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.

Correlating the Effects of Antimicrobial Preservatives on Conformational Stability, Aggregation Propensity, and Backbone Flexibility of an IgG1 mAb.

Multidose formulations of biotherapeutics, which offer better dosage management and reduced production costs, require the addition of antimicrobial preservatives (APs). APs have been shown, however, to decrease protein stability in solution and cause protein aggregation. In this report, the effect of 4 APs, m-cresol, phenol, phenoxyethanol, and benzyl alcohol on conformational stability, aggregation propensity, and backbone flexibility of an IgG1 mAb, mAb-4, is investigated. Compared with no preservative control, each of the APs decreased the conformational stability of mAb-4 as measured by differential scanning calorimetry and extrinsic fluorescence spectroscopy. The addition of APs resulted in increased monomer loss and aggregate accumulation at 50°C over 28 days, as monitored by size-exclusion chromatography. The extent of conformational destabilization and protein aggregation of mAb-4 induced by APs followed their calculated octanol-water partition coefficients. Increases in backbone flexibility, as measured by hydrogen exchange, of a region located in the CH2 domain of the mAb (heavy chain 237-254) in the presence of APs also correlated with hydrophobicity. Based on these results, the destabilizing effect of APs on mAb-4 correlates with the increased hydrophobicity of the APs and their ability to enhance the local backbone flexibility of an aggregation hot spot within the CH2 domain of the mAb.

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity.

Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in CH3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.

Site-Specific Hydrolysis Reaction C-Terminal of Methionine in Met-His during Metal-Catalyzed Oxidation of IgG-1.

The metal-catalyzed oxidation by [Fe(II)(EDTA)](2-)/H2O2 of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1-34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics 2013, 10 (2), 739-755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3-6%) were, however, observed in the oxidized samples by UV-visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI).

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody.

There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the VH domain, the constant domain (CH2), and the hinge region (CH1-CH2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.

Minimizing carry-over in an online pepsin digestion system used for the H/D exchange mass spectrometric analysis of an IgG1 monoclonal antibody.

Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30% of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5%)/isopropanol (5%)/acetic acid (20%) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.

Bacillus cereus: the forgotten pathogen.

BACKGROUND: Infection with Bacillus cereus is generally associated with gastrointestinal effects of food poisoning linked to infected rice. Isolates of B. cereus in hospital and clinical settings from any material other than vomitus or feces are commonly dismissed as contaminants. CASE REPORT: We report a case of B. cereus surgical site infection after fasciotomy in a healthy 31 year-old man admitted to the orthopedic ward with a comminuted fracture of the tibia. No source was identified. CONCLUSIONS: This report highlights the risk of surgical site infection with an unlikely bacterium known to contaminate surgical materials. It stresses the importance of vigilance against this infrequent but potentially serious non-gastrointestinal bacillary infection, as organisms dismissed initially as contaminants may lead to rapid clinical deterioration. The use of antimicrobial agents with nosocomial coverage, even of non-nosocomial pathogens, is considered in the treatment of postoperative surgical site infections.

Transulnar styloid palmar scapho-lunate dislocation with median nerve injury.

A patient with a transulnar styloid palmar scapho-lunate dislocation with median nerve injury is described. The dislocation could be reduced by closed manipulation under anaesthesia, and the scapho-lunate ligament was repaired subsequently using a Mytek Micro bone anchor. This case is reported for its rarity and its management. Although closed reduction can be achieved by manipulation, scapho-lunate ligament repair is essential to prevent rotatory instability of the scaphoid with this pattern of injury.