Physicochemical characterization of biological and synthetic forms of two lipid A-based TLR4 agonists.

Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4' phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules' biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants.

Conformational Changes and Drivers of Monoclonal Antibody Liquid-Liquid Phase Separation.

Liquid-liquid phase separation is a phenomenon within biology whereby proteins can separate into dense and more dilute phases with distinct properties. Three antibodies that undergo liquid-liquid phase separation were characterized in the protein-rich and protein-poor phases. In comparison to the protein-poor phase, the protein-rich phase demonstrates more blue-shift tryptophan emissions and red-shifted amide I absorbances. Large changes involving conformational isomerization around disulfide bonds were observed using Raman spectroscopy. Amide I and protein fluorescence differences between the phases persisted to temperatures above the critical temperature but ceased at the temperature at which aggregation occurred. In addition, large changes occurred in the structural organization of water molecules within the protein-rich phase for all three antibodies. It is hypothesized that as the proteins have the same chemical potential in both phases, the protein viscosity is higher in the protein-rich phase resulting in slowed diffusion dependent protein aggregation in this phase. For all three antibodies we performed accelerated stability studies and found that the protein-rich phase aggregated at the same rate or slower than the protein-poor phase.

Intra-Micellar and Extra-Micellar Oxidation in Phosphate and Histidine Buffers Containing Polysorbate 80.

Polysorbate is a key excipient included in formulations of therapeutic proteins to help prevent aggregation and surface adsorption. The stability of both polysorbate and therapeutic proteins can be compromised by oxidative degradation. In general, polysorbate is added to formulations at concentrations above the critical micelle concentration (cmc). To date, however, few experiments have quantitatively addressed the extent of extra- and intra-micellar oxidation of polysorbate in pharmaceutically relevant buffers. This study utilizes 2,2'-azobis(2-methylpropionamidine)dihydrochloride (AAPH), a peroxyl radical-generating initiator, C11-BODIPY(581/591), a lipid peroxidation probe, and fluorescence spectroscopy to reveal that both intra- and extra-micellar oxidation proceed in pharmaceutically relevant phosphate and histidine buffers. It is further demonstrated that the relative extent of oxidation observed in the intra- and extra-micellar compartments is similar irrespective of the buffer system.

The structural basis of histone modifying enzyme specificity and promiscuity: Implications for metabolic regulation and drug design.

Histone modifying enzymes regulate chromatin architecture through covalent modifications and ultimately control multiple aspects of cellular function. Disruption of histone modification leads to changes in gene expression profiles and may lead to disease. Both small molecule inhibitors and intermediary metabolites have been shown to modulate histone modifying enzyme activity although our ability to identify successful drug candidates or novel metabolic regulators of these enzymes has been limited. Using a combination of large scale in silico screens and in vivo phenotypic analysis, we identified several small molecules and intermediary metabolites with distinctive HME activity. Our approach using unsupervised learning identifies the chemical fingerprints of both small molecules and metabolites that facilitate recognition by the enzymes active sites which can be used as a blueprint to design novel inhibitors. Furthermore, this work supports the idea that histone modifying enzymes sense intermediary metabolites integrating genes, environment and cellular physiology.

Observing reorientation dynamics with Time-Resolved fluorescence and molecular dynamics in varying periodic boundary conditions.

This work presents a combined study of time-resolved fluorescence spectroscopy and all-atom molecular dynamics simulation to investigate periodic boundary conditions' and water models' influence on the orientation dynamics and translational and rotational diffusion of peptides in solution. We have characterized the effects of solvent box size and water model choice on the dynamics of two peptide systems, NATA and WK5. Computationally, translational, and rotational diffusion and internal fluctuations are investigated through all-atom molecular dynamics simulation with two water models and different box sizes. These results are compared with time-resolved fluorescence anisotropy decay (FAD) measurements. The associated time constant and orientation dynamics from FAD measurement along the 1Lb axis provided baseline data to validate molecular dynamics simulation. The modeling results show that diffusion rates vary roughly in inverse proportion to water model viscosity, as one would expect. Corrections for finite box size are significant for translational diffusion and insignificant for rotational diffusion. This study also finds that internal dynamics described by autocorrelation functions and kinetic network models are relatively insensitive to both box size and water model properties. Our observation suggests that different peptide properties respond differently to a change in simulation conditions.Communicated by Ramaswamy H. Sarma.

pH-Dependent Phase Behavior and Stability of Cationic Lipid-mRNA Nanoparticles.

