Modulation of the Fibrillation Kinetics and Morphology of a Therapeutic Peptide by Cucurbit[7]uril.

Fibrillation is a challenge commonly encountered in the formulation and development of therapeutic peptides. Cucurbit[7]urils (CB[7]), a group of water soluble macrocycles, have been reported to suppress fibrillation in insulin and human calcitonin through association with Phe and Tyr residues which drive fibril formation. Here, we report the effect of CB[7] on the fibrillation behavior of the HIV fusion inhibitor enfuvirtide (ENF) that contains N-terminal Tyr and C-terminal Phe residues. Thioflavin T fluorescence, CD spectroscopy, and transmission electron microscopy were used to monitor fibrillation behavior. Fibrillation onset showed a strong pH dependency, with pH 6.5 identified as the condition most suitable to monitor the effects of CB[7]. Binding of CB[7] to wild-type ENF was measured by isothermal titration calorimetry and was consistent with a single site (Ka = 2.4 × 105 M-1). A weaker interaction (Ka = 2.8 × 103 M-1) was observed for an ENF mutant with the C-terminal Phe substituted for Ala (ENFm), suggesting that Phe was the specific site for CB[7] recognition. The onset of ENF fibrillation onset was delayed, rather than fully suppressed, in the presence of CB[7]. The ENFm mutant showed a greater delay in fibrillation onset but with no observable effect on fibrillation kinetics in the presence of CB[7]. Interestingly, ENF/CB[7] and ENFm fibrils exhibited comparable morphologies, differing from those observed for ENF alone. The results indicate that CB[7] is capable of modulating fibrillation onset and the resulting ENF fibrils by specifically binding to the C-terminal Phe residue. The work reinforces the potential of CB[7] as an inhibitor of fibrillation and highlights its role in determining fibril morphologies.

Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Investigating the Orientation of an Interfacially Adsorbed Monoclonal Antibody and Its Fragments Using Neutron Reflection.

Interfacial adsorption is a molecular process occurring during the production, purification, transport, and storage of antibodies, with a direct impact on their structural stability and subsequent implications on their bioactivities. While the average conformational orientation of an adsorbed protein can be readily determined, its associated structures are more complex to characterize. Neutron reflection has been used in this work to investigate the conformational orientations of the monoclonal antibody COE-3 and its Fab and Fc fragments at the oil/water and air/water interfaces. Rigid body rotation modeling was found to be suitable for globular and relatively rigid proteins such as the Fab and Fc fragments but less so for relatively flexible proteins such as full COE-3. Fab and Fc fragments adopted a 'flat-on' orientation at the air/water interface, minimizing the thickness of the protein layer, but they adopted a substantially tilted orientation at the oil/water interface with increased layer thickness. In contrast, COE-3 was found to adsorb in tilted orientations at both interfaces, with one fragment protruding into the solution. This work demonstrates that rigid-body modeling can provide additional insights into protein layers at various interfaces relevant to bioprocess engineering.

Acidification-Induced Structure Evolution of Lipid Nanoparticles Correlates with Their In Vitro Gene Transfections.

The rational design of lipid nanoparticles (LNPs) for enhanced gene delivery remains challenging because of incomplete knowledge of their formulation-structure relationship that impacts their intracellular behavior and consequent function. Small-angle neutron scattering has been used in this work to investigate the structure of LNPs encapsulating plasmid DNA upon their acidification (from pH 7.4 to 4.0), as would be encountered during endocytosis. The results revealed the acidification-induced structure evolution (AISE) of the LNPs on different dimension scales, involving protonation of the ionizable lipid, volume expansion and redistribution of aqueous and lipid components. A similarity analysis using an LNP's structural feature space showed a strong positive correlation between function (measured by intracellular luciferase expression) and the extent of AISE, which was further enhanced by the fraction of unsaturated helper lipid. Our findings reveal molecular and nanoscale changes occurring during AISE that underpin the LNPs' formulation-nanostructure-function relationship, aiding the rational design of application-directed gene delivery vehicles.

