2 research outputs found
LC/MS/MS Bioanalysis of Protein–Drug ConjugatesThe Importance of Incorporating Succinimide Hydrolysis Products
Bioanalysis of antibody–drug
conjugates (ADCs) is challenging
due to the complex, heterogeneous nature of their structures and their
complicated catabolism. To fully describe the pharmacokinetics (PK)
of an ADC, several analytes are commonly quantified, including total
antibody, conjugate, and payload. Among them, conjugate is the most
challenging to measure, because it requires detection of both small
and large molecules as one entity. Existing approaches to quantify
the conjugated species of ADCs involve a ligand binding assay (LBA)
for conjugated antibody or hybrid LBA/liquid chromatography/tandem
mass spectrometry (LC/MS/MS) for quantitation of conjugated drug.
In our current work for a protein–drug conjugate (PDC) using
the Centyrin scaffold, a similar concept to ADCs but with smaller
protein size, an alternative method to quantify the conjugate by using
a surrogate peptide approach, was utilized. The His-tagged proteins
were isolated from biological samples using immobilized metal affinity
chromatography (IMAC), followed by trypsin digestion. The tryptic
peptide containing the linker attached to the payload was used as
a surrogate of the conjugate and monitored by LC/MS/MS analysis. During
method development and its application, we found that hydrolysis of
the succinimide ring of the linker was ubiquitous, taking place at
many stages during the lifetime of the PDC including in the initial
drug product, in vivo in circulation in the animals, and ex vivo during
the trypsin digestion step of the sample preparation. We have shown
that hydrolysis during trypsin digestion is concentration-independent
and consistent during the work flowî—¸therefore, having no impact
on assay performance. However, for samples that have undergone extensive
hydrolysis prior to trypsin digestion, significant bias could be introduced
if only the non-hydrolyzed form is considered in the quantitation.
Therefore, it is important to incorporate succinimide hydrolysis products
in the quantitation method in order to provide an accurate estimation
of the total conjugate level. More importantly, the LC/MS/MS-based
method described here provides a useful tool to quantitatively evaluate
succinimide hydrolysis of ADCs in vivo, which has been previously
reported to have significant impact on their stability, exposure,
and efficacy
Understanding How the Stability of the Thiol-Maleimide Linkage Impacts the Pharmacokinetics of Lysine-Linked Antibody–Maytansinoid Conjugates
Antibody-drug
conjugates (ADCs) have become a widely investigated
modality for cancer therapy, in part due to the clinical findings
with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine
utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized
maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab)
via the maleimide moiety of the heterobifunctional SMCC linker. The
pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a
faster clearance for the ADC than for total antibody. Cytotoxic agent
release in plasma has been reported with nonmaytansinoid, cysteine-linked
ADCs via thiol-maleimide exchange, for example, brentuximab vedotin.
For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1,
the expected product of intracellular antibody proteolysis. To understand
these observations better, we conducted a series of studies to examine
the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting
conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made
with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH<sub>2</sub>)<sub>3</sub>-DM). We employed radiolabeled ADCs to directly
measure both the antibody and the ADC components in plasma. The PK
properties of the conjugated antibody moiety of the two conjugates,
J2898A-SMCC-DM1 and J2898A-(CH<sub>2</sub>)<sub>3</sub>-DM (each with
an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear
to be similar to that of the unconjugated antibody. Clearance values
of the intact conjugates were slightly faster than those of the Ab
components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than
J2898A-(CH<sub>2</sub>)<sub>3</sub>-DM, suggesting that there is a
fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through
a thiol-maleimide dependent mechanism. Experiments on ex vivo stability
confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates
can occur via thiol elimination, but at a slower rate than the corresponding
rate of loss reported for thiol-maleimide links formed at thiols derived
by reduction of endogenous cysteine residues in antibodies, consistent
with expected differences in thiol-maleimide stability related to
thiol p<i>K</i><sub>a</sub>. These findings inform the design
strategy for future ADCs