Assessing Immunogenicity in the Presence of Excess Protein

Anal. Chem. 2008, 80, 6907–6914

Assessing Immunogenicity in the Presence of Excess Protein Therapeutic Using Immunoprecipitation and Quantitative Mass Spectrometry Hendrik Neubert,* Christopher Grace, Klaus Rumpel, and Ian James Pfizer Global Research and Development, Sandwich, U.K. 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.

ing a protein product.2 The nature of immune responses induced by therapeutic proteins can also vary widely and is more often than not of only temporary appearance and of no clinical significance.3 However, severe and harmful clinical consequences often related to induced autoimmunity have been reported, and hence, immunogenicity remains a major safety concern that needs to be addressed during drug development and postmarketing surveillance.1,4,5 Regulatory agencies request the pharmaceutical industry to examine immunogenicity using a risk-based approach, and specific strategies for developing “fit-for-purpose” bioanalytical approaches are now being discussed.6 The ability to measure ADA is predominantly dependent on the study design, sampling time points, and assay format employed. Established methodologies include ligand binding assays, e.g., bridging and direct binding ELISAs, as well as cell-based assays and surface plasmon resonance.7,8 All of these methods are routinely used to measure free ADA but are subject to interference from high circulating concentrations of the protein therapeutic. Higher sensitivity detection modalities such as electrochemiluminescence9 and dissociation-enhanced lanthanide fluorescence immunoassay show a greater tolerance to excess protein therapeutic but still suffer from drug interference. Further complications in the assessment of ADA responses can result from modifications to the therapeutic protein, such as pegylation,10 that may alter the immunogenic potential and may dramatically increase the circulating half-life, possibly resulting in peak blood protein therapeutic concentrations in the micrograms per milliliter

The administration of biological protein therapeutics can lead to unwanted immune responses due to the generation of antidrug antibodies (ADA). The issue of immunogenicity is complex and depends on many factors such as the nature of the protein therapeutic and its relationship to endogenous proteins, the target patient population, the treatment regimen, and the formulation.1 ADA responses have been reported to alter the efficacy of a protein therapeutic in a number of ways, including increased clearance, maintenance of the drug in the circulation, or neutralization.1 Knowledge about ADAs can be helpful in explaining unusual pharmacokinetics and contribute to efforts in re-engineer-

(2) Tangri, S.; Mothe, B. R.; Eisenbraun, J.; Sidney, J.; Southwood, S.; Briggs, K.; Zinckgraf, J.; Bilsel, P.; Newman, M.; Chesnut, R.; LiCalsi, C.; Sette, A. J. Immunol. 2005, 174, 3187–3196. (3) Shankar, G.; Shores, E.; Wagner, C.; Mire-Sluis, A. Trends Biotechnol. 2006, 24, 274–280. (4) Gershon, S.; Luksenburg, H.; Cote, T.; Braun, M.; Soko, l.L.; Prchal, J.; Casadevall, N.; Mayeux, P.; Bunn, H. N. Engl. J. Med. 2002, 346, 1584–1586. (5) Rossert, J.; Casadevall, N.; Eckardt, K.-U. J. Am. Soc. Nephrol. 2004, 15, 398–406. (6) Shankar, G.; Pendley, C.; Stein, K. E. Nat. Biotechnol. 2007, 25, 555–561. (7) Mire-Sluis, A. R.; Barrett, Y. C.; Devanarayan, V.; Koren, E.; Liu, H.; Maia, M.; Parish, T.; Scott, G.; Shankar, G.; Shores, E.; Swanson, S. J.; Taniguchi, G.; Wierda, D.; Zuckerman, L. A. J. Immunol. Methods 2004, 289, 1–16. (8) Wadhwa, M.; Bird, C.; Dilger, P.; Gaines-Das, R.; Thorpe, R. J. Immunol. Methods 2003, 278, 1–17. (9) Moxness, M.; Tatarewicz, S.; Weeraratne, D.; Murakami, N.; Wullner, D.; Mytych, D.; Jawa, V.; Koren, E.; Swanson, S. J. Clin. Chem. 2005, 51, 1983– 1985. (10) Delgado, C.; Francis, G.; Fisher, D. Crit. Rev. Ther. Drug Carrier Syst. 1992, 9, 249–304.

