Anouk Donners

36 Chapter 2 Sample purification Arguably, the most laborious step in the development of a successful bioanalytical method is sample purification. Sample purification is necessary to eliminate interfering proteins and reduce sample complexity (Table 3) and over the last decades a myriad of strategies have been reported. Target-specific sample purification in a human biomatrix utilizes an anti-idiotypic antibody or ligand fixed to a solid support such as a magnetic bead or 96 well plate (Figure 6A). Here, only the active therapeutic mAb fraction with at least one free epitope can be purified. This principle was used by our group to purify active infliximab in human serum with its antigen tumor necrosis factor alpha bound to a 96-well plate by means of biotin-streptavidin interaction [72]. A similar principle was reported for the purification of bimagrumab in human serum using activin receptor type 2B which was cross-liked to sepharose magnetic beads by means of NHS (Nhydroxysuccinimide) reagent [77]. The use of anti-idiotypic antibodies, which bind the complementarity-determining regions (CDRs) of the therapeutic mAb, is exemplified with the purification of trastuzumab in human serum with anti-trastuzumab idiotypic antibodies [42]. Purifications with anti-human Fc antibodies in animal bio-matrices can also be considered ‘targeted’. Here, only the human/humanized therapeutic antibody will be captured, resulting in the quantification of total therapeutic antibody [48, 56, 59]. When combining a targeted sample pre-treatment with LC-MS/MS measurement, low detection levels (<0.5 mg/L) can be achieved through background noise reduction (Table 3). In contrast, a generic sample work-up aimed to capture the entire IgG fraction in serum can be used. Protein A and G are bacterial cell wall proteins that bind IgG via their Fc region, thus protecting it in vivo from the immune system. These proteins achieve relatively clean extracts and have been used with success to purify the total therapeutic mAb (Figure 6B) [45, 53, 65, 68-71, 76, 80]. Figure 6. Sample purification methods using; TNF alpha to selectively capture infliximab (A), Protein A or G to capture FC portion of the antibodies (B) and IgG pellet precipitation with ammoniumsulfate or methanol (C)[72]. Experiments performed in-house and others have shown that the ammonium sulfate (AS) precipitation method is highly efficient in the removal of albumin fraction, which comprises of around 60% of total plasma protein (Figure 6C) [85, 94]. Proteins with low solubility (usually large proteins), are precipitated first at increasing AS concentration, leaving the highly soluble, smaller proteins and molecules in solution [95]. This is a cost