Y. John Wang

Trehalose: Current Use and Future Applications

Trehalose, a disaccharide of glucose, has been reported to accumulate in many organisms that can withstand extended periods of inanimation. Since this discovery, the properties of trehalose have been examined extensively to understand its role and abundance in nature. The unique features of this sugar became clearer with each new finding which demonstrated its ability to sustain and preserve a wide array of biological molecules. Trehalose has been used in a variety of research applications and is contained in several commercially available therapeutic products, including Herceptin®, Avastin®, Lucentis®, and Advate®. Currently, there is a growing interest in the use of trehalose in solid dosage formulations, most notably in quick-dissolving tablets. Furthermore, trehalose has found its use in several food and cosmetic products, and new applications capitalizing on its unique properties are being developed and implemented in everyday-use products. As trehalose is an approved ingredient in all major markets, there is no significant barrier to its use. Extensive work with trehalose has been conducted in the three major industries, however with little overlap. Further understanding of the role of trehalose in the various applications may lead to an increase in the number of trehalose-containing products.

Physicochemical Stability of the Antibody−Drug Conjugate Trastuzumab-DM1: Changes due to Modification and Conjugation Processes

In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1. DSC, SEC, CE-SDS, and LC-MS were used to study the stability of Tmab, T-MCC, and T-DM1 to thermal stress. The DSC thermograms show a decrease in melting temperature for the CH2 transition, in the order Tmab > T-MCC > T-DM1. As per SEC analysis, a significant increase in level of aggregation was detected in T-MCC (∼32%) and T-DM1 (∼5%) after 14 days at 40 °C. Tmab did not show significant aggregate formation. CE-SDS and LC-MS data demonstrate that the aggregation in the case of T-MCC is largely covalent and involves mechanisms other than formation of intermolecular disulfide cross-links. The aggregation observed for T-MCC was significantly inhibited upon addition of amino acids with nucleophilic side chains containing thiol (Cys) and hydroxyl moieties (Ser, Tyr). The covalent aggregation observed for T-MCC and the ability of nucleophilic amino acids, particularly Cys, to inhibit it indicate that the maleimide moiety in the MCC linker may react to form intermolecular covalent cross-links between T-MCC molecules, possibly through a Michael addition mechanism. In addition, DSC results demonstrate that the conjugation of the drug moiety DM1 to Tmab results in destabilization of the CH2 domain of the antibody.

Asparagine Deamidation Dependence on Buffer Type, pH, and Temperature

The deamidation of asparagine into aspartate and isoaspartate moieties is a major pathway for the chemical degradation of monoclonal antibodies (mAbs). It can affect the shelf life of a therapeutic antibody that is not formulated or stored appropriately. A new approach to detect deamidation using ion exchange chromatography was developed that separates papain-digested mAbs into Fc and Fab fragments. From this, deamidation rates of each fragment can be calculated. To generate kinetic parameters useful in setting shelf life, buffers prepared at room temperature and then placed at the appropriate stability temperatures. Solution pH was not adjusted to the same at different temperatures. Deamidation rate at 40°C was faster in acidic buffers than in basic buffers. However, this trend is reversed at 5°C, attributed to the change in hydroxide ion concentration influenced by buffer and temperature. The apparent activation energy was higher for rates generated in an acidic buffer than in a basic buffer. The rate-pH profile for mAb1 can be deconvoluted to Fc and Fab. The Fc deamidation showed a V-shaped profile: deamidation of PENNY peptide is responsible for the rate at high-pH, whereas deamidation of a new site, Asn323, may be responsible for the rate at low-pH. The profile for Fab is a straight line without curvature.

Compatibility, Physical Stability, and Characterization of an IgG4 Monoclonal Antibody After Dilution into Different Intravenous Administration Bags

The physical stability of an immunoglobulin G4 monoclonal antibody (mAb) upon dilution into intravenous (i.v.) bags containing 0.9% saline was examined. Soluble aggregates and subvisible particles were observed by size-exclusion high-performance liquid chromatography (SE-HPLC) and light obscuration when formulated with suboptimal levels of polysorbate 20. The formation of soluble aggregates and particulates was further characterized by a combination of SE-HPLC, nanoparticle tracking analysis (NTA), microflow-digital imaging (MFI), and turbidity measurements. With sufficient PS20 levels, particle formation was minimized, although quantification of submicron sized particles by NTA was not possible because of the interference from PS20. Intravenous bags composed of polyvinyl chloride caused more protein particle formation than polyolefin bags. Differences between bag types were affected by removing headspace and by transferring the saline solution into glass vials. Characterization studies with Fourier transform infrared microscopy and extrinsic fluorescence spectroscopy demonstrated that isolated particles contained native-like secondary structure with partially altered tertiary structure, compared with heat-denatured and nonstressed controls. Transmission electron microscopy and MFI analysis showed particles had an amorphous morphology of varying sizes. Particles contained some non-native disulfide bond crosslinks, potentially initiated by low levels of free thiol in the native mAb. The critical role of proper formulation design to stabilize proteins against physical instability during i.v. administration is discussed.