Lipid nanoparticles (LNPs) containing mRNA can deliver genetic material to cells for use as vaccines or protein replacement therapies. We characterized the effect of solution pH on cationic LNPs containing green fluorescent protein (EGFP) mRNA and their transfection efficiency. We compared the structural and colloidal properties of mRNA LNPs with LNPs not containing mRNA and mRNA free in solution. We used a combination of biophysical technique to build a picture of the structure of the lipids and mRNA across pH and temperature in the form of an empirical phase diagram (EPD). A combination of Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry was used to investigate lipid phase behavior. The mRNA-LNPs transition from an inverse hexagonal phase at pH values below the pKa of the cationic lipid to a lamellar phase above the pKa. At higher temperatures the mRNA-LNPs also transitioned from an inverse hexagonal phase to a lamellar phase indicating the inverse hexagonal phase is more thermodynamically favorable. Based on circular dichroism, the mRNA within the LNP has more A form structure at pH values below the lipid pKa than above it. Optical density, zeta potential and dynamic light scattering measurements were used to probe the colloidal stability of the mRNA-LNPs. The particles were larger and more prone to aggregation below the pKa. A stability study was performed to relate the biophysical characteristics to the storage of the particles in solution at 4 and 25 °C. mRNA-LNPs had the highest transfection efficiency and stability at pH values below the pKa. However, there was a trade-off between the stability and aggregation propensity since at very low pH the particles were most prone to aggregation. We performed kinetic experiments to show that the time scale of the pH-dependent phase behavior is slow (6 hour transition) and the transition from lamellar to inverse hexagonal phases is irreversible. This suggests that the lamellar phase is less stable and kinetically trapped. Our findings deepen our structural understanding of mRNA-LNPs and will aid the development of related formulations.

Oligomeric States and Hydrodynamic Properties of Lysyl Oxidase-Like 2.

Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius (Rh) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its Rh is in good agreement with the predicted value. The Rh values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in Rh between fl- and Δ1-2SRCR-LOXL2s in solution.

Probing the Internal Dynamics and Shape of Simple Peptides in Urea, Guanidinium Hydrochloride, and Proline Solutions with Time-Resolved Fluorescence Anisotropy and Atomistic Cosolvent Simulations.

Picosecond time-resolved fluorescence anisotropy was used to measure the effect of denaturants and osmolytes on the reorientation dynamics of the simplest dipeptide. The solvent denaturants guanidinium hydrochloride (gdm), urea, and the osmolyte proline were used at several concentrations. Analysis of the concentration dependence of denaturants at a fixed temperature showed faster and slower reorientation time in two different denaturants at a nearly identical solvent viscosity (η). The reorientation time τ significantly deviates from Kramers' theory (τ ∝ η1) in the high friction limit for guanidinium and urea with r ≈ 0.4 and r ≈ 0.6 at pH 7.2, respectively. In proline, τ is nearly proportional to η. Atomistic molecular dynamics simulations of the dipeptide in identical cosolvents showed excellent agreement with the measured rotational orientation time. The dipeptide dihedral (ϕ, ψ) isomerization times in water and 6 M urea are almost identical and significantly slower in guanidinium. If a faster and slower reorientation time can be associated with the compact and expanded shapes, the fractional viscosity dependence for guanidinium and urea may result from the fact that internal dynamics of peptides in these cosolvents involve higher and lower internal friction within the dynamic elements.

Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl-l-tryptophan Amide.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).

Dissecting Multiple Pathways in the Relaxation Dynamics of Helix <==> Coil Transitions with Optimum Dimensionality Reduction.

