Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.     

High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial or complete enzymatic removal of the N-linked Fc glycan, were compared with the untreated mAb over a wide range of temperature (10 °C-90 °C) and solution pH (3-8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams. Subtle-to-larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0-6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in the physical stability of a mAb glycoform. On the basis of these results, a two-step methodology was used in which conformational stability of a mAb glycoform is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques, and data visualization methods. With this approach, a high-throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated.     

Structural basis of the interaction between immunoglobulins and Fc receptors provided by NMR spectroscopy

Fc gamma Receptors (Fc gamma R) are membrane glycoproteins that bind the Fc portion of immunoglobulin G (IgG). The cross linking of Fc gamma R-bound IgG by multivalent antigens allows clustering of the Fc gamma R and initiates a variety of effector mechanisms which play a key role in immune defenses against pathogens. The Fc region is composed of two identical polypeptide chains, which are related to each other by a two-fold axis. Recent elucidation of the crystal structure of human Fc gamma RII provided two distinct views of modes of IgG-Fc gamma R interactions, which is controversial against each other. Nuclear magnetic resonance (NMR) spectroscopy provides a unique and irreplaceable tool to solve these issues. We recently studied the interaction between the Fc fragment of mouse IgG2b and the extracellular domain of mouse Fc gamma RII by this method. We showed that Fc gamma RII binds to a negatively charged area of the CH2 domain, corresponding to the lower hinge region, and that the binding of Fc gamma RII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. This conformational change may account for the 1:1 stoichiometry that we and others observed between Fc gamma R and Fc. We therefore propose a model that explains why the interaction between IgG molecules and Fc gamma R does not trigger cellular responses in the absence of cross linking by multivalent antigens and does not lead to spontaneous inflammatory responses that would be deleterious for the organism.     

A Detailed Protocol for Generation of Therapeutic Antibodies with Galactosylated Glycovariants at Laboratory Scale Using In-Vitro Glycoengineering Technology

N-linked glycosylation is an important post translational modification that occurs on Asparagine 297 residue or a homologous position on the Fc portion of monoclonal antibodies (mAbs). mAb Fc glycans play important roles in antibody structure, stability, and function including effector function and pharmacokinetics. The Fc glycans are made up of a wide variety of sugars including galactose, mannose, and sialic acid. The role of galactose in mediating antibody effector functions is not well understood. Hence, there is widespread interest in the antibody research community to understand the role of galactose in antibody effector functions as galactose is a major constituent of antibody glycans. This requires generation of highly enriched galactosylated variants that has been very challenging via cell culture process. To tackle this challenge, we developed a laboratory scale biochemical process to produce highly enriched galactosylated variants. In this article, we report optimized lab-scale workflows and detailed protocols for generation of deglycosylated, hypo-galactosylated and hyper-galactosylated variants of IgG therapeutic antibodies using the in-vitro glycoengineering technology. The optimized workflows offer short turnaround time and produce highly enriched deglycosylated/hypo-galactosylated/hyper-galactosylated IgG glycovariants, with high purity & molecular integrity as demonstrated by data from an example IgG.     

Stability of Monoclonal Antibodies at High-Concentration: Head-to-Head Comparison of the IgG1 and IgG4 Subclass

Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG₁ and one IgG₄) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences between the IgG subclasses. Both physical and chemical stability were evaluated by applying a range of methods to measure formation of protein aggregates [sizeexclusion chromatography (SEC)–HPLC and UV340 nm], structural integrity (circular dichroism and FTIR), thermodynamic stability (differential scanning calorimetry), colloidal interactions (dynamic light scattering), and fragmentation and deamidation (SEC–HPLC and capillary isoelectric focusing). The impact of pH (4–9) and ionic strength (10 and 150 mm) was investigated using highlyconcentrated (150 mg/mL) mAb formulations. Lower conformational stability was identified for the IgG₄ resulting in increased levels of soluble aggregates. The IgG₁ was chemically less stable as compared with the IgG₄, presumably because of the higher flexibility in the IgG₁ hinge region. The thermodynamic stability of individual mAb domains was also addressed in detail. The stability of our mAb molecules is clearly affected by the IgG framework, and this study suggests that subclass switching may alter aggregation propensity and aggregation pathway and thus potentially improve the overall formulation stability while retaining antigen specificity. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:115–127, 2014     