Effect of Individual Fc Methionine Oxidation on FcRn Binding: Met252 Oxidation Impairs FcRn Binding More Profoundly than Met428 Oxidation

The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies. However, the quantitative effect of individual Met oxidation on Fc-FcRn binding has not been addressed. This information is valuable for defining critical quality attributes. In the present study, two sets of homodimeric site-directed IgG1 mutations were generated to understand how individual Fc Met oxidation affects FcRn binding. The first approach used Met to Leu mutants to block site-specific Met oxidation. In the other approach, Met to Gln mutants were designed to mimic site-specific Met oxidation. Both mutagenesis approaches show that either Met252 or Met428 oxidation alone significantly impairs Fc-FcRn binding. Met252 oxidation has a more deleterious effect on FcRn binding than M428 oxidation, whereas Met428 oxidation has a bigger destabilization effect on the thermal stability. Our results also show that Met358 oxidation does not affect FcRn binding. In addition, our study suggests that Met to Gln mutation may serve as an important tool to understand Met oxidation.

Increasing IgG Concentration Modulates the Conformational Heterogeneity and Bonding Network that Influence Solution Properties

Multiple molecular driving forces mediate protein stability, association, and recognition in concentrated solutions. Here we investigate the interactions that modulate the nonideal solution behavior of two immunoglobulins (IgG1s) in highly concentrated solutions using two-dimensional vibrational correlation spectroscopy (2D-COS) and principal components analysis (PCA). A specific sequence of changes is observed in the concentration-dependent vibrational spectra of the highly viscous IgG solution that deviates from ideality, whereas that sequence is reversed for all other conditions examined. The asynchronous spectra reveal variation in beta-sheet and turn regions occur before intensity variations in disordered and alpha-helical regions as the concentration is increased for the highly viscous regime. This is in contrast to the sequence observed for all other conditions studied and to the idea that beta-sheet regions are resistant to concentration-dependent affects. Finally, we show that increased hydrogen bonding and electrostatics primarily modulate the intermolecular association and nonideal behavior. Specifically, 2D-COS and PCA analysis of the amide II region suggests that Glu and Asp residues trigger the change resulting in increased viscosity and association of one IgG.

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 molecules 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.

On developing a process for conducting extractable–leachable assessment of components used for storage of biopharmaceuticals

Extractables and leachables are product-related impurities that result from product contact with components such as gaskets, stoppers, storage bags, cartridges, and prefilled syringes that are used for processing, storage, and/or delivery of biopharmaceuticals. These impurities are a concern for patients due to potential effects on product quality and safety. It is possible that such an impurity could directly impact the patient or indirectly impact the patient by interacting with the protein therapeutics and forming protein adducts. Adducts and leachables may or may not be detected as product-related impurities in routine stability indicating assays depending on the rigor of the analytical program. The need for the development of a thorough and holistic extractable and leachable program based on risk assessment, review of existing literature, and consolidation of industry best practices is discussed. Standardizing component use within an organization enables streamlining of the extractable-leachable program. Our strategy for an extractable-leachable program is divided into different stages, each stage detailing the activities and the department within the organization that is responsible for execution of these activities. The roles and responsibilities of the key stakeholders are identified. The integration of analytical activities with health-based risk-assessment information into the design of an extractable-leachable program is highlighted.

Isomerization of Asp–Asp Motif in Model Peptides and a Monoclonal Antibody Fab Fragment

Isomerization of aspartyl (Asp or D) residues is a critical degradation route to consider for stable monoclonal antibody formulations. Among the known hotspot sequences, the DD motif is relatively understudied. To gain mechanistic insights, we used model hexapeptides, YADXFK, YADDXK, and DIDDDM, as surrogates for the hotspots in a Fab protein (YADDFK and DIDDDM), to characterize the rate-pH profile of Asp isomerization. Compared with the YADGFK peptide, isomerization of D3 (the first D in the DD pair) in YADDFK was highly pH dependent. Comparison of rate-pH profiles of YADDFK, YADNFK, and YADHFK revealed a charge effect of the n + 1 residue-isomerization rate is accelerated by the positive side chain and reduced by negative side chain at n + 1 residue. Studies on YADDFK, YADDAK, and YADDGK indicated a mutual impact of D3 and D4 on their respective isomerization rates through charge effect. Comparison of rate-pH profile of DIDDDM sequence in peptide models with that in the complementary determining region of the Fab showed a faster rate in the Fab than in peptides, presumably because of contribution from structural factors in the former.

The Relative Rate of Immunoglobulin Gamma 1 Fragmentation

The physicochemical stability of protein therapeutics is of significant pharmaceutical interest. Immunoglobulin gamma (IgG) hinge region fragmentation has recently garnered attention as an important degradation route of therapeutic monoclonal antibodies. In this work, the rates and relative amount of fragment species are compared for five different IgGs (IgG1-5) with widely varying solution properties. Native size-exclusion chromatography (SEC), sodium dodecyl sulfate (SDS)-based SEC, and capillary electrophoresis-SDS were used to characterize IgG1 fragmentation after storage at 30°C, 40°C, and 50°C. Two-dimensional correlation analysis of the chromatograms as a function of time was used to illustrate the relative rates of cleavage. Interestingly, the relative rate of Fab cleavage was greater than that of other species. An average apparent energy of activation for IgG1 fragmentation was also measured for all five molecules. This work suggests that IgG1 fragmentation is primarily hinge sequence dependent and other IgG1 molecules should behave similarly within the limits of the solution conditions used.