Selective Uptake of Macromolecules to the Brain in Microfluidics and Animal Models Using the HAVN1 Peptide as a Blood-Brain Barrier Modulator.

Monoclonal antibodies (mAbs) possess favorable pharmacokinetic properties, high binding specificity and affinity, and minimal off-target effects, making them promising therapeutic agents for central nervous system (CNS) disorders. However, their development as effective therapeutic and diagnostic agents for brain disorders is hindered by their limited ability to efficiently penetrate the blood-brain barrier (BBB). Therefore, it is crucial to develop efficient delivery methods that enhance the penetration of antibodies into the brain. Previous studies have demonstrated the potential of cadherin-derived peptides (i.e., ADTC5, HAVN1 peptides) as BBB modulators (BBBMs) to increase paracellular porosities for penetration of molecules across the BBB. Here, we test the effectiveness of the leading BBBM peptide, HAVN1 (Cyclo(1,6)SHAVSS), in enhancing the permeation of various monoclonal antibodies through the BBB using both in vitro and in vivo systems. In vitro, HAVN1 has been shown to increase the permeability of fluorescently labeled macromolecules, such as a 70 kDa dextran, 50 kDa Fab1, and 150 kDa mAb1, by 4- to 9-fold in a three-dimensional blood-brain barrier (3D-BBB) microfluidics model using a human BBB endothelial cell line (i.e., hCMEC/D3). HAVN1 was selective in modulating the BBB endothelial cell, compared to the pulmonary vascular endothelial (PVE) cell barrier. Co-administration of HAVN1 significantly improved brain depositions of mAb1, mAb2, and Fab1 in C57BL/6 mice after 15 min in the systemic circulation. Furthermore, HAVN1 still significantly enhanced brain deposition of mAb2 when it was administered 24 h after the administration of the mAb. Lastly, we observed that multiple doses of HAVN1 may have a cumulative effect on the brain deposition of mAb2 within a 24-h period. These findings offer promising insights into optimizing HAVN1 and mAb dosing regimens to control or modulate mAb brain deposition for achieving desired mAb dose in the brain to provide its therapeutic effects.

Exploring How Antibody Format Drives Clearance from the Brain.

Monoclonal antibodies (mAbs) have high binding specificity and affinity, making them attractive for treating brain diseases. However, their effectiveness is limited by poor blood-brain barrier (BBB) penetration and rapid central nervous system (CNS) clearance. Our group identified blood-brain barrier modulator (BBBM) peptides that improved mAb penetration across the BBB into the brain. In this study, we investigated the pharmacokinetics of a mAb delivered to the brain using BBBMs after intravenous (IV) administration and explored the impact of antibody format (size, neonatal Fc receptor (FcRn) binding, hyaluronic acid binding) on brain clearance following direct injection into the central nervous system (CNS) via intracerebroventricular (ICV) injection. IRDye800CW-labeled antibodies were administered into C57BL/6 mice via ICV or IV injection, and organ concentrations were measured after various time points. When a mAb was coadministered with a BBBM peptide, the permeation of mAb across the BBB was increased compared to mAb alone at early time points; however, the mAb was cleared within 2 h from the brain. ICV experiments revealed that an antibody Fab fragment had a higher brain exposure than a mAb, and that a Fab fused to a hyaluronic acid binding domain (Fab-VG1) showed remarkable improvement in brain exposure. These findings suggest that BBBMs and antibody format optimization may be promising strategies for enhancing brain retention of therapeutic antibodies.

Translatability of In Vitro Stress for Predicting Deamidation and Oxidation Biotransformation on Biotherapeutics.

