T2 heterogeneity as an in vivo marker of microstructural integrity in medial temporal lobe subfields in ageing and mild cognitive impairment.

A better understanding of early brain changes that precede loss of independence in diseases like Alzheimer's disease (AD) is critical for development of disease-modifying therapies. Quantitative MRI, such as T2 relaxometry, can identify microstructural changes relevant to early stages of pathology. Recent evidence suggests heterogeneity of T2 may be a more informative MRI measure of early pathology than absolute T2. Here we test whether T2 markers of brain integrity precede the volume changes we know are present in established AD and whether such changes are most marked in medial temporal lobe (MTL) subfields known to be most affected early in AD. We show that T2 heterogeneity was greater in people with mild cognitive impairment (MCI; n = 49) compared to healthy older controls (n = 99) in all MTL subfields, but this increase was greatest in MTL cortices, and smallest in dentate gyrus. This reflects the spatio-temporal progression of neurodegeneration in AD. T2 heterogeneity in CA1-3 and entorhinal cortex and volume of entorhinal cortex showed some ability to predict cognitive decline, where absolute T2 could not, however further studies are required to verify this result. Increases in T2 heterogeneity in MTL cortices may reflect localised pathological change and may present as one of the earliest detectible brain changes prior to atrophy. Finally, we describe a mechanism by which memory, as measured by accuracy and reaction time on a paired associate learning task, deteriorates with age. Age-related memory deficits were explained in part by lower subfield volumes, which in turn were directly associated with greater T2 heterogeneity. We propose that tissue with high T2 heterogeneity represents extant tissue at risk of permanent damage but with the potential for therapeutic rescue. This has implications for early detection of neurodegenerative diseases and the study of brain-behaviour relationships.

Detection of Isopeptide Bonds in Monoclonal Antibody Aggregates.

PURPOSE: A major difficulty in monoclonal antibody (mAb) therapeutic development is product aggregation. In this study, intermolecular isopeptide bonds in mAb aggregates were characterized for the first time. We aim to propose a mechanism of covalent aggregation in a model antibody using stressed studies at raised temperatures to aid in the understanding of mAb aggregation pathways. METHODS: Aggregate fractions were generated using raised temperature and were purified using size-exclusion chromatography (SEC). The fractions were tryptically digested and characterized using liquid chromatography hyphenated to tandem mass-spectrometry (LC-MS/MS). RESULTS: An increased amount of clipping between aspartic acid and proline in a solvent accessible loop in the constant heavy 2 (CH2) domain of the mAb was observed under these conditions. Detailed peptide mapping revealed 14 isopeptide bonds between aspartic acid at that cleavage site and lysine residues on adjacent antibodies. Two additional isopeptide bonds were identified between the mAb HC N-terminal glutamic acid or a separate aspartic acid to lysine residues on adjacent antibodies. CONCLUSIONS: Inter-protein isopeptide bonds between the side chains of acidic amino acids (aspartate and glutamate) and lysine were characterized for the first time in mAb aggregates. A chemical mechanism was presented whereby spontaneous isopeptide bond formation could be facilitated via either the aspartic acid side chain or C-terminus.

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift.

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a 'fingerprint' of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (µg-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

Picometer Resolution Structure of the Coordination Sphere in the Metal-Binding Site in a Metalloprotein by NMR.

Most of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10-4-10-5 Å. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity. In contrast, the resolution of X-ray structures in proteins is limited typically to the order of 10-1 Å. This resolution is often not sufficient to develop precise structure-activity relations for the metal sites in proteins, because the uncertainty in positions can cover all of the known ranges of bond lengths and bond angles for a given type of metal complex. Here we introduce a new approach that enables the determination of a high-definition structure of the active site of a metalloprotein from a powder sample, by combining magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, tailored radio frequency (RF) irradiation schemes, and computational approaches. This allows us to overcome the "blind sphere" in paramagnetic proteins, and to observe and assign 1H, 13C, and 15N resonances for the ligands directly coordinating the metal center. We illustrate the method by determining the bond lengths in the structure of the CoII coordination sphere at the core of human superoxide dismutase 1 (SOD) with 0.7 pm precision. The coordination geometry of the resulting structure explains the nonreactive nature of the CoII/ZnII centers in these proteins, which allows them to play a purely structural role.

Multimodal Response to Copper Binding in Superoxide Dismutase Dynamics.

Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme that has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metalated forms of SOD, namely, the fully mature functional Cu,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (1H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms. We determine that metal ion uptake does not act as a rigidification element but as a switch redistributing motional processes on different time scales, with coupling of the dynamics of histidine side chains and those of remote key backbone elements of the protein.

T2 Relaxometry and Diffusion Tensor Indices of the Hippocampus and Entorhinal Cortex Improve Sensitivity and Specificity of MRI to Detect Amnestic Mild Cognitive Impairment and Alzheimer's Disease Dementia.

BACKGROUND: Quantitative T2 and diffusion MRI indices inform about tissue state and microstructure, both of which may be affected by pathology before tissue atrophy. PURPOSE: To evaluate the capability of both volumetric and quantitative MRI (qMRI) of the hippocampus and entorhinal cortex (EC) for classification of amnestic mild cognitive impairment (aMCI) and Alzheimer's disease dementia (ADD). STUDY TYPE: Retrospective cross-sectional study. POPULATION: Consecutive cohorts of healthy age-matched controls (n = 62), aMCI patients (n = 25), and ADD patients (n = 14). FIELD STRENGTH/SEQUENCE: 3T using T1-weighted imaging, T2-weighted imaging, T2 relaxometry and diffusion tensor imaging (DTI). ASSESSMENT: Montreal Cognitive Assessment and paired associate learning tests for cognitive state. Hippocampal subfield volumes by the automated segmentation of hippocampal subfields system from structural brain images. T2 relaxation time and DTI indices quantified for hippocampal subfields. The fraction of voxels with high T2 values (>20 ms above subfield median) was calculated and regionalized for hippocampus and EC. STATISTICAL TESTS: Support vector machine and receiver operating characteristic analyses from cognitive and MRI data. RESULTS: qMRI classified aMCI and ADD with excellent sensitivity (79.0% and 94.5%, respectively) and specificity (85.6% and 86.1%, respectively), superior to volumes alone (70.0% and 84.5% for respective sensitivities; 82.2 and 91.1 for respective specificities) and similar to cognitive tests (61.7% and 87.5% for respective sensitivities; 88.2% and 90.7% for respective specificities). Regions of high T2 are dispersed throughout each hippocampal subfield in aMCI and ADD with higher concentration than controls, and was most pronounced in the EC. No other individual qMRI marker than EC volume can separate aMCI from ADD, however. DATA CONCLUSION: qMRI markers of hippocampal and entorhinal tissue states are sensitive and specific classifiers of aMCI and ADD. They may serve as markers of a neurodegenerative state preceding volume loss. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 3 J. Magn. Reson. Imaging 2019;49:445-455.

Magnetic Resonance Relaxation Anisotropy: Physical Principles and Uses in Microstructure Imaging.

Magnetic resonance imaging (MRI) provides an excellent means of studying tissue microstructure noninvasively since the microscopic tissue environment is imprinted on the MRI signal even at macroscopic voxel level. Mesoscopic variations in magnetic field, created by microstructure, influence the transverse relaxation time (T2) in an orientation-dependent fashion (T2 is anisotropic). However, predicting the effects of microstructure upon MRI observables is challenging and requires theoretical insight. We provide a formalism for calculating the effects upon T2 of tissue microstructure, using a model of cylindrical magnetic field perturbers. In a cohort of clinically healthy adults, we show that the angular information in spin-echo T2 is consistent with this model. We show that T2 in brain white matter of nondemented volunteers follows a U-shaped trajectory with age, passing its minimum at an age of ∼30 but that this depends on the particular white matter tract. The anisotropy of T2 also interacts with age and declines with increasing age. Late-myelinating white matter is more susceptible to age-related change than early-myelinating white matter, consistent with the retrogenesis hypothesis. T2 mapping may therefore be incorporated into microstructural imaging.

Quantitative T1 and T2 MRI signal characteristics in the human brain: different patterns of MR contrasts in normal ageing.

