Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation-exchange HPLC

A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab–Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs.     

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 V_H domain, the constant domain (C_H2), and the hinge region (C_H1-C_H2 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.     

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 V_H domain, the constant domain (C_H2), and the hinge region (C_H1-C_H2 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.     

The interplay of non-specific binding,target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies

The application of protein engineering technologies toward successfully improving antibody pharmacokinetics has been challenging due to the multiplicity of biochemical factors that influence monoclonal antibody (mAb) disposition in vivo. Physiological factors including interactions with the neonatal Fc receptor (FcRn) and specific antigen binding properties of mAbs, along with biophysical properties of the mAbs themselves play a critical role. It has become evident that applying an integrated approach to understand the relative contribution of these factors is critical to rationally guide and apply engineering strategies to optimize mAb pharmacokinetics. The study presented here evaluated the influence of unintended non-specific interactions on the disposition of mAbs whose clearance rates are governed predominantly by either non-specific (FcRn) or target-mediated processes. The pharmacokinetics of 8 mAbs representing a diverse range of these properties was evaluated in cynomolgus monkeys. Results revealed complementarity-determining region (CDR) charge patch engineering to decrease charge-related non-specific binding can have a significant impact on improving the clearance. In contrast, the influence of enhanced in vitro FcRn binding was mixed, and related to both the strength of charge interaction and the general mechanism predominant in governing the clearance of the particular mAb. Overall, improved pharmacokinetics through enhanced FcRn interactions were apparent for a CDR charge-patch normalized mAb which was affected by non-specific clearance. The findings in this report are an important demonstration that mAb pharmacokinetics requires optimization on a case-by-case basis to improve the design of molecules with increased therapeutic application.     

The interplay of non-specific binding,target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies

The application of protein engineering technologies toward successfully improving antibody pharmacokinetics has been challenging due to the multiplicity of biochemical factors that influence monoclonal antibody (mAb) disposition in vivo. Physiological factors including interactions with the neonatal Fc receptor (FcRn) and specific antigen binding properties of mAbs, along with biophysical properties of the mAbs themselves play a critical role. It has become evident that applying an integrated approach to understand the relative contribution of these factors is critical to rationally guide and apply engineering strategies to optimize mAb pharmacokinetics. The study presented here evaluated the influence of unintended non-specific interactions on the disposition of mAbs whose clearance rates are governed predominantly by either non-specific (FcRn) or target-mediated processes. The pharmacokinetics of 8 mAbs representing a diverse range of these properties was evaluated in cynomolgus monkeys. Results revealed complementarity-determining region (CDR) charge patch engineering to decrease charge-related non-specific binding can have a significant impact on improving the clearance. In contrast, the influence of enhanced in vitro FcRn binding was mixed, and related to both the strength of charge interaction and the general mechanism predominant in governing the clearance of the particular mAb. Overall, improved pharmacokinetics through enhanced FcRn interactions were apparent for a CDR charge-patch normalized mAb which was affected by non-specific clearance. The findings in this report are an important demonstration that mAb pharmacokinetics requires optimization on a case-by-case basis to improve the design of molecules with increased therapeutic application.     

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational,structure-guided Fv engineering

Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.     

Investigation of cathepsin D–mAb interactions using a combined experimental and computational tool set