Biotransformation leading to single residue modifications (e.g., deamidation, oxidation) can contribute to decreased efficacy/potency, poor pharmacokinetics, and/or toxicity/immunogenicity for protein therapeutics. Identifying and characterizing such liabilities in vivo are emerging needs for biologics drug discovery. In vitro stress assays involving PBS for deamidation or AAPH for oxidation are commonly used for predicting liabilities in manufacturing and storage and are sometimes considered a predictive tool for in vivo liabilities. However, reports discussing their in vivo translatability are limited. Herein, we introduce a mass spectrometry workflow that characterizes in vivo oxidation and deamidation in pharmacokinetically relevant compartments for diverse protein therapeutic modalities. The workflow has low bias of <10% in quantitating degradation in the relevant pharmacokinetic concentration range for monkey and rabbit serum/plasma (1-100 µg/mL) and allows for high sequence coverage (∼85%) for discovery/monitoring of amino acid modifications. For oxidation and deamidation, the assay was precise, with percent coefficient of variation of <8% at 1-100 µg/mL and ≤6% method-induced artifacts. A high degree of in vitro and in vivo correlation was observed for deamidation on the six diverse protein therapeutics (seven liability sites) tested. In vivo translatability for oxidation liabilities were not observed for the 11 molecules tested using in vitro AAPH stress. One of the molecules dosed in eyes resulted in a false positive and a false negative prediction for in vivo oxidation following AAPH stress. Finally, peroxide stress was also tested but resulted in limited success (1 out of 4 molecules) in predicting oxidation liabilities.

Metal Ion Interactions with mAbs: Part 2. Zinc-Mediated Aggregation of IgG1 Monoclonal Antibodies.

PURPOSE: To evaluate the physical and chemical degradation of monoclonal antibodies in the presence of Zn2+. METHODS: A full length IgG1 monoclonal antibody (mAb1) was formulated with various amounts of Zn2+. The resulting mixture was incubated for several weeks at room temperature and analyzed using a variety of biochemical techniques to look for various physical (e.g. aggregation) and chemical (e.g. fragmentation) degradation pathways. RESULTS: mAb1 of the IgG1 subclass undergoes aggregation in the presence of Zn2+ in a concentration dependent manner. Up to hexamers were characterized using SEC-MALS. No fragmentation was noticed in the presence of Zn2+ as opposed to that found in our previous report when IgG1 mAbs were incubated in the presence of Cu2+ ions. Site directed mutagenesis indicated the involvement of Fc histidine (His 310) in Zn2+ mediated aggregation. CONCLUSIONS: A novel metal ion mediated isodesmic aggregation mechanism was found in IgG1 class of monoclonal antibodies. Histidine residues in the Fc region were determined to be the binding site and implicated in Zn2+ mediated aggregation.

A Comprehensive Assessment of All-Oleate Polysorbate 80: Free Fatty Acid Particle Formation, Interfacial Protection and Oxidative Degradation.

PURPOSE: Enzymatic polysorbate (PS) degradation and resulting free fatty acid (FFA) particles are detrimental to biopharmaceutical drug product (DP) stability. Different types and grades of polysorbate have varying propensity to form FFA particles. This work evaluates the homogenous all-oleate (AO) PS80 alongside heterogeneous PS20 and PS80 grades in terms its propensity to form FFA particles and other important attributes like interfacial protection and oxidation susceptibility. METHODS: FFA particle formation rates were compared by degrading PS using non-immobilized hydrolases and fast degrading DP formulations. Interfacial protection of monoclonal antibodies (mAbs) was assessed by agitation studies in saline using non-degraded and degraded PS. Several antioxidants were assessed for their ability to mitigate AO PS80 oxidation and subsequent mAb oxidation by a 40°C placebo stability study and a 2, 2'-Azobis (2-amidinopropane) dihydrochloride stress model, respectively. RESULTS: Visible and subvisible particles were significantly delayed in AO PS80 formulations compared with heterogeneous PS20 and PS80 formulations. Non-degraded AO PS80 was less protective of mAbs against the air-water interface compared with heterogeneous PS20. Interfacial protection by AO PS80 improved upon degradation owing to high surface activity of FFAs. Diethylenetriaminepentaacetic acid (DTPA) completely mitigated AO PS80 oxidation unlike L-methionine and N-Acetyl-DL-Tryptophan. However, DTPA did not mitigate radical mediated mAb oxidation. CONCLUSION: AO PS80 is a promising alternative to reduce FFA particle formation compared with other PS types and grades. However, limitations observed here---such as lower protection against interfacial stresses and higher propensity for oxidation---need to be considered in assessing the risk/benefit ratio in using AO PS80.

Protein Stability and Photostability under In Vitro Vitreal Conditions - Implications for Long Acting Delivery of Protein Therapeutics for Ocular Disease.