Discovering Novel Small Molecule Compound for Prevention of Monoclonal Antibody Self-Association.

Designing an antibody with the desired affinity to the antigen is challenging, often achieved by lengthening the hydrophobic CDRs, which can lead to aggregation and cause major hindrance to the development of successful biopharmaceutical products. Aggregation can cause immunogenicity, viscosity and stability issues affecting both the safety and quality of the product. As the hydrophobic residues on the CDR are required for direct binding to antigens, it is not always possible to substitute these residues for aggregation-reduction purposes. Therefore, discovery of specific excipients to prevent aggregation is highly desirable for formulation development. Here, we used a combination of in silico screening methods to identify aggregation-prone regions on an aggregation-prone therapeutic antibody. The most aggregation-prone region on the antibody was selected to conduct virtual screening of compounds that can bind to such regions and act as an aggregation breaker. The most promising excipient candidate was further studied alongside plain buffer formulations and formulations with trehalose using coarse-grained molecular dynamics (CGMD) simulations with MARTINI force field. Mean interaction value between two antibody molecules in each formulation was calculated based on 1024 replicates of 512 ns of such CGMD simulations. Corresponding formulations with an excipient:antibody ratio of 1:5 were compared experimentally by measuring the diffusion interaction parameter kD and accelerated stability studies. Although the compound with the highest affinity score did not show any additional protective effects compared with trehalose, this study proved using a combination of in silico tools can aid excipient design and formulation development.

Report on Webinar Series Cell and Gene Therapy: From Concept to Clinical Use.

With the launch of the UK Academy of Pharmaceutical Sciences Advanced Therapy Medicinal Products Focus Group in late 2020, a webinar series reviewing the current and emerging trends in cell and gene therapy was held virtually in May 2021. This webinar series was timely given the recent withdrawal of the United Kingdom from the European Union and the global COVID-19 pandemic impacting all sectors of the pharmaceutical sciences research landscape globally and in the UK. Delegates from the academic, industry, regulatory and NHS sectors attended the session where challenges and opportunities in the development and clinical implementation of cell and gene therapies were discussed. Globally, the cell and gene therapy market has reached a value of 4.3 billion dollars in 2020, having increased at a compound annual growth rate of 25.5% since 2015. This webinar series captured all the major developments in this rapidly evolving area and highlighted emerging concepts warranting cross-sector efforts from across the community in the future.

Formulation Considerations for Autologous T Cell Drug Products.

Genetically modified autologous T cells have become an established immunotherapy in the fight against cancer. The manufacture of chimeric antigen receptor (CAR) and αß-T cell receptor (TCR) transduced T cells poses unique challenges, including the formulation, cryopreservation and fill-finish steps, which are the focus of this review. With an increasing number of marketing approvals for CAR-T cell therapies, comparison of their formulation design and presentation for administration can be made. These differences will be discussed alongside the emergence of automated formulation and fill-finish processes, the formulation design space, Monte Carlo simulation applied to risk analysis, primary container selection, freezing profiles and thaw and the use of dimethyl sulfoxide and alternative solvents/excipients as cryopreservation agents. The review will conclude with a discussion of the pharmaceutical solutions required to meet the simplification of manufacture and flexibility in dosage form for clinical treatment.

Cluster Percolation Causes Shear Thinning Behavior in Concentrated Solutions of Monoclonal Antibodies.

High-concentration (>100 g/L) solutions of monoclonal antibodies (mAbs) are typically characterized by anomalously large solution viscosity and shear thinning behavior for strain rates ≥103 s-1. Here, the link between protein-protein interactions (PPIs) and the rheology of concentrated solutions of COE-03 and COE-19 mAbs is studied by means of static and dynamic light scattering and microfluidic rheometry. By comparing the experimental data with predictions based on the Baxter sticky hard-sphere model, we surprisingly find a connection between the observed shear thinning and the predicted percolation threshold. The longest shear relaxation time of mAbs was much larger than that of model sticky hard spheres within the same region of the phase diagram, which is attributed to the anisotropy of the mAb PPIs. Our results suggest that not only the strength but also the patchiness of short-range attractive PPIs should be explicitly accounted for by theoretical approaches aimed at predicting the shear rate-dependent viscosity of dense mAb solutions.