OBJECTIVE: The objective of this study was to examine age-dependent changes in both T1-weighted and T2-weighted image contrasts and spin-echo T2 relaxation time in the human brain during healthy ageing. METHODS: A total of 37 participants between the ages of 49 and 87 years old were scanned with a 3 Tesla system, using T1-weighted, T2 weighted and quantitative spin-echo T2 imaging. Contrast between image intensities and T2 values was calculated for various regions, including between individual hippocampal subfields. RESULTS: The T1 contrast-to-noise (CNR) and gray:white signal intensity ratio (GWR) did not change in the hippocampus, but it declined in the cingulate cortex with age. In contrast, T2 CNR and GWR declined in both brain regions. T2 relaxation time was almost constant in gray matter and most (but not all) hippocampal subfields, but increased substantially in white matter, pointing to an age effect on water relaxation in white matter. CONCLUSIONS: Changes in T1 and T2 MR characteristics influence the appearance of brain images in later life and should be considered in image analyses of aged subjects. It is speculated that alterations at the cell biology level, with concomitant alterations to the local magnetic environment, reduce dephasing and subsequently prolong spin-echo T2 through reduced diffusion effects in later life.

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR.

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with (1)H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of (15)N and (13)C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu(+) (diamagnetic) or Cu(2+) (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to (1)H-(1)H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning.

Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary (13)C/(15)N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

The yeast enzyme Eht1 is an octanoyl-CoA:ethanol acyltransferase that also functions as a thioesterase.

Fatty acid ethyl esters are secondary metabolites that are produced during microbial fermentation, in fruiting plants and in higher organisms during ethanol stress. In particular, volatile medium-chain fatty acid ethyl esters are important flavour compounds that impart desirable fruit aromas to fermented beverages, including beer and wine. The biochemical synthesis of medium-chain fatty acid ethyl esters is poorly understood but likely involves acyl-CoA:ethanol O-acyltransferases. Here, we characterize the enzyme ethanol hexanoyl transferase 1 (Eht1) from the brewer's yeast Saccharomyces cerevisiae. Full-length Eht1 was successfully overexpressed from a recombinant yeast plasmid and purified at the milligram scale after detergent solubilization of sedimenting membranes. Recombinant Eht1 was functional as an acyltransferase and, unexpectedly, was optimally active toward octanoyl-CoA, with k(cat) = 0.28 ± 0.02/s and K(M) = 1.9 ± 0.6 µm. Eht1 was also revealed to be active as a thioesterase but was not able to hydrolyse p-nitrophenyl acyl esters, in contrast to the findings of a previous study. Low-resolution structural data and site-directed mutagenesis provide experimental support for a predicted α/ß-hydrolase domain featuring a Ser-Asp-His catalytic triad. The S. cerevisiae gene YBR177C/EHT1 should thus be reannotated as coding for an octanoyl-CoA:ethanol acyltransferase that can also function as a thioesterase.

Magic angle spinning NMR of paramagnetic proteins.

Metal ions are ubiquitous in biochemical and cellular processes. Since many metal ions are paramagnetic due to the presence of unpaired electrons, paramagnetic molecules are an important class of targets for research in structural biology and related fields. Today, NMR spectroscopy plays a central role in the investigation of the structure and chemical properties of paramagnetic metalloproteins, linking the observed paramagnetic phenomena directly to electronic and molecular structure. A major step forward in the study of proteins by solid-state NMR came with the advent of ultrafast magic angle spinning (MAS) and the ability to use (1)H detection. Combined, these techniques have allowed investigators to observe nuclei that previously were invisible in highly paramagnetic metalloproteins. In addition, these techniques have enabled quantitative site-specific measurement of a variety of long-range paramagnetic effects. Instead of limiting solid-state NMR studies of biological systems, paramagnetism provides an information-rich phenomenon that can be exploited in these studies. This Account emphasizes state-of-the-art methods and applications of solid-state NMR in paramagnetic systems in biological chemistry. In particular, we discuss the use of ultrafast MAS and (1)H-detection in perdeuterated paramagnetic metalloproteins. Current methodology allows us to determine the structure and dynamics of metalloenzymes, and, as an example, we describe solid-state NMR studies of microcrystalline superoxide dismutase, a 32 kDa dimer. Data were acquired with remarkably short times, and these experiments required only a few milligrams of sample.

13C-detected through-bond correlation experiments for protein resonance assignment by ultra-fast MAS solid-state NMR.