Cathepsin D has been identified as a challenge to remove in downstream bioprocessing of monoclonal antibodies (mAbs) due to interactions with some mAbs. This study focused on investigating the mechanisms of interaction between cathepsin D and two industrial mAbs using a combined experimental and computational approach. Surface plasmon resonance was used to study the impact of pH and salt concentration on these protein–protein interactions. While salt had a moderate effect on the interactions with one of the mAbs, the other mAb demonstrated highly salt-dependent association behavior. Cathepsin D binding to the mAbs was also seen to be highly pH dependent, with operation at pH 9 resulting in a significant decrease in the binding affinity. Protein–protein docking simulations identified three interaction sites on both mAbs; near the complementarity determining region (CDR), in the hinge, and in the C_H3 domain. In contrast, only one face of cathepsin D was identified to interact with all the three sites on the mAbs. Surface property analysis revealed that the binding regions on the mAbs contained strong hydrophobic clusters and were predominantly negatively charged. In contrast, the binding site on cathepsin D was determined to be highly positively charged and hydrophobic, indicating that these protein–protein interactions were likely due to a combination of hydrophobic and electrostatic interactions. Finally, covalent crosslinking coupled with mass spectrometry was used to validate the docking predictions and to further investigate the regions of interaction involved in mAb–cathepsin D binding. A strong agreement was observed between the two approaches, and the CDR loops were identified to be important for cathepsin D interactions. This study establishes a combined experimental and computational platform that can be used to probe mAb–host cell protein (HCP) interactions of importance in biomanufacturing.     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography

In this article, a systematic workflow was formulated and implemented to understand selectivity differences and preferred binding patches for bispecific monoclonal antibodies (mAbs) and their parental mAbs on three multimodal cation exchange resin systems. This workflow incorporates chromatographic screening of the parent mAbs and their fragments at various pH followed by surface property mapping and protein footprinting using covalent labeling followed by liquid chromatography–mass spectrometry analysis. The chromatography screens on multimodal resins with the intact mAbs indicated enhanced selectivity as compared to single-mode interaction systems. While the bispecific antibody (bsAb) eluted between the two parental mAbs on most of the resins, the retention of the bispecific transitioned from co-eluting with one parental mAb to the other parental mAb on Capto MMC. To investigate the contribution of different domains, mAb fragments were evaluated and the results indicated that the interactions were likely dominated by the Fab domain at higher pH. Protein surface property maps were then employed to hypothesize the potential preferred binding patches in the solvent-exposed regions of the parental Fabs. Finally, protein footprinting was carried out with the parental mAbs and the bsAb in the bound and unbound states at pH 7.5 to identify the preferred binding patches. Results with the intact mAb analysis supported the hypothesis that interactions with the resins were primarily driven by the residues in the Fab fragments and not the Fc. Furthermore, peptide mapping data indicated that the light chain may be playing a more important role in the higher binding of Parent A as compared with Parent B in these resin systems. Finally, results with the bsAb indicated that both halves of the molecule contributed to binding with the resins, albeit with subtle differences as compared to the parental mAbs. The workflow presented in this paper lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules which can provide insights into improved biomanufacturability and expedited downstream bioprocess development.     

The use of native cation-exchange chromatography to study aggregation and phase separation of monoclonal antibodies

This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation‐exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size‐exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co‐incubating Fab and Fc fragments with their respective full‐length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX‐based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein–protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb‐based therapeutics.     

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.     

Rotational dynamics of type I Fc epsilon receptors on individually-selected rat mast cells studied by polarized fluorescence depletion.

We report the first application of polarized fluorescence depletion (PFD), a technique which combines the sensitivity of fluorescence detection with the long lifetimes of triplet probes, to the measurement of membrane protein rotational diffusion on individually selected, intact mammalian cells. We have examined the rotation of type I Fc epsilon receptors (Fc epsilon RI) on rat mucosal mast cells of the RBL-2H3 line in their resting monomeric and differently oligomerized states using as probes IgE and three monoclonal antibodies (mAbs; H10, J17, and F4) specific for the Fc epsilon RI. PFD experiments using eosin (EITC)-IgE show that individual Fc epsilon RI on cells have a rotational correlation time (RCT) at 4 degrees C of 79 +/- 4 microseconds. Similarly, Fc epsilon RI-bound EITC-Fab fragments of the J17 Fc epsilon RI-specific mAb exhibit an RCT of 76 +/- 6 microseconds. These values agree with previous measurements of Fc epsilon RI-bound IgE rotation by time-resolved phosphorescence anisotropy methods. Receptor-bound EITC-conjugated divalent J17 antibody exhibits an increased RCT of 140 +/- 6 microseconds. This is consistent with the ability of this mAb to form substantial amounts of Fc epsilon RI dimers on these cell surfaces. The ratio of limiting to initial anisotropy in these experiments remains constant at about 0.5 from 5 degrees C through 25 degrees C for IgE, Fab, and intact mAb receptor ligands. Extensive cross-linking by second antibody of cell-bound IgE, of intact Fc epsilon RI-specific mAbs or of their Fab fragments, however, produced large fixed anisotropies demonstrating, under these conditions, receptor immobilization in large aggregates. PFD using the mAbs H10 and F4 as receptor probes yielded values for triplet lifetimes, RCT values, and anisotropy parameters essentially indistinguishable from those obtained with the mAb J17 clone. Possible explanations for these observations are discussed.     