PURPOSE: To evaluate the stability of a model Mab1 and Fab1 under in vitro vitreal conditions in the presence of various metabolites and in the presence of light at λ > 400 nM. METHODS: A full length IgG1 monoclonal antibody (Mab1) and a fab fragment (Fab1) were formulated with various metabolites typically found in the vitreous humor and subjected to visible light treatment. Both proteins were analyzed using a variety of biochemical techniques. Furthermore, Fab1 was also tested for antigen binding ability using surface plasmon resonance. RESULTS: Our data shows that some vitreal metabolites such as riboflavin and ascorbic acid affect protein stability, via formation of reactive oxygen species (ROS) and advanced glycation end products (AGE) s respectively. In contrast, metabolites such as glutathione may protect these proteins from light-induced degradation to some extent. CONCLUSIONS: Ascorbic acid and riboflavin were found to photosensitize therapeutic proteins especially when exposed to light. Ascorbic acid reacted with proteins even in the absence of light. Antioxidants such as glutathione helped limit photooxidation under ambient or blue light exposure. Since antioxidant capacity in the eye decreases with age we recommend understanding long term stability, including photooxidation and photosensitization, of new candidate proteins in the context of controlled or sustained release technologies for ocular diseases.

Trehalose Limits Fragment Antibody Aggregation and Influences Charge Variant Formation in Spray-Dried Formulations at Elevated Temperatures.

The preparation of PLGA rods for sustained release applications via a hot-melt extrusion process employs heat and mechanical shear. Understanding protein stability and degradation mechanisms at high temperature in the solid state is therefore important for the preparation of protein-loaded PLGA rods. The stability of a model protein, labeled Fab2, has been investigated in solid-state formulations containing trehalose at elevated temperatures. Spray-dried formulations containing varying levels of trehalose were exposed to temperatures ranging from 90 to 120 °C. Measurement of aggregation and chemical degradation rates suggests that trehalose limits Fab2 degradation in a concentration-dependent manner, but the effect tends to saturate when the mass ratio of trehalose to protein is around 1 in the solid formulation. The Fab2 secondary structure and spray-dried particle morphology were studied using circular dichroism and scanning electron microscopy techniques, respectively. On the basis of temperature and trehalose-dependent aggregation kinetics as well as changes in spray-dried particle morphology, a mechanism is proposed for the trehalose stabilization of proteins in solid state at elevated temperatures. The results reported here suggest that when fragment antibodies in the solid state are formulated with trehalose as excipient, a high temperature process such as hot-melt extrusion can be successfully accomplished with minimal degradation.

Photodisruption of the Structurally Conserved Cys-Cys-Trp Triads Leads to Reduction-Resistant Scrambled Intrachain Disulfides in an IgG1 Monoclonal Antibody.

Photostability conditions as prescribed by ICH guidelines induced highly reduction-resistant scrambled disulfides that contribute to the population of apparent nonreducible aggregates in an IgG1 mAb. Photoinduced cross-linked species were isolated under reducing conditions using an organic phase size exclusion chromatography (OP-SEC) method, followed by O18-labeling tryptic mapping to identify cross-linked peptides. Disulfide scrambling was observed within the IgG1 structurally conserved-intrachain cysteine-cysteine-tryptophan triads (Cys-Cys-Trp), and correlated with Trp-to-kynurenine (Kyn) photodegradation within these triads. We hypothesize that intrachain disulfides protect the proximal Trp within the Cys-Cys-Trp triads from photodegradation by enabling dissipation of Trp-absorbed UV energy via electron transfer to the disulfide bond. Finally, we propose three distinct mechanisms of photochemical degradation of monoclonal antibodies mediated by Trp residues.

Fragmentation of a Monoclonal Antibody by Peroxotungstate.

PURPOSE: Tungsten and tungsten oxide leachates found in glass pre-filled syringes were identified to initiate protein precipitation and aggregation. Here, we tested the possibility of tungsten and tungsten oxide to induce the chemical degradation of proteins via reaction with hydrogen peroxide, a possible impurity present in protein formulations, to yield peroxotungstate. METHODS: A monoclonal antibody (mAb) was incubated with various concentrations of peroxotungstate and the reaction mixtures analyzed by SDS-PAGE and mass spectrometry. RESULTS: Exposure of a mAb to 1.07-1070 ppm peroxotungstate (based on tungsten content) at temperatures of 4°C and 22°C (pH 5-7) induced protein fragmentation. The extent of fragmentation increased with higher temperatures, lower pH and higher peroxotungstate concentrations. The mAb fragments were identified to contain different combinations of heavy chains (H) and light chains (L). Analogous mAb fragments were generated when the protein was exposed to H2O2 and orthotungstate at levels as low as 5 ppm. In addition, extracts from tungsten pins used to manufacture glass pre-filled syringes, in combination with H2O2 caused comparable fragmentation of the mAb. Mass spectrometric identification of the fragments suggests fragment generation by oxidative disulfide bond cleavage between the heavy and light chains, confirmed by mass spectrometry data on product formation. The mechanism of oxidative fragmentation was separately confirmed with insulin. CONCLUSION: Fragmentation of the mAb by peroxotungstate is proposed to occur through inter-chain disulfide bond oxidation to form thiosulfinate (CyS(═O)SCy) and thiosulfonate [CyS(═O)2SCy], followed by hydrolysis.

