Immunoconjugates, including antibody-drug conjugates and Fc-conjugates, represent a rapidly growing class of therapeutics undergoing clinical development. Despite their growing popularity, the high intrinsic heterogeneity of immunoconjugates often complicates the development process and limits their widespread application. In particular, immunoconjugate charge variants exhibit markedly different colloidal stabilities, solubilities, pharmacokinetics and tissue distributions. Charge variants arise spontaneously due to degradation and, depending on the type of drug, linker and conjugation site, through drug conjugation. Electrostatic changes in naked antibodies often result in poor performance characteristics, and therefore charge alterations due to degradation are critical to control. Charge properties are expected to be equally important to producing well-behaved ADCs. Charge-based methods of analysis, such as isoelectric focusing and ion exchange chromatography, are capable of probing the underlying complexities within immunoconjugate drug products. Despite the utility of these methods, there are only a few published reports of charge-based assays applied to immunoconjugates. In the present study, we sought to identify the effects of chemical conjugation on the electrostatic properties of Fc-conjugates. In order to minimize the effects of post-translational modifications (e.g. deamidation), a single Fc charge variant was isolated prior to conjugation of a fluorescent probe, Alexa Fluor 350, to the side chains of lysine residues. The resulting Fc-conjugates were assessed by a variety of analytical techniques, including isoelectric focusing and ion exchange chromatography, to determine their charge properties.
Antibody drug conjugates (ADCs) are an emerging class of chemotherapeutic cancer treatment agents that combine the targeting specificity of antibodies with the efficient cell-killing potential of cytotoxic drugs. Unlike their protein and small-molecule therapeutic counterparts, the stability and degradation properties of ADCs are relatively unknown. Theoretically, ADC stability could be governed by properties and processes stemming from both the antibody and the linker-toxin chemistry. Recently, systematic studies of intrinsic ADC molecule stability have been presented in the primary literature. As there are burgeoning industrial and academic efforts aimed at developing optimized conjugation chemistries and antibody engineering approaches for next-generation ADCs, it is important to capture the current state of understanding of ADC stability. In this minireview, we discuss aspects of physical and chemical stability of ADCs gathered from a survey of the literature and illustrate how investigations into stability enable the development of more effective ADC molecules for the future.
During synthesis, purification, and especially storage, antibody-drug conjugates (ADCs) may be exposed to various types of light. Several of the drugs commonly conjugated to antibodies contain photosensitive functional groups. Exposure to light could generate an excited state of the drug that subsequently triggers drug and/or protein degradation. To mimic and study photoinduced ADC degradation, we designed a model ADC in which the monoclonal antibody (mAb) trastuzumab was treated with the amine-reactive probe eosin-5-isothiocyanate to yield an antibody-eosin conjugate (T-EO). Photoinduced degradation was monitored by size exclusion chromatography (SEC), dynamic light scattering (DLS), SDS-PAGE under reducing and nonreducing conditions, and MS/MS analysis. SEC analysis of the model ADC showed the formation of higher molecular weight species directly following a 20 W-hr/m(2) exposure of UVA light. DLS analysis of these samples showed the formation of larger soluble particles, and precipitate was observed 24 h post light exposure. These results were not seen in control samples of the model ADC that were shielded from light. Furthermore, these results were not seen in control samples containing mAb alone, suggesting that aggregation was the result of light exposure of the conjugate. Importantly, when eosin-5-isothiocyanate was added separately to solutions containing mAb (i.e., without conjugation), the extent of photoinduced aggregation was substantially less, indicating that the conjugation of the photosensitizer to the mAb specifically promoted photoinduced aggregation. Reducing and nonreducing SDS-PAGE suggested that photoinduced interchain covalent cross-linking occurred through a mechanism other than disulfide formation. Using peptide mapping and MS/MS analysis, we identified key peptides in the T-EO sequence that undergo photodegradation. Finally, we also show that cross-linking products formed in as little as 1 h of exposure to ambient light. These findings suggest that precautions should be taken to ensure minimal exposure to light during the synthesis, purification, and storage of ADCs containing photosensitive drugs.
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