We present two sequences which combine ((1)H,(15)N) and ((15)N,(13)C) selective cross-polarization steps with an efficient variant of the J-based homonuclear transfer scheme, in which a spin-state-selective (S(3)E) block is incorporated to improve both resolution and sensitivity in the direct (13)C dimension. We propose these two sequences as a part of a suite of four N-C correlation experiments allowing for the assignment of protein backbone resonances in the solid state. We illustrate these experiments under ultra-fast magic angle spinning conditions on two samples of microcrystalline dimeric human superoxide dismutase (SOD, 153×2 amino acids), in its diamagnetic ("empty", Zn(II)) and paramagnetic (Cu(II), Zn(II)) states.

Computational modelling of diatom silicic acid transporters predicts a conserved fold with implications for their function and evolution.

Diatoms are an important group of algae that can produce intricate silicified cell walls (frustules). The complex process of silicification involves a set of enigmatic integral membrane proteins that are thought to actively transport the soluble precursor of biosilica, dissolved silicic acid. Full-length silicic acid transporters are found widely across the diatoms while homologous shorter proteins have now been identified in a range of other organisms. It has been suggested that modern silicic acid transporters arose from the union of such partial sequences. Here, we present a computational study of the silicic acid transporters and related transporter-like sequences to help understand the structure, function and evolution of this class of membrane protein. The AlphaFold software predicts that all of the protein sequences studied here share a common fold in the membrane domain which is entirely different from the predicted folds of non-homologous silicic acid transporters from plants. Substrate docking reveals how conserved polar residues could interact with silicic acid at a central solvent-accessible binding site, consistent with an alternating access mechanism of transport. The structural conservation between these proteins supports a model where modern silicon transporters evolved from smaller ancestral proteins by gene fusion.

Expression, purification, and reconstitution of a diatom silicon transporter.

The synthesis and manipulation of silicon materials on the nanoscale are core themes in nanotechnology research. Inspiration is increasingly being taken from the natural world because the biological mineralization of silicon results in precisely controlled, complex silica structures with dimensions from the millimeter to the nanometer. One fascinating example of silicon biomineralization occurs in the diatoms, unicellular algae that sheath themselves in an ornate silica-based cell wall. To harvest silicon from the environment, diatoms have developed a unique family of integral membrane proteins that bind to a soluble form of silica, silicic acid, and transport it across the cell membrane to the cell interior. These are the first proteins shown to directly interact with silicon, but the current understanding of these specific silicon transport proteins is limited by the lack of in vitro studies of structure and function. We report here the recombinant expression, purification, and reconstitution of a silicon transporter from the model diatom Thalassiosira pseudonana. After using GFP fusions to optimize expression and purification protocols, a His(10)-tagged construct was expressed in Saccharomyces cerevisiae, solubilized in the detergent Fos-choline-12, and purified by affinity chromatography. Size-exclusion chromatography and particle sizing by dynamic light scattering showed that the protein was purified as a homotetramer, although nonspecific oligomerization occurred at high protein concentrations. Circular dichroism measurements confirmed sequence-based predictions that silicon transporters are α-helical membrane proteins. Silicic acid transport could be established in reconstituted proteoliposomes, and silicon uptake was found to be dependent upon an applied sodium gradient. Transport data across different substrate concentrations were best fit to the sigmoidal Hill equation, with a K(0.5) of 19.4 ± 1.3 µM and a cooperativity coefficient of 1.6. Sodium binding was noncooperative with a K(m)(app) of 1.7 ± 1.0 mM, suggesting a transport silicic acid:Na(+) stoichiometry of 2:1. These results provide the basis for a full understanding of both silicon transport in the diatom and protein-silicon interactions in general.

Rapid measurement of pseudocontact shifts in metalloproteins by proton-detected solid-state NMR spectroscopy.

Pseudocontact shifts (PCSs) arise in paramagnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the paramagnetic center and the position relative to the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of (1)H-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved (1)H PCSs, in a large metalloprotein, Co(2+)-substituted superoxide dismutase (Co(2+)-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with (1)H-(1)H distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.

Combination of DQ and ZQ coherences for sensitive through-bond NMR correlation experiments in biosolids under ultra-fast MAS.