Isolation and characterization of a monoclonal antibody containing an extra heavy-light chain Fab arm

Isolation and characterization of monoclonal antibody (mAb) variants to understand the impact of their structure on function is a typical activity during early-stage candidate selection that contributes to derisking clinical development. In particular, efforts are devoted to characterizing oligomeric variants, owing to their potential immunogenic nature. We report here a mAb variant consisting of a canonical mAb monomer associated in a non-covalent fashion with an antigen-binding fragment (Fab) arm amputated from its Fc domain. The truncated heavy chain is encoded in the cell line genome and is the likely product of a genomic recombination during cell line generation. The addition of the Fab arm results in severe loss of potency, indicating its interaction with the Fab domain of the monomer. The presence of such a variant can easily be mitigated by an adequate purification step.     

A recombinant Fab neutralizes dengue virus in vitro.

A recombinant Fab that recognizes a neutralizing epitope located in the (296-400) region of protein E of dengue virus was obtained from cloned hybridoma cells secreting the mouse monoclonal antibody (mAb) 4E11. The Fd and light chain antibody genes were amplified by polymerase chain reaction, cloned into the phagemid vector pMad, expressed in bacteria to produce Fab fragments and sequenced. The mAb 4E11, in particular its light chain complementary-determining regions, shared homologies with two other anti-viral mAbs. The affinity of the parental mAb and the cloned Fab to the MalE-E(296-400) fusion protein were shown to be of the same magnitude, i.e. nanomolar. Fab 4E11 neutralization capacity was found between 8 and 4-times or less lower than that of mAb 4E11, depending on serotypes, thus the Fab could have a smaller antiviral activity than the mAb in vitro.     

Molecular structural and functional characterization of tumor suppressive anti-ErbB-2 monoclonal antibody by phage display system

To investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with (87)AHNQVRQVPLQR(98) in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2.     

An inhibitory monoclonal antibody binds at the turn of the helix-turn-helix motif in the N-terminal domain of HIV-1 integrase

With the increase in our understanding of its structure and enzymatic mechanism, HIV-1 integrase (IN) has become a promising target for designing drugs to treat patients with AIDS. To investigate the structure and function of IN, a panel of monoclonal antibodies (mAbs) directed against HIV-1 IN was raised and characterized previously in this laboratory. Among them, mAbs17, -4, and -33 were found to inhibit IN activity in vitro. In this study, we investigated the interaction of N-terminal-specific mAb17 and its isolated Fab fragment with full-length HIV-1 IN(1-288) and its isolated N-terminal, Zn(2+)-binding domain IN(1-49). Our results show that binding of Zn(2+) to IN(1-49) stabilizes the mAb17-IN complex and that dimer dissociation is not required for binding of the Fab. To identify the epitope recognized by mAb17, we developed a protein footprinting technique based on controlled proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Binding was mapped to a region within amino acids Asp(25)-Glu(35). This peptide corresponds to the end of a helix-turn-helix motif in the IN(1-55) NMR structure and contributes to the dimerization of the N-terminal domain. Antibody binding also appears to destabilize the N-terminal helix in this domain. A molecular model of the [IN(1-49)](2).(Fab)(1) complex shows Fab binding across the dimer protein and suggests a potential target for drug design. These data also suggest that mAb17 inhibits integrase activity by blocking critical protein-protein interactions and/or by distorting the orientation of the N-terminal alpha-helix. The relevance of our results to an understanding of IN function is discussed.     

