"Negative" Impact: The Role of Payload Charge in the Physicochemical Stability of Auristatin Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) tend to be less stable than their parent antibodies, which is often attributed to the hydrophobic nature of their drug payloads. This study investigated how the payload charge affects ADC stability by comparing two interchain cysteine ADCs that had matched drug-to-antibody ratios and identical linkers but differently charged auristatin payloads, vcMMAE (neutral) and vcMMAF (negative). Both ADCs exhibited higher aggregation than their parent antibody under shaking stress and thermal stress conditions. However, conjugation with vcMMAF increased the aggregation rates to a greater extent than conjugation with uncharged but more hydrophobic vcMMAE. Consistent with the payload logD values, ADC-vcMMAE showed the greatest increase in hydrophobicity but minor changes in charge compared with the parent antibody, as indicated by hydrophobic interaction chromatography and capillary electrophoresis data. In contrast, ADC-vcMMAF showed a decrease in net charge and isoelectric point along with an increase in charge heterogeneity. This charge alteration likely contributed to a reduced electrostatic repulsion and increased surface activity in ADC-vcMMAF, thus affecting its aggregation propensity. These findings suggest that not only the hydrophobicity of the payload, but also its charge should be considered as a critical factor affecting the stability of ADCs.

Agitation-Induced Aggregation of Lysine- And Interchain Cysteine-Linked Antibody-Drug Conjugates.

Drug conjugation to an antibody can affect its stability, which depends on factors such as the conjugation technique used, drug-linker properties, and stress encountered. This study focused on the effects of agitation stress on the physical stability of two lysine (ADC-K) and two interchain cysteine (ADC-C) conjugates of an IgG1 monoclonal antibody (mAb) linked to either ∼4 MMAE or DM1 payloads. During agitation, all antibody-drug conjugates (ADCs) exhibited higher aggregation than the mAb, which was dependent on the conjugation technique (aggregation of ADC-Ks > ADC-Cs) and drug-linker (aggregation of ADCs with MMAE > ADCs with DM1). The aggregation propensities correlated well with higher self-interaction, hydrophobicity, and surface activity of ADCs relative to the mAb. The intermediate reduced mAb (mAb-SH) showed even higher aggregation than the final product ADC-Cs. However, blocking mAb-SH's free thiols with N-ethylmaleimide (NEM) strongly reduced its aggregation, suggesting that free thiols should be minimized in cysteine ADCs. Further, this study demonstrates that a low-volume surface tension method can be used for estimating agitation-induced aggregation of ADCs in early development phases. Identifying liabilities to agitation stress and their relationship to biophysical properties may help optimize ADC stability.

Stability of freeze-dried products subjected to microcomputed tomography radiation doses.

OBJECTIVES: Microcomputed tomography (µCT) is a powerful analytical tool for non-invasive structural analysis. The stability of drug substances and formulations subjected to X-ray radiation may be a concern in the industry. This study examines the effect of X-ray radiation on the stability of freeze-dried pharmaceuticals. The investigation is a proof of concept study for the safety of µCT X-ray radiation doses during the non-destructive investigation of freeze-dried products. METHODS: Different formulations of clotrimazole, insulin and l-lactate dehydrogenase were freeze-dried and the products exposed to a defined dose of radiation by µCT. Conservative freeze-drying conditions were used. Irradiated and normal samples were analysed for their stability directly after freeze-drying and after stability testing. KEY FINDINGS: The stability of model compounds was well maintained during freeze-drying. Some degradation of all compounds occurred during accelerated stability testing. The results showed no differences between the irradiated and normal state directly after freeze-drying and accelerated stability testing. CONCLUSIONS: No evidence of a detrimental effect of 100 Gy X-ray exposure on a model small molecule, peptide and protein compound was found while useful structural information could be obtained. Consequently, the technology may be useful as a non-destructive tool for product inspections if the formulation proves stable.