Fast kinetic experiments with dramatically improved time resolution have contributed significantly to understanding the fundamental processes in protein folding pathways involving the formation of a-helices and b-hairpin, contact formation, and overall collapse of the peptide chain. Interpretation of experimental results through application of a simple statistical mechanical model was key to this understanding. Atomistic description of all events observed in the experimental findings was challenging. Recent advancements in theory, more sophisticated algorithms, and a true long-term trajectory made way for an atomically detailed description of kinetics, examining folding pathways, validating experimental results, and reporting new findings for a wide range of molecular processes in biophysical chemistry. This review describes how optimum dimensionality reduction theory can construct a simplified coarse-grained model with low dimensionality involving a kinetic matrix that captures novel insights into folding pathways. A set of metastable states derived from molecular dynamics analysis generate an optimally reduced dimensionality rate matrix following transition pathway analysis. Analysis of the actual long-term simulation trajectory extracts a relaxation time directly comparable to the experimental results and confirms the validity of the combined approach. The application of the theory is discussed and illustrated using several examples of helix <==> coil transition pathways. This paper focuses primarily on a combined approach of time-resolved experiments and long-term molecular dynamics simulation from our ongoing work.

Prediction Machines: Applied Machine Learning for Therapeutic Protein Design and Development.

The rapid growth in technological advances and quantity of scientific data over the past decade has led to several challenges including data storage and analysis. Accurate models of complex datasets were previously difficult to develop and interpret. However, improvements in machine learning algorithms have since enabled unparalleled classification and prediction capabilities. The application of machine learning can be seen throughout diverse industries due to their ease of use and interpretability. In this review, we describe popular machine learning algorithms and highlight their application in pharmaceutical protein development. Machine learning models have now been applied to better understand the nonlinear concentration dependent viscosity of protein solutions, predict protein oxidation and deamidation rates, classify sub-visible particles and compare the physical stability of proteins. We also applied several machine learning algorithms using previously published data and describe models with improved predictions and classification. The authors hope that this review can be used as a resource to others and encourage continued application of machine learning algorithms to problems in pharmaceutical protein development.

Optimization of RG1-VLP vaccine performance in mice with novel TLR4 agonists.

Current human papilloma virus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain that lack vaccine coverage. The novel RG1-VLP (virus-like particle) vaccine candidate utilizes the HPV16-L1 subunit as a backbone to display an inserted HPV16-L2 17-36 a.a. "RG1" epitope; the L2 RG1 epitope is conserved across many HPV types and the generation of cross-neutralizing antibodies (Abs) against which has been demonstrated. In an effort to heighten the immunogenicity of the RG1-VLP vaccine, we compared in BALB/c mice adjuvant formulations consisting of novel bacterial enzymatic combinatorial chemistry (BECC)-derived toll-like receptor 4 (TLR4) agonists and the aluminum hydroxide adjuvant Alhydrogel. In the presence of BECC molecules, consistent improvements in the magnitude of Ab responses to both HPV16-L1 and the L2 RG1 epitope were observed compared to Alhydrogel alone. Furthermore, neutralizing titers to HPV16 as well as cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39 were augmented in the presence of BECC agonists as well. Levels of L1 and L2-specific Abs were achieved after two vaccinations with BECC/Alhydrogel adjuvant that were equivalent to or greater than levels achieved with 3 vaccinations with Alhydrogel alone, indicating that the presence of BECC molecules resulted in accelerated immune responses that could allow for a decreased dose schedule for VLP-based HPV vaccines. In addition, dose-sparing studies indicated that adjuvantation with BECC/Alhydrogel allowed for a 75% reduction in antigen dose while still retaining equivalent magnitudes of responses to the full VLP dose with Alhydrogel. These data suggest that adjuvant optimization of HPV VLP-based vaccines can lead to rapid immunity requiring fewer boosts, dose-sparing of VLPs expensive to produce, and the establishment of a longer-lasting humoral immunity.

Short-term and longer-term protective immune responses generated by subunit vaccination with smallpox A33, B5, L1 or A27 proteins adjuvanted with aluminum hydroxide and CpG in mice challenged with vaccinia virus.