Identification of D-Amino Acids in Light Exposed mAb Formulations.

PURPOSE: We previously demonstrated that D-amino acids can form as a result of photo-irradiation of a monoclonal antibody (mAb) at both λ = 254 nm and λ > 295 nm (λmax = 305 nm), likely via reversible hydrogen transfer reactions of intermediary thiyl radicals. Here, we investigate the role of various excipients (sucrose, glucose, L-Arg, L-Met and L-Leu) on D-amino acid formation, and specifically the distribution of D-amino acids in mAb monomers and aggregates present after light exposure. METHODS: The mAb-containing formulations were photo-irradiated at λ = 254 nm and λmax = 305 nm, followed by fractionation of aggregate and monomer fractions using size exclusion chromatography. These aggregate and monomer fractions were subjected to hydrolysis and subsequent amino acid analysis. RESULTS: Both aggregate and monomer fractions collected from all formulations showed the formation of D-Glu and D-Val, whereas the formation of D-Ala was limited to the aggregate fraction collected from an L-Arg-containing formulation. Interestingly, quantitative analysis revealed higher yields of D-amino acids in the L-Arg-containing formulation. CONCLUSIONS: Generally, D-amino acids accumulated to similar extents in monomers and aggregates.

Photo-oxidation of IgG1 and Model Peptides: Detection and Analysis of Triply Oxidized His and Trp Side Chain Cleavage Products.

PURPOSE: Triply oxidized histidine in an IgG1 monoclonal antibody was noticed when exposed to ICH light conditions. In order to understand the role of light source, irradiation wavelengths and primary sequence, specifically those of a nearby tryptophan, we synthesized and exposed several peptides to ICH light conditions and analyzed the products using LC-MS analysis. METHODS: Protein and peptide samples were photo-irradiated under ICH conditions as well as with monochromatic light at λ = 254 nm and analyzed using either LTQ Orbitrap or a LTQ-FT ion cyclotron resonance mass spectrometer respectively. RESULTS: A triply oxidized His residue was detected along with a second doubly oxidized His residue in an IgG1. Both of these oxidized His residues are located near Trp residues. In order to investigate the role of Trp photosensitization in His oxidation we synthesized model peptides and Ala mutants. Peptides exposed to ICH light stress conditions revealed a small percent of triply oxidized His in the Trp-containing peptide sequences but not in their corresponding Ala mutants. CONCLUSIONS: The differences in product formation under different photo-irradiation conditions underline the importance of light source, irradiation wavelengths and primary sequence in the photosensitivity of proteins.

Mechanistic Insights into Radical-Mediated Oxidation of Tryptophan from ab Initio Quantum Chemistry Calculations and QM/MM Molecular Dynamics Simulations.

An assessment of the mechanisms of (•)OH and (•)OOH radical-mediated oxidation of tryptophan was performed using density functional theory calculations and ab initio plane-wave Quantum Mechanics/Molecular Mechanics (QM/MM) molecular dynamics simulations. For the (•)OH reactions, addition to the pyrrole ring at position 2 is the most favored site with a barrierless reaction in the gas phase. The subsequent degradation of this adduct through a H atom transfer to water was intermittently observed in aqueous-phase molecular dynamics simulations. For the (•)OOH reactions, addition to the pyrrole ring at position 2 is the most favored pathway, in contrast to the situation in the model system ethylene, where concerted addition to the double bond is preferred. From the (•)OOH position 2 adduct QM/MM simulations show that formation of oxy-3-indolanaline occurs readily in an aqueous environment. The observed transformation starts from an initial rupture of the O-O bond followed by a H atom transfer with the accompanying loss of an (•)OH radical to solution. Finally, classical molecular dynamics simulations were performed to equate observed differential oxidation rates of various tryptophan residues in monoclonal antibody fragments. It was found that simple parameters derived from simulation correlate well with the experimental data.