Freeze-Drying From Organic Co-Solvent Systems, Part 2: Process Modifications to Reduce Residual Solvent Levels and Improve Product Quality Attributes.

The use of co-solvent systems can benefit the freeze-drying process and product performance. In this study, cycle designs were applied based on existing recommendations for water-based formulations. Modifications thereof and the influence on the process (e.g., drying times) and product quality attributes (e.g., product appearance, residual solvent) were tested for various cosolvent systems. It was found that fast freezing was associated with the formation of large crystals for 50 mg/g polyvinylpyrrolidone in 40% 1,4-dioxane (w/w), resulting in a 7% reduction of primary drying. The application of high shelf temperatures during primary drying for 50 mg/g polyvinylpyrrolidone in 70% tert-butanol was feasible, resulting in shorter primary drying times but high residual solvent levels (7.7%). Most notable was that the inclusion of an evaporation step after freezing improved the product appearance for low-melting co-solvents (10% ethanol and 10% acetone). No ice or solvent nucleation occurred in the case of 50 mg/g mannitol in 50% N,N-dimethylacetamide during the normal freezing stage. Instead, the solution viscosity significantly increased after cooling to low shelf temperatures, followed by product evaporation (rather than sublimation) during the drying phase and failure to form a product cake after drying. The application of annealing enabled nucleation and sublimation.

Oxidation-Induced Destabilization of Model Antibody-Drug Conjugates.

Oxidation of biopharmaceutics represents a major degradation pathway, which may impact bioactivity, serum half-life, and colloidal stability. This study focused on the quantification of oxidation and its effects on structure and colloidal stability for a model antibody and its lysine (ADC-L) and cysteine (ADC-C) conjugates. The effects of oxidation were evaluated by a forced degradation study using H2O2 and a shelf-life simulation, which used degrading polysorbate 80 as source for reactive oxygen species. Differential scanning fluorimetry revealed decreasing transition temperatures of the CH2 domain with rising oxidation, resulting in a loss of colloidal stability as assessed by size-exclusion high pressure liquid chromatography. The conjugation technique influences structural changes of the monoclonal antibody (mAb) and subsequently alters the impact of oxidation. ADC-C was most effected by oxidation as the CH2 domain showed the biggest destabilization on conjugation compared to the mAb and ADC-L. Quantification of Fc methionine oxidation by analytical protein A chromatography revealed 4-fold higher oxidation after 8 weeks for the ADC-C compared to the mAb. Payload degradation was observed independently of the conjugation technique used or if free in solution by ultraviolet-visible. In addition, adding antioxidants can be a suitable approach to prevent oxidation and achieve baseline stabilization of the proteins.

Impact of Payload Hydrophobicity on the Stability of Antibody-Drug Conjugates.

In silico screening of toxin payloads typically employed in ADCs revealed a wide range of hydrophobicities and sizes as measured by log P and topological polar surface area (tPSA) values. These descriptors were used to identify three nontoxic surrogate payloads that encompass the range of hydrophobicity defined by the ADC toxin training set. The uniform drug to antibody ratio (DAR) ADCs were prepared for each surrogate payload by conjugation to the interchain cysteine residues of a model IgG1 subtype mAb. Linkage of these surrogate payloads to a common mAb with a matched DAR value allowed for preliminary analytical interrogation of the influence of payload hydrophobicity on global structure, self-association, and aggregation properties. The results of differential scanning fluorimetry and dynamic light scattering experiments clearly revealed a direct correlation between the destabilization of the native mAb structure and the increasing payload hydrophobicity. Also, self-association/aggregation propensity examined by self-interaction biolayer interferometry or size exclusion HPLC was consistent with increased conversion of the monomeric mAb to higher order aggregated species, with the degree of conversion directly proportional to the payload hydrophobicity. In summary, these findings prove that the payload-dependent structure destabilization and enhanced propensity to self-associate/aggregate driven by the increasing payload hydrophobicity contribute to reduced ADC stability and more complex behavior when assessing exposure and safety/efficacy relationships.