Biophysical Characterization and Stability of Modified IgG1 Antibodies with Different Hexamerization Propensities

The hexamerization of natural, human IgG antibodies after cell surface antigen binding can induce activation of the classical complement pathway. Mutations stimulating Fc domain-mediated hexamerization can potentiate complement activation and induce the clustering of cell surface receptors, a finding that was applied to different clinically investigated antibody therapeutics. Here, we biophysically characterized how increased self-association of IgG1 antibody variants with different hexamerization propensity may impact their developability, rather than functional properties. Self-Interaction Chromatography, Dynamic Light Scattering and PEG-induced precipitation showed that IgG variant self-association at neutral pH increased in the order wild type (WT) < E430G < E345K < E345R < E430G-E345R-S440Y, consistent with functional activity. Self-association was strongly pH-dependent, and single point mutants were fully monomeric at pH 5. Differential Scanning Calorimetry and Fluorimetry showed that mutation E430G decreased conformational stability. Interestingly, heat-induced unfolding facilitated by mutation E430G was reversible at 60°C, while a solvent-exposed hydrophobic mutation caused irreversible aggregation. Remarkably, neither increased dynamic self-association propensity at neutral pH nor decreased conformational stability substantially affected the stability of concentrated variants E430G or E345K during storage for two years at 2-8°C. We discuss how these findings may inform the design and development of IgG-based therapeutics.     

Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions

A systematic approach is presented to characterize and stabilize the higher order structural integrity of an immunoglobulin G (IgG1) monoclonal antibody (mAb) formulated at both low concentrations and as a highly concentrated solution. The conformational and colloidal stabilities of a recombinant humanized IgG1κ mAb at both 1 and 100 mg/mL were investigated as a function of solution temperature (10°C-87.5°C) and pH (3-8). Protein secondary structure was characterized using circular dichroism, whereas intrinsic (tryptophan) and extrinsic (8-anilino-1-naphthalenesulfonic acid) fluorescence spectroscopy measurements were used to evaluate the tertiary structure of the protein. Light scattering analysis was employed to monitor mAb aggregation behavior as a function of temperature and solution pH. These biophysical data sets were analyzed and summarized using a previously described empirical phase diagrams (EPDs) approach. The different phases observed in the EPD were correlated with the individual physical states of the IgG1 in solution (aggregated, native, unfolded, etc.). The temperature-dependent conformational stability profile of the mAb, at both 1 and 100 mg/mL, generally followed the order pH 6 ≥ pH 7 ≥ pH 8 > pH 5 > pH 4 ≥ pH 3. Analysis of the EPD apparent phase boundaries identified solution conditions of pH 4.5 near 60°C for the development of an excipient screening assay. A supplemented generally regarded as safe excipient library was screened using an aggregation assay (optical density at 350 nm) at low mAb concentrations (4 mg/mL) and potential stabilizers were identified. The ability of these excipients to prevent conformational alterations in high concentration mAb solutions (100 mg/mL) was determined by monitoring tertiary structure changes using an intrinsic fluorescence method. The results suggest that substantial increases in the onset temperature of thermal transitions (>5°C) are obtained in the presence of (a) 20% dextrose, (b) 20% sorbitol, and (c) 5% dextrose + 10% sorbitol. Similar stabilization effects were obtained at an intermediate (50 mg/mL) as well as low mAb concentrations (1 mg/mL).     