Screening methods to identify indole derivatives that protect against reactive oxygen species induced tryptophan oxidation in proteins.

Oxidative damage to proteins is one of the most prominent chemical degradation pathways that are of concern for drug product development in the biotechnology industry. Especially susceptible to oxidation are the Met and Trp residues in proteins. While L-Met and L-Trp have been shown to act as antioxidants typically protecting proteins against Met and Trp oxidation, respectively, l-Trp has been shown to be particularly sensitive to light, thereby producing various reactive oxygen species (ROS), including H2O2. There is hence a need to identify nonphotosensitive molecules that can protect Trp oxidation in proteins so that they can be easily handled under drug product manufacturing conditions. A combination of screening methods, namely, cyclic voltammetry (CV) and hydrogen peroxide generation upon photoirradiation, was used to screen several molecules to identify compounds that can act as antioxidants. Specifically, indole and tryptophan with hydroxy groups on the six-membered aromatic ring were found to have lower oxidation potentials than the parent compounds and produced the least amount of H2O2 upon light exposure. These derivatives were also found to sufficiently protect tryptophan oxidation in mAb1 against a variety of reactive oxygen species such as alkyl peroxides, hydroxyl radicals, and singlet oxygen and may be useful as part of the formulation toolkit to protect against protein degradation via oxidation.

Reactions of benzene and 3-methylpyrrole with the •OH and •OOH radicals: an assessment of contemporary density functional theory methods.

A high-level quantum chemistry investigation has been carried out for the addition and abstraction reactions by the radicals (•)OH and (•)OOH to and from the model alkenes 3-methylpyrrole and benzene. These models were chosen to reflect the functionalities contained in the side chain of the amino acid tryptophan. The W1BD procedure was used to calculate benchmark barriers and reaction energies for the smaller model system of (•)OOH addition to ethylene. It was found that the CBS-QB3 methodology compares best with the W1BD benchmark, demonstrating a mean absolute deviation (MAD) from W1BD of 3.9 kJ mol(-1). For the reactions involving the (•)OH radical and benzene or 3-methylpyrrole, addition is favored over abstraction in all cases. In particular the CBS-QB3 calculations suggest a barrierless addition reaction of the (•)OH radical to position two of 3-methylpyrrole. For the analogous addition and abstraction reactions involving the (•)OOH radical, the same order of reactivity was found, albeit with higher barriers. A number of other processes involving the addition of the (•)OOH radical were also investigated. The main findings of these studies determined that the initial (•)OOH barrier of stepwise addition to 3-methylpyrrole (+18.8 kJ mol(-1)) is significantly smaller than the concerted addition barrier (+71.5 kJ mol(-1)). This conclusion contrasts starkly with the situation for ethylene in which it is well established that the concerted process has the smaller barrier. A considerable variety of contemporary density functional theory procedures have been tested to examine their accuracy in predicting the CBS-QB3 results. It was found that the best overall performing method was UBMK with an MAD of 7.3 kJ mol(-1). A number of other functionals additionally performed well. They included UM06, RM06, UXYG3 and RXYG3, all of which have MADs of less than 8 kJ mol(-1).

Role of surface exposed tryptophan as substrate generators for the antibody catalyzed water oxidation pathway.

The reaction of singlet oxygen with water to form hydrogen peroxide was catalyzed by antibodies and has been termed as the antibody catalyzed water oxidation pathway (ACWOP) (Nieva and Wentworth, Trends Biochem. Sci. 2004, 29, 274-278; Nieva et al. Immunol. Lett. 2006, 103, 33-38). While conserved and buried tryptophans in the antibody are thought to play a major role in this pathway, our studies with a monoclonal antibody, mAb-1 and its mutant W53A, clearly demonstrate the role of surface-exposed tryptophans in production of hydrogen peroxide, via the photo-oxidation pathway. Reactive oxygen species (ROS) such as singlet oxygen and superoxide were detected and site-specific tryptophan (Trp53) oxidation was observed under these conditions using RP-HPLC and mass spectrometry. The single mutant of the surface exposed Trp53 to Ala53 (W53A) results in a 50% reduction in hydrogen peroxide generated under these conditions, indicating that surface exposed tryptophans are highly efficient in transferring light energy to oxygen and contribute significantly to ROS generation. ACWOP potentially leads to the chemical instability of mAb-1 via the generation of ROS and is important to consider during clinical and pharmaceutical development of mAbs.