Smallpox, a contagious and deadly disease caused by variola virus, was eradicated by a strategy that included vaccination with vaccinia virus, a live-virus vaccine. Because the threat of bioterrorism with smallpox persists and infections with zoonotic poxvirus infections like monkeypox continue, and there may be a time when an alternative vaccine platform is needed, recombinant-subunit vaccine strategies for poxviruses have been pursued. Our prior work focused on understanding the immune responses generated to vaccine-formulations containing the virus protein L1. In this work, we examine vaccine-formulations with additional key protein targets: A33 and B5 (components of the extracellular virus) and another protein on the mature virus (A27) adjuvanted with aluminum hydroxide (AH) with and without CpG- oligonucleotide. Each vaccine was formulated to allow either adsorption or non-adsorption of the protein (and CpG) to AH. Mice given a prime and single boost produced long-lasting antibody responses. A second boost (given ~5-months after the first) further increased antibody titers. Similar to our prior findings with L1 vaccine-formulations, the most protective A33 vaccine-formulations included CpG, resulted in the generation of IgG2a-antibody responses. Unlike the prior findings with L1 (where formulations that adsorbed both the protein and the CpG to AH resulted in 100% survival after challenge and minimal weight loss), the AH-adsorption status of A33 and CpG did not play as important a role, since both AH-adsorbed and non-adsorbed groups lost weight after challenge and had similar survival. Vaccination with B5-formulations gave different results. While CpG-containing formulations were the only ones that generated IgG2a-antibody responses, the vaccine-formulation that adsorbed B5 to AH (without CpG) was as equally effective in protecting mice after challenge. These results indicate that the mechanism of how antibodies against A33 and B5 protect differ. The data also show the complexity of designing optimized vaccine-formulations containing multiple adjuvants and recombinant protein-based antigens.

Synthetic Cationic Autoantigen Mimics Glatiramer Acetate Persistence at the Site of Injection and Is Efficacious Against Experimental Autoimmune Encephalomyelitis.

A synthetic peptide, K-PLP, consisting of 11-unit poly-lysine (K11) linked via polyethylene glycol (PEG) to proteolipid protein epitope (PLP) was synthesized, characterized, and evaluated for efficacy in ameliorating experimental autoimmune encephalomyelitis (EAE) induced by PLP. K-PLP was designed to mimic the cationic nature of the relapsing-remitting multiple sclerosis treatment, glatiramer acetate (GA). With a pI of ~10, GA is able to form visible aggregates at the site of injection via electrostatic interactions with the anionic extracellular matrix. Aggregation further facilitates the retention of GA at the site of injection and draining lymph nodes, which may contribute to its mechanism of action. K-PLP with a pI of ~11, was found to form visible aggregates in the presence of glycosaminoglycans and persist at the injection site and draining lymph nodes in vivo, similar to GA. Additionally, EAE mice treated with K-PLP showed significant inhibition of clinical symptoms compared to free poly-lysine and to PLP, which are the components of K-PLP. The ability of the poly-lysine motif to retain PLP at the injection site, which increased the local exposure of PLP to immune cells may be an important factor affecting drug efficacy.

Amino/Amido Conjugates Form to Nanoscale Cobalt Physiometacomposite (PMC) Materials Functionally Delivering Nucleic Acid Therapeutic to Nucleus Enhancing Anticancer Activity via Ras-Targeted Protein Interference.

Aberrant splicing and protein interaction of Ras binding domain (RBD) are associated with melanoma drug resistance. Here, cobalt or nickel doped zinc oxide (ZnO) physiometacomposite (PMC) materials bind to RNA and peptide shown by Ninhydrin staining, UV-vis, Fourier transform infrared, and circular dichroism spectroscopy. PMCs deliver splice switching oligomer (SSO) into melanoma cells or 3-D tumor spheroids shown by flow cytometry, fluorescence microscopy, and bioluminescence. Stability in serum, liver, or tumor homogenate up to 48 h and B16F10 melanoma inhibition ≥98-99% is shown. These data suggest preclinical potential of PMC for delivery of SSO, RBD, or other nucleic acid therapeutic and anticancer peptides.

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 2.