* To whom correspondence should be addressed. E-mail: hendrik.neubert@ pfizer.com (1) Schellekens, H. Clin. Ther. 2002, 24, 1720–1740. 10.1021/ac8005439 CCC: $40.75  2008 American Chemical Society Published on Web 08/15/2008

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range. Concerns about data reliability arise when protein therapeutic is still present in the sample; hence, it is common practice to measure ADAs in patient samples taken following a washout period, approximately five half-lives or greater after the last administered dose of the protein therapeutic. In an attempt to overcome the issue of drug interference and to facilitate a more rigorous evaluation of ADAs, drug depletion strategies have been investigated and various approaches that employ acid dissociation have been shown to increase the ability to detect ADAs in samples containing excess drug.11-13 Although such strategies can improve the ability of immunoassay methodologies to detect ADAs, each application should be evaluated on a case by case basis as concerns remain relating to acid-mediated loss of ADA activity. In order to improve the assay tolerance to excessive circulating blood concentrations of protein therapeutic, we have explored alternative sample preparation and detection strategies. To this end, we have developed and characterized a magnetic bead based immunoprecipitation method followed by quantitative LC/MS and applied this to determine anti-protein therapeutic antibodies in human and cynomolgus monkey serum in the presence of high concentrations of the protein therapeutic. The method uses lowmicroliter volumes of serum that is spiked with high concentrations of the protein therapeutic in order to saturate available binding sites on ADAs. A magnetic bead based protein G affinity enrichment step facilitates retention of IgG antibodies and their bound antigens followed by suitable wash steps. Isolated antibodyantigen complexes are then eluted from the magnetic beads and stable isotope labeled peptide standards mimicking sequences of the targeted therapeutic protein are added. Following chemical proteolysis, multiple peptides of the target therapeutic proteins are quantified by LC/MS inferring the presence of total antidrug IgG in the presence of high drug concentrations. We chose to employ MALDI mass spectrometry as an end-point detection taking advantage of its sensitivity for the targeted peptides and also the potential to archive MALDI sample plates for retrospective and iterative MS experiments. Several quantitative workflows based on MALDI mass spectrometry, with and without the use of stable isotope labeled standards, have been previously employed, underlining the general utility this method.14-17 This approach adds to the current methodologies for the assessment of immunogenicity responses and currently supports clinical programs addressing the safety and tolerability of a pegylated human growth hormone analogue (hGHA). It is also expected to be transferable to other protein therapeutic programs and technology platforms. This paper illustrates the methodology and presents relevant examples from assay characterization. (11) Smith, H. W.; Butterfield, A.; Sun, D. Regul. Toxicol. Pharmacol. 2007, 49, 230–237. (12) Bourdage, J. S.; Cook, C. A.; Farrington, D. L.; Chain, J. S.; Konrad, R. J. J. Immunol. Methods 2007, 327, 10–17. (13) Patton, A.; Mullenix, M. C.; Swanson, S. J.; Koren, E. J. Immunol. Methods 2005, 304, 189–195. (14) Gutierrez, J.; Dorocke, J.; Knierman, M.; Gelfanova, V.; Higgs, R.; Koh, N.; Hale, J. Biotechniques 2005, 13–17, Suppl. (15) Jiang, J.; Parker, C. E.; Fuller, J. R.; Kawula, T. H.; Borchers, C. H. Anal. Chim. Acta 2007, 605, 70–79. (16) Jiang, J.; Parker, C. E.; Hoadley, K. A.; Perou, C. M.; Boysen, G.; Borchers, C. H. Proteomics Clin. Appl. 2007, 1, 1651–1659. (17) Baechle, D.; Sparbier, K.; Dihazi, H.; Blaschke, S.; Mueller, G.-A.; Kostrzewa, M.; Flad, T. Proteomics Clin. Appl. 2007, 1, 1280–1284.