Characterization of the isomerization products of aspartate residues at two different sites in a monoclonal antibody.

PURPOSE: To identify and understand isomerization products and degradation profile of different aspartate residues in an IgG1 monoclonal antibody. METHODS: Recombinant IgG1 was incubated for extended periods of time in a formulation buffer at recommended and accelerated storage temperatures. Isomerization reaction products were analyzed using ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), peptide mapping, and LC-MS. Model peptides with sequences containing specific aspartate residues in IgG1 were synthesized and incubated under accelerated conditions. Products of isomerization reactions of peptides were analyzed by reverse phase chromatography (RP-HPLC) and LC-MS. X-ray crystallography data from Fab of IgG1 were used to understand mechanism of isomerization reactions. RESULTS: A MAb containing labile Asp32-Gly sequence in CDR I region undergoes rapid isomerization reaction and leads to formation of isoaspartate (IsoAsp) and cyclic imide (Asu) forms. Isomerization of aspartate residues was observed in a non-CDR region containing Asp74-Ser sequence. Isomerization reaction at Asp74-Ser led to formation of Asu74 and trace isoAsp74. While isoAsp32 increased linearly with time, isoAsp74 did not increase during storage. Asu32 and Asu74 followed non-linear degradation kinetics and reached steady state over time. Isomerization reaction of two different model peptides containing Asp32-Gly or Asp74-Ser with neighboring amino acid sequences as those found in the MAb result in formation of IsoAsp. CONCLUSIONS: Observed levels of Asu and trace IsoAsp at the Asp74 site are unusual for typical isomerization reactions. In addition to primary sequences, pKa, solvent exposure and high order structure around aspartate residues may have influenced isomerization reaction at Asp74 in MAbI. Different degradation profiles from the two Asp residues can influence shelf life and should be carefully evaluated during product development.

Physicochemical and biological impact of metal-catalyzed oxidation of IgG1 monoclonal antibodies and antibody-drug conjugates via reactive oxygen species.

Biotherapeutics are exposed to common transition metal ions such as Cu(II) and Fe(II) during manufacturing processes and storage. IgG1 biotherapeutics are vulnerable to reactive oxygen species (ROS) generated via the metal-catalyzed oxidation reactions. Exposure to these metal ions can lead to potential changes to structure and function, ultimately influencing efficacy, potency, and potential immunogenicity of the molecules. Here, we stress four biotherapeutics of the IgG1 subclass (trastuzumab, trastuzumab emtansine, anti-NaPi2b, and anti-NaPi2b-vc-MMAE) with two common pharmaceutically relevant metal-induced oxidizing systems, Cu(II)/ ascorbic acid and Fe(II)/ H2O2, and evaluated oxidation, size distribution, carbonylation, Fc effector functions, antibody-dependent cellular cytotoxicity (ADCC) activity, cell anti-proliferation and autophaghic flux. Our study demonstrates that the extent of oxidation was metal ion-dependent and site-specific, leading to decreased FcγRIIIa and FcRn receptor binding and subsequently potentially reduced bioactivity, though antigen binding was not affected to a great extent. In general, the monoclonal antibody (mAb) and corresponding antibody-drug conjugate (ADC) showed similar impacts to product quality when exposed to the same metal ion, either Cu(II) or Fe(II). Our study clearly demonstrates that transition metal ion binding to therapeutic IgG1 mAbs and ADCs is not random and that oxidation products show unique structural and functional ramifications. A critical outcome from this study is our highlighting of key process parameters, route of degradation, especially oxidation (metal catalyzed or via ROS), on the CH1 and Fc region of full-length mAbs and ADCs.Abbreviations: DNPH 2,4-dinitrophenylhydrazine; ADC Antibody drug conjugate; ADCC Antibody-dependent cellular cytotoxicity; CDR Complementary determining region; DTT Dithiothreitol; HMWF high molecular weight form; LC-MS Liquid chromatography-mass spectrometry; LMWF low molecular weight forms; MOA Mechanism of action; MCO Metal-catalyzed oxidation; MetO Methionine sulfoxide; mAbs Monoclonal antibodies; MyBPC Myosin binding protein C; ROS Reactive oxygen species; SEC Size exclusion chromatography.

Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality.

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.

End-to-End Approach to Surfactant Selection, Risk Mitigation, and Control Strategies for Protein-Based Therapeutics.

A survey performed by the AAPS Drug Product Handling community revealed a general, mostly consensus, approach to the strategy for the selection of surfactant type and level for biopharmaceutical products. Discussing and building on the survey results, this article describes the common approach for surfactant selection and control strategy for protein-based therapeutics and focuses on key studies, common issues, mitigations, and rationale. Where relevant, each section is prefaced by survey responses from the 22 anonymized respondents. The article format consists of an overview of surfactant stabilization, followed by a strategy for the selection of surfactant level, and then discussions regarding risk identification, mitigation, and control strategy. Since surfactants that are commonly used in biologic formulations are known to undergo various forms of degradation, an effective control strategy for the chosen surfactant focuses on understanding and controlling the design space of the surfactant material attributes to ensure that the desired material quality is used consistently in DS/DP manufacturing. The material attributes of a surfactant added in the final DP formulation can influence DP performance (e.g., protein stability). Mitigation strategies are described that encompass risks from host cell proteins (HCP), DS/DP manufacturing processes, long-term storage, as well as during in-use conditions.

Insights into ultra-low affinity lipase-antibody noncovalent complex binding mechanisms.

Detection of host cell protein (HCP) impurities is critical to ensuring that recombinant drug products, including monoclonal antibodies (mAbs), are safe. Mechanistic characterization as to how HCPs persist in drug products is important to refining downstream processing. It has been hypothesized that weak lipase-mAb interactions enable HCP lipases to evade drug purification processes. Here, we apply state-of-the-art methods to establish lipase-mAb binding mechanisms. First, the mass spectrometry (MS) approach of fast photochemical oxidation of proteins was used to elucidate putative binding regions. The CH1 domain was identified as a conserved interaction site for IgG1 and IgG4 mAbs against the HCPs phospholipase B-like protein (PLBL2) and lysosomal phospholipase A2 (LPLA2). Rationally designed mutations in the CH1 domain of the IgG4 mAb caused a 3- to 70-fold KD reduction against PLBL2 by surface plasmon resonance (SPR). LPLA2-IgG4 mutant complexes, undetected by SPR and studied using native MS collisional dissociation experiments, also showed significant complex disruption, from 16% to 100%. Native MS and ion mobility (IM) determined complex stoichiometries for four lipase-IgG4 complexes and directly interrogated the enrichment of specific lipase glycoforms. Confirmed with time-course and exoglycosidase experiments, deglycosylated lipases prevented binding, and low-molecular-weight glycoforms promoted binding, to mAbs. This work demonstrates the value of integrated biophysical approaches to characterize micromolar affinity complexes. It is the first in-depth structural report of lipase-mAb binding, finding roles for the CH1 domain and lipase glycosylation in mediating binding. The structural insights gained offer new approaches for the bioengineering of cells or mAbs to reduce HCP impurity levels.Abbreviations: CAN, Acetonitrile; AMAC, Ammonium acetate; BFGS, Broyden-Fletcher-Goldfarb-Shanno; CHO, Chinese Hamster Ovary; KD, Dissociation constant; DTT, Dithiothreitol; ELISA, Enzyme-linked immunosorbent assay; FPOP, Fast photochemical oxidation of proteins; FA, Formic acid; F(ab'), Fragment antibodies; HCP, Host cell protein; IgG, Immunoglobulin; IM, Ion mobility; LOD, Lower limit of detection; LPLA2, Lysosomal phospholipase A2; Man, Mannose; MS, Mass spectrometry; MeOH, Methanol; MST, Microscale thermophoresis; mAbs, Monoclonal antibodies; PPT1, Palmitoyl protein thioesterase; ppm, Parts per million; PLBL2, Phospholipase B-like protein; PLD3, Phospholipase D3; PS-20, Polysorbate-20; SP, Sphingomyelin phosphodiesterase; SPR, Surface plasmon resonance; TFA, Trifluoroacetic acid.