Recent Advances in Studying Interfacial Adsorption of Bioengineered Monoclonal Antibodies.

Monoclonal antibodies (mAbs) are an important class of biotherapeutics; as of 2020, dozens are commercialized medicines, over a hundred are in clinical trials, and many more are in preclinical developmental stages. Therapeutic mAbs are sequence modified from the wild type IgG isoforms to varying extents and can have different intrinsic structural stability. For chronic treatments in particular, high concentration (≥ 100 mg/mL) aqueous formulations are often preferred for at-home administration with a syringe-based device. MAbs, like any globular protein, are amphiphilic and readily adsorb to interfaces, potentially causing structural deformation and even unfolding. Desorption of structurally perturbed mAbs is often hypothesized to promote aggregation, potentially leading to the formation of subvisible particles and visible precipitates. Since mAbs are exposed to numerous interfaces during biomanufacturing, storage and administration, many studies have examined mAb adsorption to different interfaces under various mitigation strategies. This review examines recent published literature focusing on adsorption of bioengineered mAbs under well-defined solution and surface conditions. The focus of this review is on understanding adsorption features driven by distinct antibody domains and on recent advances in establishing model interfaces suitable for high resolution surface measurements. Our summary highlights the need to further understand the relationship between mAb interfacial adsorption and desorption, solution aggregation, and product instability during fill-finish, transport, storage and administration.

Inhibiting the fibrillation of a GLP-1-like peptide.

Aggregation, including the formation of fibrils, poses significant challenges for the development of therapeutic peptides. To prepare stable peptide formulations, some understanding of the mechanisms underpinning the fibrillation process is required. A thioflavin T fluorescence assay was first used to determine the fibrillation profile of a GLP-1-like peptide (G48) at conditions being considered to formulate the peptide. G48 concentrations ranged from 0 to 600 µM and three pH values (pH 3.7, 7.4 and 8.5) were evaluated. Kinetic data demonstrate that G48 displays a pH-dependent aggregation profile. At pH 3.7, which is below the isoelectric point of G48 (pI ~ 5), kinetics representative of amorphous aggregates forming via a nucleation-independent mechanism were seen. At pH 7.4 and 8.5 (pH > pI) typical nucleation-dependent aggregation kinetics were observed. The weight concentration of ß-sheet rich aggregates (FLmax) correlated inversely with net charge, so lower FLmax values were observed at pH 3.7 and 8.5 than at pH 7.4. Incorporation of a non-ionic surfactant (polysorbate 80) into the peptide solution suppressed the fibrillation of G48 at all pH values and maintained the native peptide conformation, whereas a phenolic co-formulant (ferulic acid) had minimal effects on fibril growth. Peptide fibrillation, which can occur within a range of formulation concentrations and pH values, can hence be inhibited by the judicious use of excipients.

Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry.

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceutical products for oncology, with the cytotoxic pyrrolobenzodiazepine (PBD) family of "warheads" well-established in the clinic. While PBDs offer high potency, they are also characterized by their hydrophobicity, which can make formulation of the ADC challenging. Several approaches have been investigated to improve the physicochemical properties of PBD-containing ADCs, and herein a supramolecular approach was explored using cucurbit[8]uril (CB[8]). The ability of CB[8] to simultaneously encapsulate two guests was exploited to incorporate a 12-mer polyethylene glycol harboring a methyl viologen moiety at one terminus (MV-PEG12), together with a PBD harboring an indole moiety at the C2' position (SG3811). This formulation approach successfully introduced a hydrophilic PEG to mask the hydrophobicity of SG3811, improving the physical stability of the ADC while avoiding any loss of potency related to chemical modification.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

Determination of Protein-Protein Interactions in a Mixture of Two Monoclonal Antibodies.