Merits and Limitations of Dynamic Vapor Sorption Studies on the Morphology and Physicochemical State of Freeze-Dried Products.

The goal of the present study was to assess the applicability of dynamic vapor sorption analysis of freeze-dried products. Water vapor sorption profiles of intact and ground cakes were recorded to determine the relevance of powder handling. Grinding prior to measurements appeared to be related with a more rapid uptake of water vapor and crystallization. Crystallization may be prevented when analyzing intact cakes. More hygroscopic materials appeared to require a longer time to achieve a constant mass. The specific surface area of different freeze-dried products was calculated from the sorption isotherms using water, organic solvents, and krypton. The specific surface areas calculated for mannitol with water and ethanol was in good agreement with krypton data. False high values were obtained from water vapor sorption of the investigated amorphous materials. The results were slightly improved by the application of vacuum. For trehalose and sucrose, no sorption and thus faulty results were detected with the studied organic solvents. The degree of crystallinity of mannitol within a binary formulation could not be determined by dynamic vapor sorption. Differences in sorption and crystallization tendencies of mannitol and sucrose that were freeze-dried separately and in a binary mixture were considered as the root cause.

High-throughput oxidation screen of antibody-drug conjugates by analytical protein A chromatography following IdeS digest.

OBJECTIVES: Oxidation of protein therapeutics is a major chemical degradation pathway which may impact bioactivity, serum half-life and stability. Therefore, oxidation is a relevant parameter which has to be monitored throughout formulation development. Methods such as HIC, RPLC and LC/MS achieve a separation of oxidized and non-oxidized species by differences in hydrophobicity. Antibody-drug conjugates (ADC) although are highly more complex due to the heterogeneity in linker, drug, drug-to-antibody ratio (DAR) and conjugation site. The analytical protein A chromatography can provide a simple and fast alternative to these common methods. METHODS: A miniature analytical protein A chromatography method in combination with an IdeS digest was developed to analyse ADCs. The IdeS digest efficiency of an IgG1 was monitored using SEC-HPLC and non-reducing SDS-PAGE. An antibody-fluorescent dye conjugate was conjugated at different dye-to-antibody ratios as model construct to mimic an ADC. KEY FINDINGS: With IdeS, an almost complete digest of a model IgG1 can be achieved (digested protein amount >98%). This enables subsequent analytical protein A chromatography, which consequently eliminates any interference of payload with the stationary phase. CONCLUSION: A novel high-throughput method for an interchain cysteine-linked ADC oxidation screens during formulation development was developed.

Freeze-Drying From Organic Cosolvent Systems, Part 1: Thermal Analysis of Cosolvent-Based Placebo Formulations in the Frozen State.

The use of cosolvent systems has been demonstrated to shorten lengthy freeze-drying processes and improve the solubility and stability of certain active pharmaceutical ingredients. The goal of the present study was to evaluate the suitability of 2 thermal characterization techniques, differential scanning calorimetry and freeze-dry microscopy, and to identify an optimal cosolvent system. Binary mixtures of a cosolvent (tert-butanol, dimethyl sulfoxide, 1,4-dioxane, acetone, or ethanol) and water were investigated. Ternary mixtures of frequently used excipients (50 mg/g mannitol, sucrose, glycine, or polyvinylpyrrolidone [PVP]) and a solvent-water system were then analyzed for their thermal properties. PVP presented a particularly high glass transition temperature (Tg') in 70% tert-butanol at -17.9°C. Large needle-shaped crystals that have been shown to be associated with improved processability were observed with mannitol and PVP in 40% 1,4-dioxane. A heterogeneous sublimation rate of the solvent and water whose impact on product stability remained unclear was observed with PVP in 40% 1,4-dioxane. Freeze-dry microscopy analysis demonstrated a possible extension of the process time for PVP in 99% dimethyl sulfoxide due to a slowly moving sublimation front. Conceivable negative consequences and the need for special treatment for low-melting cosolvents, such as ethanol and acetone, were predicted and discussed.