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METHODS Materials and Reagents. Phosphate-buffered saline, guanidine hydrochloride, Tween 20, trifluoroacetic acid for protein sequence analysis (>99.5%), cyanogen bromide (g98.5), HPLC grade acetonitrile, and water were purchased from Sigma Aldrich (Poole, UK). Cyanogen bromide (CNBr) is toxic and volatile; hence, all operations involving CNBr should be performed in a hood. Protein G-coupled magnetic beads (Dynabeads protein G) were purchased from Invitrogen (Paisley, UK). The protein therapeutics recombinant hGH and a pegylated hGHA were obtained from Pfizer Pharmaceuticals. HGHA is a chemically modified version of hGH, and both proteins share an identical C terminal sequence. Stable isotope labeled peptides FPTIP-[13C6,15N-L]-SRLFDNAML-acid (hGH, N terminus) and MDK-[13C5,15N-V]-ETFLRIVQCRSVEGSCGF-acid [disulfide 13-20] (hGH, C terminus) were custom synthesized, and the net peptide content of each preparation was determined by amino acid analysis (Cambridge Research Biochemicals Ltd., Cleveland, UK). These peptide sequences were subjected to a protein BLAST search for short and nearly exact matches in protein and gene databases to confirm that they exclusively occur in human growth hormone. Affinity-purified polyclonal goat anti-hGH antibody (R&D Systems, Abingdon, UK, Catalog No. AF1067) was used as a positive control and calibrant. It was prepared to an ascribed value of 1000 µg/mL based on the manufacturer’s protein determination. The affinity constant KD of the antibody preparation for hGH was 0.045 nM (determined by Biacore). Normal human serum, for the preparation of standard and quality control samples, was obtained from Sigma (Poole, UK). Twenty-four cynomolgus serum samples for the determination of negative cutoff values as well as a pool of normal cynomolgus sera were obtained from an in-house source, stored at -20 °C, and thawed immediately prior to use. Preparation of Standards and Samples. To investigate drug effect, pooled human serum was spiked with anti-hGH positive control antibody at 0, 10, and 100 µg/mL and with hGHA at 1, 5, 25, 75, 100, 150, 200, and 500 µg/mL. In order to illustrate the analysis of anti-hGH antibodies in cynomolgus serum, anti-hGH antibody calibrants were prepared at 0, 50, 100, 200, 500, 1000, 2000, and 5000 ng/mL by diluting the stock antibody solution in cynomolgus serum. These samples were also spiked with hGH at a final concentration of 150 µg/mL. Twenty-four normal individual cynomolgus sera spiked with hGH at 150 µg/mL were also analyzed on three occasions on different days to define a negative cut point for the assay. A number of cynomolgus sera from a hGHA immunogenicity study were selected to demonstrate the principle of this MS methodology. The obtained sera were spiked with hGH to a final concentration of 150 µg/mL. Samples were available from baseline, two time points during the dosing period, and an additional time point from the recovery period. All serum samples were prepared as described above, left at +4 °C for 16 h, diluted with 0.05% Tween 20/PBS to a final serum dilution of 1:50, aliquoted into 125-µL portions in polypropylene 96-well plates, and stored at -80 °C until use. On each testing occasion, fresh aliquots were thawed and used once. Any remaining material was discarded and not refrozen.

Figure 1. Schematic workflow image of the magnetic bead based immunoprecipitation procedure for sample preparation in the MS immunogenicity assay.