The coformulation of monoclonal antibody (mAb) mixtures provides an attractive route to achieving therapeutic efficacy where the targeting of multiple epitopes is necessary. Controlling and predicting the behavior of such mixtures requires elucidating the molecular basis for the self- and cross-protein-protein interactions and how they depend on solution variables. While self-interactions are now beginning to be well understood, systematic studies of cross-interactions between mAbs in solution do not exist. Here, we have used static light scattering to measure the set of self- and cross-osmotic second virial coefficients in a solution containing a mixture of two mAbs, mAbA and mAbB, as a function of ionic strength and pH. mAbB exhibits strong association at a low ionic strength, which is attributed to an electrostatic attraction that is enhanced by the presence of a strong short-ranged attraction of nonelectrostatic origin. Under all solution conditions, the measured cross-interactions are intermediate self-interactions and follow similar patterns of behavior. There is a strong electrostatic attraction at higher pH values, reflecting the behavior of mAbB. Protein-protein interactions become more attractive with an increasing pH due to reducing the overall protein net charges, an effect that is attenuated with an increasing ionic strength due to the screening of electrostatic interactions. Under moderate ionic strength conditions, the reduced cross-virial coefficient, which reflects only the energetic contribution to protein-protein interactions, is given by a geometric average of the corresponding self-coefficients. We show the relationship can be rationalized using a patchy sphere model, where the interaction energy between sites i and j is given by the arithmetic mean of the i-i and j-j interactions. The geometric mean does not necessarily apply to all mAb mixtures and is expected to break down at a lower ionic strength due to the nonadditivity of electrostatic interactions.

Observation of high-temperature macromolecular confinement in lyophilised protein formulations using terahertz spectroscopy.

Characterising the structural dynamics of proteins and the effects of excipients are critical for optimising the design of formulations. In this work we investigated four lyophilised formulations containing bovine serum albumin (BSA) and three formulations containing a monoclonal antibody (mAb, here mAb1), and explored the role of the excipients polysorbate 80, sucrose, trehalose, and arginine on stabilising proteins. By performing temperature variable terahertz time-domain spectroscopy (THz-TDS) experiments it is possible to study the vibrational dynamics of these formulations. The THz-TDS measurements reveal two distinct glass transition processes in all tested formulations. The lower temperature transition, T g , ß , is associated with the onset of local motion due to the secondary relaxation whilst the higher temperature transition, T g , α , marks the onset of the α -relaxation. For some of the formulations, containing globular BSA as well as mAb1, the absorption at terahertz frequencies does not increase further at temperatures above T g , α . Such behaviour is in contrast to our previous observations for small organic molecules as well as linear polymers where absorption is always observed to steadily increase with temperature due to the stronger absorption of terahertz radiation by more mobile dipoles. The absence of such further increase in absorption with higher temperatures therefore suggests a localised confinement of the protein/excipient matrix at high temperatures that hinders any further increase in mobility. We found that subtle changes in excipient composition had an effect on the transition temperatures T g , α and T g , ß as well as the vibrational confinement in the solid state. Further work is required to establish the potential significance of the vibrational confinement in the solid state on formulation stability and chemical degradation as well as what role the excipients play in achieving such confinement.

Interfacial Adsorption of a Monoclonal Antibody and Its Fab and Fc Fragments at the Oil/Water Interface.

