The bioanalysis of large molecules has continued to grow in significance with the increase in development of proteins as therapeutic agents. In particular, the development of monoclonal antibody therapeutics is in large part due to their high target specificity and long serum half-lives. The predominant assay platform of choice for the bioanalysis of biopharmaceuticals utilizes LBA (Ligand Binding Assay) techniques. The high-throughput workflows and lower instrumentation investments of LBAs make them a practical and economical solution compared to other methodologies. However, the increasing complexity of biotherapeutic structures has also increased the data requirements necessary to achieve biotherapeutic development that lie outside the standard applications of LBAs. Specifically, issues associated with nonspecific binding, cross-reactivity and immunogenicity limit the versatility of LBA platforms.
In order to supplement LBA deficiencies, the development of LC/MS (liquid chromatography/mass spectrometry) techniques have become the most flexible alternative for mAb analysis. LC/MS methods are able to generate a broader picture by providing selectivity for compounds in complex biological matrices with the added benefit of multiplexing capabilities. LC/MS assay development is not without challenges of its own, however. The most pressing of these is the lack of developed workflows that address the method optimization necessary to achieve a viable analytical procedure.
The LB-MSIA (Ligand Binding – Mass Spectrometric Immunoassay) approach is a broadly applicable workflow that helps to streamline the bioanalysis of therapeutic mAbs. An extremely versatile IA-LC/MS platform, the LB-MSIA workflow facilitates intact or digested peptide analysis that can be tailored to meet specific data requirements. The following is an example of how assay sensitivity can be enhanced by using an IA-LC/MS/MS approach for the analysis of adalimumab, achieving a dynamic range of 5ng/mL to 10µg/mL with a 200µL sample size.
Improving Sensitivity
While LC/MS approaches are at the forefront of emerging protein analysis technology, there remains a continuous struggle to improve assay sensitivity. The enzymatic digestion of high molecular weight proteins (>10 kDa) prior to LC/MS analysis is generally several fold more sensitive compared to the alternative of intact protein analysis. In comparison, the use of intact protein LC/MS analysis simplifies the sample preparation by eliminating the protein digestion, resulting in a reduced potential for data loss. The ability to assay intact proteins may even become necessary when an appropriate digestion strategy cannot be developed that provides all of the peptide regions of interest of a particular protein. Performing intact protein analysis also requires specific MS instrumentation that is capable of detecting extended mass ranges, such as Thermo Scientific™ Q Exactive™ BioPharma Platform of mass spectrometers.
Improved sensitivity may also be achieved through the use of a hybrid approach that combines IA (immunoaffinity) sample enrichment with LC/MS protein analysis (abbreviated as IA-LC/MS). IA-LC/MS platforms provide the enhanced selectivity and reduced background of LBAs and the versatility of LC/MS analysis. The addition of an IA capture to the sample preparation requires more optimization to develop a viable method, further increasing the need for developed workflows for IA-LC/MS platforms. Specifically, the IA capture requires a high affinity capture reagent. The addition of an IA capture also introduces the potential for binding interference, which occurs when a protein complex is formed with the target protein. A common example of binding interference is the development of ADAs (antidrug antibodies) by the treated animal or human. The formation of these antibodies may greatly decrease therapeutic function and can block access to the epitopes needed for effective analysis. In this regard, IA-LC/MS provides a significant benefit over LBAs as the method allows for the use of aggressive sample pre-treatment to disrupt these protein complexes prior to performing the IA capture (e.g. acidification, the use of detergent solutions, extraction with organic or high salt).
LB-MSIA, an IA-LC/MS/MS Workflow for the Bioanalysis of Monoclonal Antibodies
The LB-MSIA workflow was developed as a complete protocol for the IA-LC/MS/MS analysis of adalimumab with high throughput capabilities. Through the use of Streptavidin MSIA D.A.R.T.’S, biotinylated-TNF-α is used as a highly specific capture reagent for unbound adalimumab in human plasma. The LB-MSIA workflow details the necessary sample preparation and digestion of the purified adalimumab followed by the detection of the digested peptides through the use of LC-MS/MS to achieve a dynamic range of 5ng/mL to 10µg/mL with a 200 µL sample size for the analysis. To access the application note, click here.
Advancing the Bioanalysis of Monoclonal Antibodies
The increased data requirements for the bioanalysis of biotherapeutics are being met by more versatile LC/MS approaches, but many of these lack pre-developed workflows to reduce the amount of labor and time necessary for assay development. LB-MSIA workflows help simplify assay development while simultaneously increasing throughput and achieving high-quality results. Discover workflows that provide innovative enablement in the areas of PKPD, DMPK, ADME, Biotransformation (DAR and payload determination), as well as routine biomarker analysis at www.thermofisher.com/MSIA.