Combined use of immunoassay and two-dimensional liquid chromatography mass spectrometry for the detection and identification of metabolites from biotherapeutic pharmacokinetic samples

Abstract

Peptides and monoclonal antibodies have both emerged as important therapeutic modalities, but each has challenges which limit their use. Non-recombinant chemical conjugation of peptides onto antibodies has the potential to minimize or eliminate altogether many of these limitations. Once such approach, pioneered by CovX has created the possibility for rapid stoichiometric fusion of pharmacophores to a single antibody platform. These molecules, called CovX-Bodies, maintain both the pharmacologic properties of a given peptide and the pharmacokinetic properties of a monoclonal antibody. The result is a new class of molecules wherein each component contributes desirable traits. In this paper, we demonstrate the use of immunoassay and two-dimensional liquid chromatography mass spectrometry (2DLC/MS) in combination to investigate the antibody conjugates of Glucagon-like peptide-1 (GLP-1) and analogs for intact protein metabolite identification directly from mouse serum. The information gained from combining these approaches has helped guide and expedite the optimization of our drug product development efforts.

Introduction

Targeted therapy using monoclonal antibodies has revolutionized medicine, with several already having demonstrated their clinical potential for a range of human diseases [1]. Peptides have advantages in terms of target affinity and specificity, and the ability to disrupt protein–protein interactions [2]. While monoclonal antibodies and peptides have emerged as important therapeutics, each has some important limitations. Each novel monoclonal antibody requires the development of a new cell line and manufacturing process. Peptides often suffer from rapid clearance and enzymatic degradation in the body. The development of highly specific chemical linkage technologies has allowed the coupling of pharmacologically active agents (specifically peptides) to an aldolase antibody in a stoichiometrically defined manner [3]. Furthermore, the platform nature of this aldolase antibody eliminates the need for new individual cell line development and optimization for each new biotherapeutic molecule [4]. This novel approach addresses the limitations of traditional peptide and antibody approaches by combining them into new bioconjugate molecules. These molecules, called CovX-Bodies, have both the pharmacologic properties of a given peptide and the pharmacokinetic properties of a monoclonal antibody. CovX-Bodies are created by covalently joining a pharmacophore via various linkers to two specific catalytic binding sites on a specially designed humanized monoclonal IgG1 antibody. The result is a well-characterized, bivalent antibody-drug conjugate that possesses the biologic actions of the peptide and the extended half-life of the antibody. We have successfully demonstrated the utility of this technology through the enhancement of both pharmacokinetic and pharmacodynamic profiles of peptides used in the treatment of several disease areas in recent human clinical trials [4]. Importantly, the use of the same antibody scaffold for multiple drug candidates allows for the use of a clone-specific anti-idiotype capture antibody which reduces drug development cycle time.

The antigenic specificity of a given antibody clone is dictated by the unique sequences of the (hypervariable) complementary determining regions (CDRs) located on the heavy and light chains of the antibody's variable domain. Collectively, these sequences form the “idiotype” of a given antibody clone. Thus, antibodies directed against the idiotypes (i.e. CDRs) of another antibody have become known as anti-idiotypic antibodies. Subsequently, anti-idiotype antibodies have been investigated as therapeutics for B-cell lymphomas and other cancers [5]. Additionally, anti-idiotypic antibodies have been used as highly specific reagents that are well suited for the detection of therapeutic antibodies in vivo [6], particularly in monkeys and humans, where the large amounts of endogenous antibodies often interfere with standard polyclonal anti-human IgG reagents. Fig. 1 displays a graphical representation of the anti-idiotype and CovX-Body interaction that is used to isolate the CovX-Body from serum.

Therapeutic drug monitoring is critical to achieving optimal patient care. Immunoassays and liquid chromatography have been used to assay small and large molecule therapeutics. The most common methods used in the clinical laboratories are immunoassays and liquid chromatography coupled with ultraviolet detection. Both methods are subject to interference by the metabolites formed in vivo, and affect the reliability of the assay performance because of the overestimation of drug concentrations due to nonspecific cross-reaction from their metabolites [7]. Liquid chromatography coupled with mass spectrometry (LC/MS) is a powerful analytical tool that provides high specificity and sensitivity. Because of the simplified sample preparation and the high sensitivity and specificity as compared to immunoassays, LC/MS has shown great potential to be the method of choice for the analysis of immunosuppressants in vivo [8]. In addition LC/MS can measure multiple ions to determine multiple drugs in a single analytical run. Like any other technique, however, LC/MS has its own limitations. The most widely used ionization source for MS (electrospray) is vulnerable to ion suppression [9], which can lead to significant sensitivity loss and erroneous results. Ion suppression occurs when co-eluting compounds suppress the ionization of the target compounds in the ionization source. The detailed mechanism of ion suppression is not clear, but the presence of complex matrices such as blood and serum often amplifies the problem. One effective way to eliminate ion suppression is to remove extraneous matrix components through sample cleanup procedures. Two-dimensional liquid chromatography reduces suppression by using two sample cleanup or separation steps prior to MS analysis. Two-dimensional liquid chromatography in combination with mass spectrometry has the ability to analyze biological samples with specificity and selectivity and is becoming the method of choice for biomarker discovery [10] and complex proteomic samples [11]. Proteolytic degradation and/or metabolite formation analysis is usually accomplished by peptide mapping or MS based sequencing techniques [12] and top-down or intact protein based methods are emerging techniques since they preserve post-translational modifications [13], [14].

An anti-idiotypic antibody was created and used to detect and quantify specific intact and metabolized forms of CovX-Bodies directly from serum. By combining mass spectrometry and immunoassay approaches, a new way to detect intact large molecule therapeutics directly from the serum of animals or humans is demonstrated. In this paper, the analysis of GLP-1 peptide is investigated, a peptide which possesses several physiological properties making it the subject of intense investigation as a potential treatment for obesity and type 2 diabetes mellitus. GLP-1 has several know and important metabolites [15]. An aminopeptidase (dipeptidyl peptidase IV) which is found both in the endothelium of the local capillary bed within the intestinal wall and in the serum, readily degrades GLP-1 to generate N-terminal truncated metabolites. Exendin-4, a 39 amino acid peptide with 53% structural homology to GLP-1, has a longer half-life in vivo and similar biological properties to GLP-1 [16]. CovX-Bodies containing GLP-1 and Exendin-4 peptides were analyzed in serum samples over time by both immunoassay and 2DLC/MS to monitor the intact antibody, as well as, various metabolites. Both the immunoassay and the first dimension in 2DLC/MS used the same anti-idiotype antibody to capture the CovX-Body from serum. The immunoassays detect the CovX-Body using an antibody specifically reactive with the intact N-terminus of the GLP-1 peptide, the C-terminus of the Exendin-4 peptide, or human IgG. The 2DLC/MS detects the captured CovX-Body by switching the affinity column fraction into a reversed phased column with a time-of-flight mass spectrometer for the analysis of the intact antibody. A new method (2DLC/MS) was developed to directly analyze intact antibodies present in the serum and was used in conjunction with immunoassay to analyze time course serum samples for the qualitative analysis of metabolites.