protein molecule. To test this hypothesis, we incorporated various pairs of charged residues at position 63 and 64 of ubiquitin and compared the stabilities of these variants. We also performed calculations of the expected changes in the charge-charge interactions. A very good correlation between experimental changes in the stability of ubiquitin variants, and changes in the energy of charge-charge interactions provides support for the hypothesis that a pair of ionizable residues next to each other in sequence modulates protein stability via long range charge-charge interactions with the rest of the protein.

Protein stability and surface electrostatics: a charged relationship.

Engineering proteins to withstand a broad range of conditions continues to be a coveted objective, holding the potential to advance biomedicine, industry, and our understanding of disease. One way of achieving this goal lies in elucidating the underlying interactions that define protein stability. It has been shown that the hydrophobic effect, hydrogen bonding, and packing interactions between residues in the protein interior are dominant factors that define protein stability. The role of surface residues in protein stability has received much less attention. It has been believed that surface residues are not important for protein stability particularly because their interactions with the solvent should be similar in the native and unfolded states. In the case of surface charged residues, it was sometimes argued that solvent exposure meant that the high dielectric of the solvent will further decrease the strength of the charge-charge interactions. In this paper, we challenge the notion that the surface charged residues are not important for protein stability. We computationally redesigned sequences of five different proteins to optimize the surface charge-charge interactions. All redesigned proteins exhibited a significant increase in stability relative to their parent proteins, as experimentally determined by circular dichroism spectroscopy and differential scanning calorimetry. These results suggest that surface charge-charge interactions are important for protein stability and that rational optimization of charge-charge interactions on the protein surface can be a viable strategy for enhancing protein stability.

Both helical propensity and side-chain hydrophobicity at a partially exposed site in alpha-helix contribute to the thermodynamic stability of ubiquitin.

Improving helical propensity of residues was proposed as one of the approaches to increase protein stability. Here the contribution of the helix propensity and hydrophobicity of residues at partially buried positions of alpha-helix to the stability of a model protein-ubiquitin- is explored. Thermodynamic stabilities of 13 ubiquitin variants with substitutions at a partially buried helical residue were measured by differential scanning calorimetry. It was found that the dynamic range of stabilities for different amino acid residues at this partially buried position is 3 times larger than that expected based on helical propensity alone. Correlation analysis shows that both helical propensity and hydrophobicity are important in defining the relative stabilities of the studied ubiquitin variants. These results provide experimental evidence that partially buried positions are potentially useful sites for engineering proteins with enhanced thermostability.

Mechanism of thermostabilization in a designed cold shock protein with optimized surface electrostatic interactions.

Using computational and sequence analysis of bacterial cold shock proteins, we designed a protein (CspB-TB) that has the core residues of mesophilic protein from Bacillus subtilis(CspB-Bs) and altered distribution of surface charged residues. This designed protein was characterized by circular dichroism spectroscopy, and found to have secondary and tertiary structure similar to that of CspB-Bs. The activity of the CspB-TB protein as measured by the affinity to a single-stranded DNA (ssDNA) template at 25 degrees C is somewhat higher than that of CspB-Bs. Furthermore, the decrease in the apparent binding constant to ssDNA upon increase in temperature is much more pronounced for CspB-Bs than for CspB-TB. Temperature-induced unfolding (as monitored by differential scanning calorimetry and circular dichroism spectroscopy) and urea-induced unfolding experiments were used to compare the stabilities of CspB-Bs and CspB-TB. It was found that CspB-TB is approximately 20 degrees C more thermostable than CspB-Bs. The thermostabilization of CspB-TB relative to CspB-Bs is achieved by decrease in the enthalpy and entropy of unfolding without affecting their temperature dependencies, i.e. these proteins have similar heat capacity changes upon unfolding. These changes in the thermodynamic parameters result in the global stability function, i.e. Gibbs energy, deltaG(T), that is shifted to higher temperatures with only small changes in the maximum stability. Such a mechanism of thermostabilization, although predicted from the basic thermodynamic considerations, has never been identified experimentally.

Contribution of surface salt bridges to protein stability: guidelines for protein engineering.

The small globular protein, ubiquitin, contains a pair of oppositely charged residues, K11 and E34, that according to the three-dimensional structure are located on the surface of this protein with a spatial orientation characteristic of a salt bridge. We investigated the strength of this salt bridge and its contribution to the global stability of the ubiquitin molecule. Using the "double mutant cycle" analysis, the strength of the pairwise interactions between K11 and E34 was estimated to be favorable by 3.6kJ/mol. Further, the salt bridge of the reverse orientation, i.e. E11/K34, can be formed and is found to have a strength (3.8kJ/mol) similar to that of the K11/E34 pair. However, the global stability of the K11/E34 variant of ubiquitin is 2.2kJ/mol higher than that of the E11/K34 variant. The difference in the contribution of the opposing salt bridge orientations to the overall stability of the ubiquitin molecule is attributed to the difference in the charge-charge interactions between residues forming the salt bridge and the rest of the ionizable groups in this protein. On the basis of these results, we concluded that surface salt bridges are stabilizing, but their contribution to the overall protein stability is strongly context-dependent, with charge-charge interactions being the largest determinant. Analysis of 16 salt bridges from six different proteins, for which detailed experimental data on energetics have been reported, support the conclusions made from the analysis of the salt bridge in ubiquitin. Implications of these findings for engineering proteins with enhanced thermostability are discussed.

Thermodynamic consequences of burial of polar and non-polar amino acid residues in the protein interior.

Effects of amino acid substitutions at four fully buried sites of the ubiquitin molecule on the thermodynamic parameters (enthalpy, Gibbs energy) of unfolding were evaluated experimentally using differential scanning calorimetry. The same set of substitutions has been incorporated at each of four sites. These substitutions have been designed to perturb packing (van der Waals) interactions, hydration, and/or hydrogen bonding. From the analysis of the thermodynamic parameters for these ubiquitin variants we conclude that: (i) packing of non-polar groups in the protein interior is favorable and is largely defined by a favorable enthalpy of van der Waals interactions. The removal of one methylene group from the protein interior will destabilize a protein by approximately 5 kJ/mol, and will decrease the enthalpy of a protein by 12 kJ/mol. (ii) Burial of polar groups in the non-polar interior of a protein is highly destabilizing, and the degree of destabilization depends on the relative polarity of this group. For example, burial of Thr side-chain in the non-polar interior will be less destabilizing than burial of Asn side-chain. This decrease in stability is defined by a large enthalpy of dehydration of polar groups upon burial. (iii) The destabilizing effect of dehydration of polar groups upon burial can be compensated if these buried polar groups form hydrogen bonding. The enthalpy of this hydrogen bonding will compensate for the unfavorable dehydration energy and as a result the effect will be energetically neutral or even slightly stabilizing.

Removal of surface charge-charge interactions from ubiquitin leaves the protein folded and very stable.

The contribution of solvent-exposed charged residues to protein stability was evaluated using ubiquitin as a model protein. We combined site-directed mutagenesis and specific chemical modifications to first replace all Arg residues with Lys, followed by carbomylation of Lys-amino groups. Under the conditions in which all carboxylic groups are protonated (at pH 2), the chemically modified protein is folded and very stable (DeltaG = 18 kJ/mol). These results indicate that surface charge-charge interactions are not an essential fundamental force for protein folding and stability.