ADC Physical Stability Analysis

As many of the cytotoxic drugs conjugated to a monoclonal antibody to form an antibody-drug conjugate (ADC) are highly hydrophobic, this covalent chemical conjugation has been shown to exert a significant effect on ADC’s physical stability, a factor that is of crucial importance for downstream ADC development. Scientists from Creative Biolabs adopt a comprehensive set of analytical approaches to fully evaluate ADC physical stability to facilitate the design and improvement of ADCs.

Studying ADC physical stability primarily refers to the understanding of the physical factors that may directly lead to mAb aggregation or those that lead to an increased propensity for ADC aggregation. Creative Biolabs has established an advanced technological platform with cutting-edge equipment for ADC physical stability analysis:

• Differential scanning calorimetry (DSC)
• Circular dichroism (CD) spectroscopy
• Liquid chromatographyemass spectrometry (LC-MS)
• Dynamic Light Scattering (DLS)
• Size-exclusion chromatography (SEC) with UV or MALS detection
• Electrophoresis, reverse-phase high performance liquid chromatography (RP-HPLC)

With that, Creative Biolabs offers a variety of assessment services for ADC physical stability, including (but not limited to):

• Physical aggregation assessment

The most direct assessment of the physical stability of an ADC is to evaluate its tendency to form aggregates. Accompanied by an increase in size, the physical aggregation of an ADC is measured by either direct observation or using analytical methods such as size-exclusion chromatography (SEC) and dynamic light scattering (DLS) to monitor the size changes.

• Thermal stability assessment

Since ADCs and the unconjugated mAbs respond differently to thermal stresses, the response to temperature change is a typical stability testing strategy used to evaluate the potential instability issues within an ADC. ADCs are more easily destabilized by thermal stress than unconjugated mAbs, although they share similar secondary and tertiary structures. This observation indicates that the conformational stability of ADCs is reduced and may lead to aggregation. Temperature-induced aggregation is correlated with an increased DAR (drug to antibody ratio). In one example, ADCs bearing different DAR values are fractionated and then incubated at 40oC, followed by the analysis of aggregation using differential scanning calorimetry (DSC), far-UV circular dichroism spectroscopy, electrophoresis, or reverse-phase high performance liquid chromatography (RP-HPLC). As a result, DAR 6 and DAR 8 species were more prone to form aggregates under stressed conditions comparing to species with lower DAR values. Temperature-induced aggregation also correlates with conjugation chemistry, with cysteine conjugates being more prone to thermal aggregation compared to lysine conjugates.

IEF electropherograms for lyophilized-stressed mAbs and ADCs in various formulations. Temperature induced increases in acidic variant (AV) and basic variant (BV) populations are observed in some cases, indicating a reduced thermal stability of ADCs comparing to unconjugated mAbs. (J Pharm Sci., 2015)

• Photic stability assessment

Some ADC payloads carry light-sensitive or light absorbing substituents. Even though light effects on the stability of the unconjugated antibody or the drug-linker complex along are negligible, it might cause some unexpected physical disturbance once conjugated. During processing and production, ADCs are intermittently exposed to light. Thus, the physical stabilities of these ADCs need to be evaluated in light exposure conditions. Light induced ADC aggregation and particulate formation are observed by size-exclusion chromatography (SEC) and dynamic light scattering, respectively.

Using our integrated analytical platform and with our experienced science team, Creative Biolabs is dedicated to provide our global customers with the most comprehensive analysis services for ADC projects. Meanwhile, with years of experience in antibodies discovery, antibody production, and full chemical synthesis, Creative Biolabs is also your one-stop service station for your other ADC development needs. Please contact us for more information and a detailed quote.

Reference：

1. Valliere-Douglass J.F.; et al. Solid-state mabs and adcs subjected to heat-stress stability conditions can be covalently modified with buffer and excipient molecules. J Pharm Sci. 2015, 104(2): 652-665.