Small-Angle X-ray Scattering Screening Complements Conventional Biophysical Analysis: Comparative Structural and Biophysical Analysis of Monoclonal Antibodies IgG1, IgG2, and IgG4

A crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development. © 2014 The Authors. Journal of Pharmaceutical Sciences published by Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1701-1710, 2014     

The comparison of BLyS-binding peptides from phage display library and computer-aided design on BLyS–TACI interaction

BLyS antagonists have become the therapeutic reagents in the treatment of autoimmune disorders. BLyS binding peptides and their Fc fusion proteins may be alternative BLyS antagonists in such application. In this study, the activity of BLyS binding peptide 814 obtained from phage display library and peptide TA designed by computer-aided modeling on the interaction of BLyS–TACI was compared. In addition, to maintain the spatial conformation and stability of the peptides, human IgG1 Fc fragment was fused to peptides 814 and TA to form peptide-Fc fusion proteins, steady and innovative peptibodies. The prokaryotic expression plasmids pET30a-814-Fc and pET30a-TA-Fc for these peptibodies were acquired by genetic engineering, and confirmed by DNA sequencing. After the right plasmids were transformed into Escherichia coli BL21 (DE3), the fusion proteins were expressed and purified by protein A affinity column. As a result of competitive ELISA, peptides 814 and TA at 100 μg/ml displayed 52.2% and 28.6% inhibition on the interaction of TACI-Fc with BLyS respectively. Moreover, 814-Fc and TA-Fc fusion proteins could bind to BLyS in a dosage-dependent manner as TACI-Fc did, and displayed 54.7% and 26.1% inhibition on the interaction of TACI-Fc-Myc with BLyS at 100 μg/ml respectively. So 814-Fc and TA-Fc proteins had the similar bioactivity as the peptides did. Furthermore, compared with TA-Fc, 814-Fc showed two-fold inhibition effect on BLyS binding to TACI, suggesting that 814-Fc could inhibit BLyS bioactivity significantly and might serve as a potential antagonist to treat autoimmune diseases associated with BLyS overexpression.     

Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure,Stability, Aggregation,and Function of a Therapeutic Monoclonal Antibody

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the C_H2 domain, whereas AAPH destabilized the variable domain in addition to C_H2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.     

Comparison of high-throughput biophysical methods to identify stabilizing excipients for a model IgG2 monoclonal antibody: conformational stability and kinetic aggregation measurements

The overall conformational stability of a model IgG2 monoclonal antibody (mAb) was examined as a function of temperature and pH using an empirical phase diagram approach. Stabilizing excipients were then identified based on high-throughput methods including (1) kinetic studies measuring aggregation via increases in optical density and (2) thermally induced structural transitions as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The kinetic profiles of antibody aggregation at 65 °C were pH dependent and correlated well with pH effects on secondary and tertiary structural transitions due to heat stress. For the screening of stabilizing excipients, the inhibition of the rate of protein aggregation at pH 4.5 at 65°C, as represented by changes in optical density, was shown to have a clear trend with a modest correlation coefficient compared with the stabilizing effect of the same excipients on the conformational stability of the antibody as measured by DSC and tryptophan fluorescence spectroscopy. These results demonstrate the utility of combining high-throughput data from protein aggregation kinetic experiments and conformational stability studies to identify stabilizing excipients that minimize the physical degradation of an IgG2 mAb.     

Effect of Ionic Strength and pH on the Physical and Chemical Stability of a Monoclonal Antibody Antigen-Binding Fragment

Monoclonal antibody (mAb) fragments are emerging as promising alternatives to full-length mAbs as protein therapeutic candidates. Antigen-binding fragments (Fabs) are the most advanced with three Fab-based drug products currently approved. This work presents preformulation characterization data on the effect of pH, NaCl concentration, and various cationic excipients on the physical and chemical stability of a Fab molecule with multiple negatively charged Asp residues in the complementarity-determining region. Conformational stability was evaluated using an empirical phase diagram approach based on circular dichroism, intrinsic Trp and extrinsic 8-anilino-1-naphthalene sulfonate (ANS) fluorescence, and static light scattering measurements. The effect of NaCl concentration, various cationic excipients and pH on the Fab molecule’s conformational stability, aggregation propensity, and chemical stability (Asp isomerization) was determined by differential scanning calorimetry, optical density measurements at 350 nm (OD₃₅₀), and ion-exchange chromatography, respectively. Increasing NaCl concentration increased the overall conformational stability, decreased aggregation rates, and lowered the rates of Asp isomerization. No such trends were noted for pH or cationic excipients. The potential interrelationships between protein conformational and chemical stability are discussed in the context of designing stable protein formulations.     

