Assessing Immunogenicity in the Presence of Excess Protein Therapeutic Using Immunoprecipitation and Quantitative Mass Spectrometry

Abstract

The administration of biological protein therapeutics can lead to an unwanted immune response resulting in the generation of anti-drug antibodies (ADA) with potentially harmful clinical consequences. Hence, to develop safe and efficacious biotherapeutics, the immunogenic potential needs to be examined during the development phase. Current assay technologies measuring ADAs are subject to interference by high circulating concentrations of the protein therapeutic, raising concerns about data reliability since protein therapeutic-free washout samples are not always available. Herein, we report the development and characterization of a magnetic bead based immunoprecipitation method followed by quantitative LC/MS to determine ADA in human and cynomolgus serum in the presence of high circulating concentrations of the protein therapeutic. Available ADA binding sites are saturated by the addition of excess therapeutic followed by magnetic bead based protein G isolation of IgG antibodies and their bound antigens before elution and digestion. Peptides of the target therapeutic proteins are then quantified by LC/MS using stable isotope labeled standards inferring the presence of total ADA. This approach complements established methodologies for the assessment of immunogenicity responses and currently supports clinical programs addressing the safety and tolerability of human growth hormone analogues.

Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen

“…Although the original SISCAPA work relied on online nanoliter immunoaffinity columns based on Poros supports linked to nanoflow LC and nanospray mass spectrometry (6 ), off-line magnetic bead-based enrichment of enzymatically released peptides followed by either nano-or capillary LC-MS/MS has become a popular approach (7)(8)(9)(10)(11)(12). Bead-based sample preparation has the advantage that the antibody capture step can be done in parallel for multiple samples, and this process can be automated on a magnetic particle processor (11 ) or liquid-handling robotics, as shown for immunoaffinity enrichment of proteins (13,14 ). An extension of the magnetic bead SISCAPA work is the use of an inline bead-trap device to allow the quantification of lower-abundance peptides in a more automated fashion and with reduced off-line handling (15 ).…”

Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

“…Our goal was to determine whether the ADA samples were positive or negative, and the spiking experiment should serve the purpose. It has been reported that 100 μg/mL of hGHA was sufficient to convert 100 μg/ mL ADA to hGH-ADA complex in human serum [21]. In the immunopurification process, the mouse mAbdrug-ADA complexes were separated from the plasma by a magnet and any endogenous monkey plasma proteins that bound non-specifically on the beads were subsequently washed off.…”

“…With traditional ADA immunoassay, only drug-free or partially drug-free ADA can be detected, and the presence of high drug levels in study samples can interfere with the detection of ADA, especially in bridging assay formats. Drug tolerance which is generally defined as the maximal amount of free drug in the samples that still allows detection of ADA at an acceptable sensitivity, may be improved using various approaches including the use of excess spiked drug to create and detect drug-ADA complexes rather than free ADA [18][19][20][21]. LC/MS has been used to assess ADA in the presence of excess protein therapeutic in support of clinical programs addressing the safety and tolerability of human growth hormone analogues [21].…”

“…Drug tolerance which is generally defined as the maximal amount of free drug in the samples that still allows detection of ADA at an acceptable sensitivity, may be improved using various approaches including the use of excess spiked drug to create and detect drug-ADA complexes rather than free ADA [18][19][20][21]. LC/MS has been used to assess ADA in the presence of excess protein therapeutic in support of clinical programs addressing the safety and tolerability of human growth hormone analogues [21]. This methodology overcame drug interference by completely saturating available ADA binding sites with the addition of excess therapeutic.…”

Theoretical Considerations and Practical Approaches to Address the Effect of Anti-drug Antibody (ADA) on Quantification of Biotherapeutics in Circulation

“…However, PK parameters remain an integral component of clinical development so it is important to select ADA and PK time points at pre-dose and trough circulating drug concentrations, and preferably after an appropriate drug washout period (8), to truly understand the impact of ADA on the PK and ensure there is minimum interference in the PK assay. Although PK sample time points are typically more numerous than ADA sampling, it is important to have some PK and ADA sampling points at the same time to truly understand the impact of the ADA on PK.…”