Process optimization and protein engineering mitigated manufacturing challenges of a monoclonal antibody with liquid-liquid phase separation issue by disrupting inter-molecule electrostatic interactions

We report a case study in which liquid-liquid phase separation (LLPS) negatively impacted the downstream manufacturability of a therapeutic mAb. Process parameter optimization partially mitigated the LLPS, but limitations remained for large-scale manufacturing. Electrostatic interaction driven self-associations and the resulting formation of high-order complexes are established critical properties that led to LLPS. Through chain swapping substitutions with a well-behaved antibody and subsequent study of their solution behaviors, we found the self-association interactions between the light chains (L_Cs) of this mAb are responsible for the LLPS behavior. With the aid of in silico homology modeling and charged-patch analysis, seven charged residues in the L_C complementarity-determining regions (CDRs) were selected for mutagenesis, then evaluated for self-association and LLPS properties. Two charged residues in the light chain (K30 and D50) were identified as the most significant to the LLPS behaviors and to the antigen-binding affinity. Four adjacent charged residues in the light chain (E49, K52, R53, and R92) also contributed to self-association, and thus to LLPS. Molecular engineering substitution of these charged residues with a neutral or oppositely-charged residue disrupted the electrostatic interactions. A double-mutation in CDR2 and CDR3 resulted in a variant that retained antigen-binding affinity and eliminated LLPS. This study demonstrates the critical nature of surface charged resides on LLPS, and highlights the applied power of in silico protein design when applied to improving physiochemical characteristics of therapeutic antibodies. Our study indicates that in silico design and effective protein engineering may be useful in the development of mAbs that encounter similar LLPS issues.     

Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

ABSTRACT

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and FcγR-binding activities, CDC activity, and FcγR-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen–deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man α1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with FcγR and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man α1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs.

Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-β-N-acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; FcγR, Fcγ receptors; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen–deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance–solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.     

Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter

Weak protein-protein interactions are thought to modulate the viscoelastic properties of concentrated antibody solutions. Predicting the viscoelastic behavior of concentrated antibodies from their dilute solution behavior is of significant interest and remains a challenge. Here, we show that the diffusion interaction parameter (k(D)), a component of the osmotic second virial coefficient (B(2)) that is amenable to high-throughput measurement in dilute solutions, correlates well with the viscosity of concentrated monoclonal antibody (mAb) solutions. We measured the k(D) of 29 different mAbs (IgG(1) and IgG(4)) in four different solvent conditions (low and high ion normality) and found a linear dependence between k(D) and the exponential coefficient that describes the viscosity concentration profiles (|R| ≥ 0.9). Through experimentally measured effective charge measurements, under low ion normality where the electroviscous effect can dominate, we show that the mAb solution viscosity is poorly correlated with the mAb net charge (|R| ≤ 0.6). With this large data set, our results provide compelling evidence in support of weak intermolecular interactions, in contrast to the notion that the electroviscous effect is important in governing the viscoelastic behavior of concentrated mAb solutions. Our approach is particularly applicable as a screening tool for selecting mAbs with desirable viscosity properties early during lead candidate selection.     

Antibody variable region binding by Staphylococcal protein A: thermodynamic analysis and location of the Fv binding site on E-domain.

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (V(H)), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3-4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5+/-0.5 x 10(5) M(-1). A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0+/-0.1, Ka = 2.0+/-0.3 x 10(5) M(-1), and deltaH = -7.1+/-0.4 kcal mol(-1). Similar binding thermodynamics are obtained for titration of the isolated V(H) domain with E-domain indicating that the E-domain binding site on Fab resides within V(H). E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2+/-0.1, Ka > 1.0 x 10(7) M(-1), and deltaH = -24.6+/-0.6 kcal mol(-1). Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the V(H) domain implicated previously in protein A binding.