Application of gas pycnometry for the density measurement of freeze-dried products.

Gas pycnometry is applied to determine the density of solid materials. The analysis of lyophilisates is particularly challenging because of their porous structure. In this study, the density of raw materials and freeze-dried products was determined using different pycnometric methodologies and gases [helium (He), nitrogen (N2 ), sulfur hexafluoride]. The number of purges was set to 60 independent of the gas used. Intact and ground lyophilisates were examined, and major differences were found between use of He and N2 . For example, the density of sucrose lyophilisates measured using He remained almost constant before (1.51 g/cm(3) ) and after (1.52 g/cm(3) ) grinding. In contrast, the density of a sucrose lyophilisate before grinding determined with N2 was 1.33 g/cm(3) . On the basis of µCT and scanning electron microscopy pictures, it appears likely that the majority of pores are interconnected, with only a small fraction of closed pores. Helium is able to penetrate deep into the freeze-dried matrix and is supposedly absorbed by the material. The N2 molecules were not able to penetrate closed pores; therefore, the skeletal density of an intact lyophilisate was determined. Reproducibility of the established method was verified, and freeze-dried orally disintegrating tablets of different compositions were analyzed.

Freeze-drying of nanosuspensions, part 3: investigation of factors compromising storage stability of highly concentrated drug nanosuspensions.

On the basis of a previously developed formulation and process guideline for lyophilized, highly concentrated drug nanosuspensions for parenteral use, it was the purpose of this study to demonstrate that the original nanoparticle size distribution can be preserved over a minimum period of 3 months, even if aggressive primary drying conditions are used. Critical factors were evaluated that were originally believed to affect storage stability of freeze-dried drug nanoparticles. It was found that the nature and concentration of the steric stabilizer, such as Poloxamer 338 and Cremophor EL, are the most important factors for long-term stability of such formulations, independent of the used drug compound. The rational choice of an adequate steric stabilizer, namely Poloxamer 338, in combination with various lyoprotectants seems crucial to prevent physical instabilities of the lyophilized drug nanoparticles during short-term stability experiments at ambient and accelerated conditions. A 200 mg/mL concentration of nanoparticles could successfully be stabilized over the investigated time interval. In the course of the present experiments, polyvinylpyrrolidone, type K15 was found superior to trehalose or sucrose in preserving the original particle size distribution, presumably based on its surface-active properties. Lastly, it was demonstrated that lower water contents are generally beneficial to stabilize such systems.

Freeze-dry microscopy: impact of nucleation temperature and excipient concentration on collapse temperature data.

The objective of this study was to investigate the impact of nucleation temperature (T(n)) and excipient concentration on the collapse temperature data obtained from freeze-dry microscopy (FDM) experiments. T(n), the temperature of the onset of collapse (T(oc)), and the full collapse temperature (T(fc)) were determined for aqueous solutions of polyvinylpyrrolidone (PVP) 40 kDa and 2-(hydroxypropyl)-beta-cyclodextrin. Concentrations were varied from 1% to 20% (w/w) for PVP and from 1% to 30% (w/w) for the 2-(hydroxypropyl)-beta-cyclodextrin. Mutual correlation coefficients were calculated for the observed T(n), T(oc), and concentrations of the solutions. In addition, outliers were detected and eliminated by applying the leaving-one-out routine and calculating correlation coefficients without it. T(n) was found to be non-correlated with concentrations and only weakly correlated with T(oc). The correlation between these two temperatures was particularly poor for the solutions of the highest and lowest concentrations. In contrast, T(oc) correlated much better with the corresponding concentrations, resulting in a quadratic fit for PVP and a linear fit for 2-(hydroxypropyl)-beta-cyclodextrin.