The physical stability of a monoclonal antibody (mAb) solution for injection in a prefilled syringe may in part depend on its behavior at the silicone oil/water interface. Here, the adsorption of a mAb (termed COE-3) and its fragment antigen-binding (Fab) and crystallizable (Fc) at the oil/water interface was measured using neutron reflection. A 1.4 ± 0.1 µm hexadecane oil film was formed on a sapphire block by a spin-freeze-thaw process, retaining its integrity upon contact with the protein solutions. Measurements revealed that adsorbed COE-3 and its Fab and Fc fragments retained their globular structure, forming layers that did not penetrate substantially into the oil phase. COE-3 and Fc were found to adsorb flat-on to the interface, with denser 45 and 42 Å inner layers, respectively, in contact with the oil and a more diffuse 17-21 Å outer layer caused by fragments adsorbing in a tilted manner. In contrast, Fab fragments formed a uniform 60 Å monolayer. Monolayers were formed under all conditions studied (10-200 ppm, using three isotopic contrasts), although changes in packing density across the COE-3 and Fc layers were observed. COE-3 had a higher affinity to the interface than either of its constituent fragments, while Fab had a lower interfacial affinity consistent with its higher net surface charge. This study extends the application of high-resolution neutron reflection measurements to the study of protein adsorption at the oil/water interface using an experimental setup mimicking the protein drug product in a siliconized prefilled syringe.

Arginine to Lysine Mutations Increase the Aggregation Stability of a Single-Chain Variable Fragment through Unfolded-State Interactions.

Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein-protein interactions. Here, we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behavior of scFv mutants harboring single or multiple site-specific arginine to lysine mutations in denaturing buffers. In 6 M guanidine hydrochloride (GdmCl) or 8 M urea, repulsive protein-protein interactions were measured for the wild-type and lysine-enriched (4RK) scFvs reflecting weakened short-range attractions and increased excluded volume. In contrast to the arginine-enriched mutant (7KR) scFv exhibited strong reversible association. In 3 M GdmCl, the minimum concentration at which the scFvs were unfolded, the hydrodynamic radius of 4RK remained constant but increased for the wild type and especially for 7KR. Studies of single-point arginine to lysine scFv mutants indicated that the observed aggregation propensity of arginine under denaturing conditions was nonspecific. Interestingly, one such swap generated a scFv with especially low aggregation rates under low/high ionic strengths and denaturing buffers; molecular modeling identified hydrogen bonding between the arginine side chain and main chain peptide groups, stabilizing the structure. The arginine/lysine ratio is not routinely considered in biopharmaceutical scaffold design or current amyloid prediction methods. This work therefore suggests a simple method for increasing the stability of a biopharmaceutical protein against aggregation.

Terahertz Spectroscopy: An Investigation of the Structural Dynamics of Freeze-Dried Poly Lactic-co-glycolic Acid Microspheres.

Biodegradable poly lactic-co-glycolic acid (PLGA) microspheres can be used to encapsulate peptide and offer a promising drug-delivery vehicle. In this work we investigate the dynamics of PLGA microspheres prepared by freeze-drying and the molecular mobility at lower temperatures leading to the glass transition temperature, using temperature-variable terahertz time-domain spectroscopy (THz-TDS) experiments. The microspheres were prepared using a water-in-oil-in-water (w/o/w) double-emulsion technique and subsequent freeze-drying of the samples. Physical characterization was performed by morphology measurements, scanning electron microscopy, and helium pycnometry. The THz-TDS data show two distinct transition processes, T g , ß in the range of 167-219 K, associated with local motions, and T g , α in the range of 313-330 K, associated with large-scale motions, for the microspheres examined. Using Fourier transform infrared spectroscopy measurements in the mid-infrared, we were able to characterize the interactions between a model polypeptide, exendin-4, and the PLGA copolymer. We observe a relationship between the experimentally determined T g , ß and T g , α and free volume and microsphere dynamics.

Interaction of a Macrocycle with an Aggregation-Prone Region of a Monoclonal Antibody.

