Brooke M. Rock

Intact mass analysis of monoclonal antibodies by capillary electrophoresis-Mass spectrometry.

Characterization of monoclonal antibody (mAb) therapeutics by intact mass analysis provides important information on sequence integrity and post-translational modifications. In order to obtain domain specific information, monoclonal antibodies are reduced to heavy and light chain components or enzymatically digested into smaller portions or peptides. Liquid chromatography (LC) is widely used for separation of the antibody fragments in line with mass spectrometry (MS) for characterization. Capillary electrophoresis (CE) is an analytical technique with high separation efficiency, high sensitivity, and minimal inter-run sample carryover. Combining the resolving power of CE with electrospray ionization (ESI) MS has great potential in regards to accurate mass characterization of protein therapeutics and has been a long sought-after approach. However, the intrinsic technical difficulty in coupling CE to MS has hindered the broad application of CE-MS across the biopharmaceutical industry. Recently, a CE-MS interface has been developed [1] and commercialized. Herein, we report implementation of this technology for coupling CE to an Agilent time-of-flight (TOF) mass spectrometer. CE-MS provides an attractive complement to LC-MS for separation and intact mass determination of mAbs and antibody-based therapeutics.

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm

Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.

Intracellular Catabolism of an Antibody Drug Conjugate with a Noncleavable Linker.

Antibody drug conjugates are emerging as a powerful class of antitumor agents with efficacy across a range of cancers; therefore, understanding the disposition of this class of therapeutic is crucial. Reported here is a method of enriching a specific organelle (lysosome) to understand the catabolism of an anti-CD70 Ab-MCC-DM1, an antibody drug conjugate with a noncleavable linker. With such techniques a higher degree of concentration-activity relationship can be established for in vitro cell lines; this can aid in understanding the resultant catabolite concentrations necessary to exert activity.

Evaluation of Near Infrared Fluorescent Labeling of Monoclonal Antibodies as a Tool for Tissue Distribution

The pharmacokinetic (PK) behavior of monoclonal antibodies (mAbs) is influenced by target-mediated drug disposition, off-target effects, antidrug antibody–mediated clearance, and interaction with fragment-crystallizable domain (Fc) receptors such as neonatal Fc receptor. All of these interactions hold the potential to impact mAb biodistribution. Near infrared (NIR) fluorescent probes offer an approach complementary to radionuclides to characterize drug disposition. Notably, the use of IRDye800 (IR800; LI-COR, Lincoln, NE) as a protein-labeling agent in preclinical work holds the potential for quantitative tissue analysis. Here, we tested the utility of the IR800 dye as a quantitative mAb tracer during pharmacokinetic analysis in both plasma and tissues using a model mouse monoclonal IgG1 (8C2) labeled with ≤1.5 molecules of IR800. The plasma PK parameters derived from a mixture of IR800-8C2 and 8C2 dosed intravenously to C57BL/6 mice at 8 mg/kg exhibited a large discrepancy in exposure depending on the method of quantitation [CLplasma = 8.4 ml/d per kilogram (NIR fluorescence detection) versus 2.5 ml/d per kilogram (enzyme-linked immunosorbent assay)]. The disagreement between measurements suggests that the PK of 8C2 is altered by addition of the IR800 dye. Additionally, direct fluorescence analysis of homogenized tissues revealed several large differences in IR800-8C2 tissue uptake when compared with a previously published study using [125I]8C2, most notably an over 4-fold increase in liver concentration. Finally, the utility of IR800 in combination with whole body imaging was examined by comparison of IR800-8C2 levels observed in animal sagittal cross-sections to those measured in homogenized tissues. Our results represent the first PK analysis in both mouse plasma and tissues of an IR800-mAb conjugate and suggest that mAb disposition is significantly altered by IR800 conjugation to 8C2.

Characterization of the active site properties of CYP4F12.

Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site.