A double-zero quantum (DZQ)-refocused INADEQUATE experiment is introduced for J-based NMR correlations under ultra-fast (60 kHz) magic angle spinning (MAS). The experiment records two spectra in the same dataset, a double quantum-single quantum (DQ-SQ) and zero quantum-single quantum (ZQ-SQ) spectrum, whereby the corresponding signals appear at different chemical shifts in ω(1). Furthermore, the spin-state selective excitation (S(3)E) J-decoupling block is incorporated in place of the second refocusing echo of the INADEQUATE scheme, providing an additional gain in sensitivity and resolution. The two sub-spectra acquired in this way can be treated separately by a shearing transformation, producing two diagonal-free single quantum (SQ-SQ)-type spectra, which are subsequently recombined to give an additional sensitivity enhancement, thus offering an improvement greater than a factor of two as compared to the original refocused INADEQUATE experiment. The combined DZQ scheme retains transverse magnetization on the initially polarized (I) spin, which typically exhibits a longer transverse dephasing time (T(2)') than its through-bond neighbour (S). By doing so, less magnetization is lost during the refocusing periods in the sequence to give even further gains in sensitivity for the J correlations. The experiment is demonstrated for the correlation between the carbonyl (CO) and alpha (CA) carbons in a microcrystalline sample of fully protonated, [(15)N,(13)C]-labelled Cu(II),Zn(II) superoxide dismutase, and its efficiency is discussed with respect to other J-based schemes.

The impact of forced degradation conditions on mAb dimer formation and subsequent influence on aggregation propensity.

Monoclonal antibody (mAb) aggregation can present major challenges for the development of biotherapeutics. An understanding of the molecular mechanisms of mAb aggregation is highly desirable both because it allows the performance of informed risk assessments regarding the criticality of mAb aggregates and because it may facilitate rational stabilization of aggregation prone regions. Here, we report the generation and isolation of dimer species of an IgG4 mAb (mAb1) that were present in stressed material under differing levels of temperature stress. We demonstrate the power of combining established higher order techniques with non-routine analysis, such as small-angle X-ray scattering, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein conformational array enzyme-linked immunosorbent assay (PCA ELISA), and show that dimer species formed under temperature stress are structurally distinct from those present in unstressed mAb1. Specifically, stress-induced dimers are shown to adopt a more elongated conformation with a greater degree of unfolding when compared to native dimers. Analysis by HDX-MS and PCA ELISA, supported by in silico shape and charge molecular docking, enabled the identification of residues in both the variable and constant domains that appear to play a significant role in the dimerization of mAb1. Furthermore, we show that dimers formed under temperature stress are significantly more long-lived than those present in unstressed mAb1. We also present evidence that mAb1 dimers can behave as aggregation nuclei, and that dimers produced under high-temperature stress do so to a greater extent. This work presents an advancement in our understanding of the molecular mechanisms of mAb aggregation and highlights the importance of structural characterization of dimer species during the development of mAb biotherapeutics.Abbreviations: 2DSA: 2-Dimensional Spectrum Analysis; CD: Circular Dichroism; CDR: Complementarity-Determining Region; CQA: Critical Quality Attribute; DSC: Differential Scanning Calorimetry; FTIR: Fourier Transform Infrared spectroscopy; HDX-MS: Hydrogen/Deuterium Exchange Mass Spectrometry; HIC: Hydrophobic interaction chromatography; HMWS: High Molecular Weight Species; HOS: Higher Order Structure; mAb: Monoclonal Antibody; MD: Molecular Dynamics PCA; ELISA: Protein Conformational Array Enzyme-Linked Immunosorbent Assay; Rg: Radius of Gyration; SAXS: Small Angle X-ray Scattering; SE-HPLC: Size Exclusion High Performance Liquid Chromatography; SV-AUC: Sedimentation Velocity-Analytical Ultracentrifugation.

Photoinduced cross-linking of formulation buffer amino acids to monoclonal antibodies.

The correct choice of formulation buffer is a critical aspect of drug development and is chosen primarily to improve the stability of a protein therapeutic and protect against degradation. Amino acids are frequently incorporated into formulation buffers. In this study we have identified and characterized light induced cross-links between the side chain of histidine residues in an IgG4 monoclonal antibody and different amino acids commonly used in formulation buffers. These reactions have the potential to impact the overall product quality of the drug. The structure of each cross-link identified was elucidated using high performance liquid chromatography (HPLC) hyphenated to tandem mass spectrometry (MS/MS) with higher energy collisional dissociation (HCD). Furthermore, we speculate on the role of amino acids in formulation buffers and their influence on mAb stability. We theorize that whilst the adduction of formulation buffer amino acids could have a negative impact on product quality, it may protect against other pathways of photo-degradation.