Colloidal stability is among the key challenges the pharmaceutical industry faces during the production and manufacturing of protein therapeutics. Self-association and aggregation processes can not only impair therapeutic efficacy but also induce immunogenic responses in patients. Aggregation-prone regions (APRs) consisting of hydrophobic patches are commonly identified as the source for colloidal instability, and rational strategies to mitigate aggregation propensity often require genetic engineering to eliminate hydrophobic amino acid residues. Here, we investigate cucurbit[7]uril (CB[7]), a water-soluble macrocycle able to form host-guest complexes with aromatic amino acid residues, as a potential excipient to mitigate protein aggregation propensity. Two monoclonal antibodies (mAbs), one harboring an APR and one lacking an APR, were first assessed for their colloidal stability (measured as the translational diffusion coefficient) in the presence and absence of CB[7] using dynamic light scattering. Due to the presence of a tryptophan residue within the APR, we were able to monitor changes in intrinsic fluorescence in response to increasing concentrations of CB[7]. Isothermal titration calorimetry and NMR spectroscopy were then used to characterize the putative host-guest interaction. Our results suggest a stabilizing effect of CB[7] on the aggregation-prone mAb, due to the specific interaction of CB[7] with aromatic amino acid residues located within the APR. This provides a starting point for exploring CB[7] as a candidate excipient for the formulation of aggregation-prone mAbs.

Application of Magnetic Resonance to Assess Lyophilized Drug Product Reconstitution.

PURPOSE: Dynamic in-situ proton (1H) magnetic resonance imaging (MRI) and 1H T2-relaxometry experiments are described in an attempt to: (i) understand the physical processes, that occur during the reconstitution of lyophilized bovine serum albumin (BSA) and monoclonal antibody (mAb) proteins; and (ii) objectify the reconstitution time. METHODS: Rapid two-dimensional 1H MRI and diffusion weighted MRI were used to study the temporal changes in solids dissolution and characterise water mass transport characteristics. One-shot T2 relaxation time measurements were also acquired in an attempt to quantify the reconstitution time. Both MRI data and T2-relaxation data were compared to standard visual observations currently adopted by industry. The 1H images were further referenced to MRI calibration data to give quantitative values of protein concentration and, percentage of remaining undissolved solids. RESULTS: An algorithmic analysis of the 1H T2-relaxation data shows it is possible to classify the reconstitution event into three regimes (undissolved, transitional and dissolved). Moreover, a combined analysis of the 2D 1H MRI and 1H T2-relaxation data gives a unique time point that characterises the onset of a reconstituted protein solution within well-defined error bars. These values compared favourably with those from visual observations. Diffusion weighted MRI showed that low concentration BSA and mAb samples showed distinct liquid-liquid phase separation attributed to two liquid layers with significant density differences. CONCLUSIONS: T2 relaxation time distributions (whose interpretation is validated from the 2D 1H MR images) provides a quick and effective framework to build objective, quantitative descriptors of the reconstitution process that facilitate the interpretation of subjective visual observations currently adopted as the standard practice industry.

19F Dark-State Exchange Saturation Transfer NMR Reveals Reversible Formation of Protein-Specific Large Clusters in High-Concentration Protein Mixtures.

Proteins frequently exist as high-concentration mixtures, both in biological environments and increasingly in biopharmaceutical co-formulations. Such crowded conditions promote protein-protein interactions, potentially leading to formation of protein clusters, aggregation, and phase separation. Characterizing these interactions and processes in situ in high-concentration mixtures is challenging due to the complexity and heterogeneity of such systems. Here we demonstrate the application of the dark-state exchange saturation transfer (DEST) NMR technique to a mixture of two differentially 19F-labeled 145 kDa monoclonal antibodies (mAbs) to assess reversible temperature-dependent formation of small and large protein-specific clusters at concentrations up to 400 mg/mL. 19F DEST allowed quantitative protein-specific characterization of the cluster populations and sizes for both mAbs in the mixture under a range of conditions. Additives such as arginine glutamate and NaCl also had protein-specific effects on the dark-state populations and cluster characteristics. Notably, both mAbs appear to largely exist as separate self-associated clusters, which mechanistically respond differently to changes in solution conditions. We show that for mixtures of differentially 19F-labeled proteins DEST NMR can characterize clustering in a protein-specific manner, offering unique tracking of clustering pathways and a means to understand and control them.