In this work, we continue to examine excipient effects on the reversible self-association (RSA) of 2 different IgG1 monoclonal antibodies (mAb-J and mAb-C). We characterize the RSA behavior of mAb-C which, similar to mAb-J (see Part 1), undergoes concentration-dependent RSA, but by a different molecular mechanism. Five additives that affect protein hydrophobic interactions to varying extents including a chaotropic salt (guanidine hydrochloride), a hydrophobic salt (trimethylphenylammonium iodide), an aromatic amino acid derivative (tryptophan amide hydrochloride), a kosmotropic salt (sodium sulfate, Na2SO4), and a less polar solvent (ethanol) were evaluated to determine their effects on the solution properties, molecular properties, and RSA of mAb-C at various protein concentrations. Four of the 5 additives examined demonstrated favorable effects on the pharmaceutical properties of high concentration mAb-C solutions (i.e., lower viscosity and weakened protein-protein interactions, PPIs) with a ranking order of guanidine hydrochloride > trimethylphenylammonium iodide > tryptophan amide hydrochloride > ethanol as measured by various biophysical techniques. Conversely, addition of Na2SO4 resulted in less desirable solution properties and enhanced PPIs. The effect of these 5 additives on mAb-C backbone dynamics were evaluated by hydrogen exchange-mass spectrometry (at high vs. low protein concentrations) to better understand their effects on the molecular sites of RSA in mAb-C.

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 1.

Many challenges limit the formulation of antibodies as high-concentration liquid dosage forms including elevated solution viscosity, decreased physical stability, and in some cases, liquid-liquid phase separation. In this work, an IgG1 monoclonal antibody (mAb-J), which undergoes concentration-dependent reversible self-association (RSA), is characterized in the presence of 4 amino acids (Arg, Lys, Asp, Glu) and NaCl using biophysical techniques and hydrogen exchange-mass spectrometry. The 5 additives disrupt RSA, prevent phase separation, and reduce solution viscosity to varying extents. These excipients also cause decreased turbidity, reduced average hydrodynamic diameter, and increased relative solubility of mAb-J in solution. The RSA disrupting efficacy of the positively charged amino acids is greater than either negatively charged amino acids or NaCl. As measured by hydrogen exchange-mass spectrometry, anionic excipients induced more alterations of mAb-J backbone dynamics at pH 6.0, and weak Fab-Fab interactions likely remained with the addition of either cationic or anionic excipients at high protein concentrations. Along with a companion paper examining a different mAb with a different molecular mechanism of RSA, these results are discussed in the context of various excipient strategies to disrupt protein-protein interactions to formulate mAbs at high protein concentrations with good stability profiles and favorable pharmaceutical properties for subcutaneous administration.

Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc.

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc2Man(8+n) [n = 0-4]), Man5 (GlcNAc2Man5), GlcNAc1, and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc1 > Man5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc1 IgG4-Fc. The affinities measured for the HM and Man5 IgG4-Fc (0.39-0.52 µM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA.

Cis/Trans Isomerization of Unsaturated Fatty Acids in Polysorbate 80 During Light Exposure of a Monoclonal Antibody-Containing Formulation.

Light exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of monounsaturated and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization mass spectrometry of intact PS80 components as well as by negative ion electrospray ionization mass spectrometry analysis of free fatty acids generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the monoclonal antibody, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide and glutathione disulfide, suggesting the involvement of thiyl radicals generated by photoinduced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid, we detected conjugated linoleic acids in PS80, which underwent light-induced cis/trans isomerization.

Comparison of Polysorbate 80 Hydrolysis and Oxidation on the Aggregation of a Monoclonal Antibody.

Polysorbates are used ubiquitously in protein therapeutic drugs to help minimize adsorption to surfaces and aggregation. It has been recognized that polysorbate can itself degrade and in turn result in loss of efficacy of therapeutic proteins. We studied the 2 main pathways of polysorbate 80 (PS80) degradation, enzymatic ester hydrolysis, and oxidation. Degraded polysorbates were quantified through mass spectrometry to identify the loss of individual components. Next Langmuir trough adsorption isotherms were used to characterize changes in the surface activity of the degraded polysorbates. PS80 degraded via hydrolysis results in slower surface adsorption rates, whereas the oxidized PS80 show increased surface activity. However, the critical micelle concentration remained unchanged. A monoclonal antibody was formulated with stock and degraded polysorbates to probe their ability to prevent aggregation. Hydrolyzed polysorbate resulted in a large increase in particle formation during shaking stress. Oxidized PS80 was still protective against aggregation for the monoclonal antibody. Monomer loss as measured by SEC was comparable in formulations without PS80 to those with esterase hydrolyzed PS80. Monomer loss for oxidized PS80 was similar to that of nondegraded PS80. Hydrolysis of PS80 resulted in free fatty acids which formed insoluble particles during mechanical agitation which stimulated protein aggregation.