Characterization of Ritonavir-Mediated Inactivation of Cytochrome P450 3A4

Ritonavir is a human immunodeficiency virus (HIV) protease inhibitor and an inhibitor of cytochrome P450 3A4, the major human hepatic drug-metabolizing enzyme. Given the potent inhibition of CYP3A4 by ritonavir, subtherapeutic doses of ritonavir are used to increase plasma concentrations of other HIV drugs oxidized by CYP3A4, thereby extending their clinical efficacy. However, the mechanism of inhibition of CYP3A4 by ritonavir remains unclear. To date, data suggests multiple types of inhibition by ritonavir, including mechanism-based inactivation by metabolic-intermediate complex formation, competitive inhibition, irreversible type II coordination to the heme iron, and more recently heme destruction. The results presented here demonstrate that inhibition of CYP3A4 by ritonavir occurs by CYP3A4-mediated activation and subsequent formation of a covalent bond to the apoprotein. Incubations of [3H]ritonavir with reconstituted CYP3A4 and human liver microsomes resulted in a covalent binding stoichiometry equal to 0.93 ± 0.04 moles of ritonavir bound per mole of inactivated CYP3A4. The metabolism of [3H]ritonavir by CYP3A4 leads to the formation of a covalent adduct specifically to CYP3A4, confirmed by radiometric liquid chromatography–trace and whole-protein mass spectrometry. Tryptic digestion of the CYP3A4-[3H]ritonavir incubations exhibited an adducted peptide (255-RMKESRLEDTQKHR-268) associated with a radiochromatic peak and a mass consistent with ritonavir plus 16 Da, in agreement with the whole-protein mass spectrometry. Additionally, nucleophilic trapping agents and scavengers of free oxygen species did not prevent inactivation of CYP3A4 by ritonavir. In conclusion, ritonavir exhibited potent time-dependent inactivation of CYP3A, with the mechanism of inactivation occurring though a covalent bond to Lys257 of the CYP3A4 apoprotein.

Altering Antibody–Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability

Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.

Multienzyme Kinetics and Sequential Metabolism

Enzymes are the catalysts of biological systems and are extremely efficient. A typical enzyme accelerates the rate of a reaction by factors of at least a million compared to the rate of the same reaction in the absence of the enzyme. In contrast to traditional catalytic enzymes, the family of cytochrome P450 (CYP) enzymes are catalytically promiscuous, and thus they possess remarkable versatility in substrates. The great diversity of reactions catalyzed by CYP enzymes appears to be based on two unique properties of these heme proteins, the ability of their iron to exist under multiple oxidation states with different reactivities and a flexible active site that can accommodate a wide variety of substrates. Herein is a discussion of two distinct types of kinetics observed with CYP enzymes. The first example is of CYP complex kinetic profiles when multiple CYP enzymes form the sample product. The second is sequential metabolism, in other words, the formation of multiple products from one CYP enzyme. Given the degree of CYP enzyme promiscuity, it is hardly surprising that there is also a high degree of complex kinetic profiles generated during the catalytic cycle.

Immunoaffinity capture coupled with capillary electrophoresis - mass spectrometry to study therapeutic protein stability in vivo

Protein engineering is at an all-time high in biopharmaceutics. As a result, absorption, distribution, metabolism and excretion (ADME) of proteins has become more important to understand in the context of engineering strategies to optimize therapeutic properties of potential lead constructs. Immunoaffinity capture coupled with a newly developed capillary electrophoresis - mass spectrometry (CE-MS) system was used to characterize intact protein mass analysis of a wild type Fc-FGF21 construct and a sequence re-engineered Fc-FGF21 construct from an in vivo study. A number of truncated forms were observed and the time courses of the various proteolytic products were identified and compared between the two constructs. The abundances of the intact and truncated forms were used to provide the basis to semi-quantify ADME properties of the two protein forms. The use of this immunoaffinity capture followed by CE-MS based intact mass analysis workflow provided a qualitative and quantitative analysis of the pharmacokinetic profiles of the two proteins. The platform presented here holds great potential in characterization of the ADME properties of proteins.

Therapeutic Monoclonal Antibody Intact Mass Analysis by Capillary Electrophoresis–Mass Spectrometry

The characterization of monoclonal antibody (mAb) therapeutics via mass spectroscopy is of important value in determining sequence integrity and identifying post-translational modifications. The monoclonal antibodies are commonly either reduced to generate heavy chain and light chain, or enzymatically cleaved to produce characteristic domains for subunit intact mass analysis. Toward this end, liquid chromatography coupled with mass spectrometry (LC-MS) is usually applied for the separation of these antibody subunits followed by on-line mass analysis. Capillary electrophoresis (CE) is an emerging separation technique that provides excellent protein separation efficiency at ambient temperature. The recent advancement on the coupling of capillary electrophoresis with mass spectrometer has essentially eliminated the technical obstacle for the broad application of CE-MS in the intact mass analysis of monoclonal antibody therapeutics. In this chapter, we will discuss several commercially available CE-MS interfaces and their applications, followed by demonstration of the CE-MS intact mass analysis procedure that has been developed for therapeutic protein characterization.