Correlating Excipient Effects on Conformational and Storage Stability of an IgG1 Monoclonal Antibody with Local Dynamics as Measured by Hydrogen/Deuterium-Exchange Mass Spectrometry

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (T_m values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (C_H2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the C_H2 domain (notably heavy chain 241–251) and the mAb's overall physical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2136–2151, 2013     

Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles

The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus-like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature-induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C-60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7-8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3-4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long-term stability studies of eVLPs and mVLPs.     

Effect of pH and light on aggregation and conformation of an IgG1 mAb

During the purification process, monoclonal antibodies may be exposed to parts of UV-C (200 to 290 nm), UV-B (290 to 320 nm) and visible light (400 to 760 nm) under a variety of buffer and pH conditions. Together, these conditions can promote both chemical and physical degradation which may result in conformational changes. To examine this possibility, an IgG1 mAb at pH 3.5, 5, and 8 was exposed to UV light at multiple protein concentrations. Exposure to 302 nm light resulted in a pH-dependent formation of high molecular weight species where the degree of oligomerization increased with increasing pH. Characterization by SDS-PAGE under reducing and nonreducing conditions and size exclusion MALS revealed that the predominant species were nonreducible dimeric, trimeric and higher order oligomeric species which occurred through processes other than intermolecular disulfide bond formation. Biophysical characterization by differential scanning calorimetry demonstrated an overall loss of heat capacity suggesting a loss of conformational integrity with light exposure. A decrease in tryptophan fluorescence was paralleled by a significant decrease in the transition temperature measured during heat-induced unfolding following light exposure, also suggesting a significant change in conformational integrity. The observations by fluorescence spectroscopy coincided with pH-dependent changes in the alterations of secondary structure characterized by Fourier transform infrared spectroscopy and far-UV circular dichroism with the most acidic pH showing the greatest degree of change in the β-sheet structure. Exposure to UV light resulted in aggregation with pH-dependent yields decreasing in the order 8.0 > 5.0 > 3.5, while the opposite trend was observed for conformational changes, with pH-dependent extents decreasing in the following order 3.5 > 5.0 > 8.0. These pH-dependent trends suggest that different strategies will be required to stabilize the protein against these modifications during processing.     

One-step synthesis of site-specific antibody–drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates

Glycosite-specific antibody?drug conjugatess (gsADCs), harnessing Asn297 N-glycan of IgG Fc as the conjugation site for drug payloads, usually require multi-step glycoengineering with two or more enzymes, which limits the substrate diversification and complicates the preparation process. Herein, we report a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to one-step synthesis of gsADCs, catalyzed by an endo-N-acetylglucosaminidase (ENGase) of Endo-S2. IgG glycoengineering via ENGases usually has two steps: deglycosylation by wild-type (WT) ENGases and transglycosylation by mutated ENGases. But in the current method, we have found that disaccharide LacNAc oxazoline can be efficiently assembled onto IgG by WT Endo-S2 without hydrolysis of the product, which enables the one-step glycoengineering directly from native antibodies. Further studies on substrate specificity revealed that this approach has excellent tolerance on various modification of 6-Gal motif of LacNAc. Within 1 h, one-step synthesis of gsADC was achieved using the LacNAc-toxin substrates including structures free of bioorthogonal groups. These gsADCs demonstrated good homogeneity, buffer stability, in vitro and in vivo anti-tumor activity. This work presents a novel strategy using LacNAc-based substrates to reprogram the multi-step IgG glycoengineering to a one-step manner for highly efficient synthesis of gsADCs.     

Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K_d values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5–32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1–8.8% and (3.0–9.0)%, respectively with the inter-assay precision and accuracy were 5.7–11.5% and (10.7–9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32–320 μg/kg).     

Characterization and Stabilization of Recombinant Human Protein Pentraxin (rhPTX-2)

The potential human therapeutic protein pentraxin (PTX-2) is a large, glycosylated plasma protein consisting of five monomers that self-associate noncovalently into a pentameric, ring-like structure. The structural integrity and conformational stability of recombinant human PTX-2 (rhPTX-2) as a function of temperature and pH is described in conjunction with the identification of stabilizing excipients that improve the protein’s physical stability. The monomer consisted primarily of two glycosylated forms (25,171 and 25,462 Da) as determined by mass spectrometry. The protein was approximately 82%–97% pentamer in solution with smaller amounts of decamer and aggregate as measured by analytical ultracentrifugation and size-exclusion chromatography (SEC). Comprehensive biophysical characterization using an empirical phase diagram approach demonstrates that rhPTX-2 is conformationally stable over a wide pH range (6.0–8.5) and at temperatures below 65°C–75°C. Formation of soluble aggregates was identified as a major physical degradation pathway of rhPTX-2 in solution under accelerated storage conditions. A series of effective stabilizers was identified by SEC analysis including disaccharides, sugar alcohols, citrate, as well as neutral and charged amino acids. The preformulation characterization data and stability profile presented in this work enable future studies for rhPTX-2 formulation development.     

Excipients differentially influence the conformational stability and pretransition dynamics of two IgG1 monoclonal antibodies

Since immunoglobulins are conformationally dynamic molecules in solution, we studied the effect of stabilizing and destabilizing excipients on the conformational stability and dynamics of two IgG1 monoclonal antibodies (mAbs; mAb-A and mAb-B) using a variety of biophysical approaches. Even though the two mAbs are of the same IgG1 subtype, the unfolding patterns, aggregation behavior, and pretransition dynamics of these two antibodies were strikingly different in response to external perturbations such as pH, temperature, and presence of excipients. Sucrose and arginine were identified as stabilizers and destabilizers, respectively, on the basis of their influence on conformational stability for both the IgG1 mAbs. The two excipients, however, had distinct effective concentrations and different effects on the conformational stability and pretransition dynamics of the two mAbs as measured by a combination of differential scanning calorimetry, high-resolution ultrasonic spectroscopy, and red-edge excitation shift fluorescence studies. Stabilizing concentrations of sucrose were found to decrease the internal motions of mAb-B, whereas arginine marginally increased its adiabatic compressibility in the pretransition region. Both sucrose and arginine did not influence the pretransition dynamics of mAb-A. The potential reasons for such differences in excipient effects between two IgG1 mAbs are discussed.     

Effect of the ADCC-Modulating Mutations and the Selection of Human IgG Isotypes on Physicochemical Properties of Fc

Monoclonal antibodies, particularly IgGs and Ig-based molecules, are a well-established and growing class of biotherapeutic drugs. In order to improve efficacy, potency and pharmacokinetics of these therapeutic drugs, pharmaceutical industries have investigated significantly in engineering fragment crystallizable (Fc) domain of these drugs to optimize the interactions of these drugs and Fc gamma receptors (FcγRs) in recent ten years. The biological function of the therapeutics with the antibody-dependent cellular cytotoxicity (ADCC) enhanced double mutation (S239D/I332E) of isotype IgG1, the ADCC reduced double mutation (L234A/L235A) of isotype IgG1, and ADCC reduced isotype IgG4 has been well understood. However, limited information regarding the effect of these mutations or isotype difference on physicochemical properties (PCP), developability, and manufacturability of therapeutics bearing these different Fc regions is available. In this report, we systematically characterize the effects of the mutations and IgG4 isotype on conformation stability, colloidal stability, solubility, and storage stability at accelerated conditions in two buffer systems using six Fc variants. Our results provide a basis for selecting appropriate Fc region during development of IgG or Ig-based therapeutics and predicting effect of the mutations on CMC development process.