Robotic Processing of Samples Using Magnetic Beads. The sample processing and magnetic bead separation was performed on an automated liquid handling robot (Microlab Starlet, Hamilton, Bonaduz, Switzerland). Magnetic bead separation was achieved by using a Magnetight HT96 stand (Novagen, Nottingham, UK). For washing, incubation, and elution steps, the plate was removed from the magnet using the CO-RE Grip tool of the robot. Beads were then resuspended using a shaker, and after a specified time, the plate containing the beads was returned to the magnet for the next processing cycle. The workflow is illustrated in Figure 1. Aliquots of a protein G magnetic bead suspension (100 µL) were transferred from a reservoir into a polypropylene 96-well plate (0.5-mL well capacity) and washed twice with 0.05% Tween 20 in PBS. A sample volume of 100 µL (equating to 2 µL of serum diluted 1:50 in 0.05% Tween20/PBS) was incubated with the beads for 1 h using a shaker. The beads were washed three times with 250 µL of 0.05% Tween 20/PBS and twice with 275 µL of PBS. Elution of bound material was achieved by mixing the beads with 100 µL of 6 M guanidine hydrochloride for 5 min. The eluate was then transferred into a polypropylene 96-well elution plate compatible with the LC autosampler. A 10-µL aliquot of a cocktail of stable isotope labeled peptides stored in Protein LoBind Eppendorf tubes (hGH N terminus, 50 fmol/µL; hGH C terminus, 120 fmol/µL) was added to each well. The samples were then acidified with 20 µL of 1.8% trifluoroacetic acid followed by the addition of 10 µL of 5 M CNBr in acetonitrile to a final volume of 120 µL. The plate was sealed immediately with a pierceable 96-well cap and left for 2 h at room temperature in the dark before being placed into the tray of the LC autosampler cooled at 8 °C. Liquid Chromatography/MALDI Target Spotting. CNBr peptide digests were separated using an Ultimate 3000 capillary HPLC system (LC Packings, Dionex, Amsterdam, Netherlands) comprising a SRD-3600 solvent rack with integrated vacuum degasser, a LPG-3600 dual-gradient pump, two FLM-3100 flow manager with thermostated column compartment, a WPS-3000 thermostated well plate autosampler, and a UVD-3000 UV detector.

The system was controlled by Chromeleon software 6.7. The analysis was performed in parallel mode such that while one column in one flow manager was performing peptide separation, the other was going through wash and equilibration cycles. Solvent A was 2% acetonitrile, 0.05% trifluoroacetic acid, and solvent B was 90% acetonitrile, 0.05% trifluoroacetic acid. A sample volume of 20 µL was injected onto an in-line precolumn (5 mm × 1 mm, 5 µm, 100 Å, C18PepMap, LC Packings, Dionex) using the loading pump at 25 µL/min solvent A. The 10-port valve was switched after 7 min, and the peptides were back-eluted onto the analytical column (150 mm × 300 µm, 3 µm, 100 Å, C18PepMap, LC Packings, Dionex) using a flow rate of 1.5 µL/min at a constant temperature of 30 °C. Peptides were separated using a linear binary gradient from 0 to 56% B (7-60 min). Finally, the flow was also directed through a 45-nL UV detector cell and UV absorption at 230 nm was recorded. The LC system was interfaced directly with a Probot MALDI target plate spotting device (LC Packings, Dionex) controlled by µCarrier software version 2.0. Depending on the peptide targeted for quantification, starting between 50 and 58 min after injection, the LC eluent was deposited onto a Prespotted AnchorChip (PAC) (Bruker Daltonics, Bremen, Germany) coated with a thin layer of R-cyano-4-hydroxycinnamic acid.18 A spot was deposited every 15 s, and a total of eight spots were collected per analysis. Once all spots were dried, the PAC target was immersed for 5 s in cold 10 mM ammonium phosphate in 0.1% trifluoroacetic acid. The target was then allowed to dry before insertion into the mass spectrometer. MALDI TOF Mass Spectrometry. Mass analysis of peptides was performed using an UltraFLEX II TOF/TOF 200 mass spectrometer with Smartbeam laser technology (Bruker Daltonics). All parameters were set in Flexcontrol version 2.4. At each sample position containing the eluted peptides of interest, the mass spectra from a total of at least 10 000 laser shots were summed over a mass range from m/z 1000 to 3000. The laser frequency (18) Schu ¨ renberg, M.; Lu ¨ bbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 3436–3442.


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was 200 Hz, and the laser power remained unchanged throughout the entire experiment. Mass deflection up to m/z 750 was employed to avoid low-mass material reaching the detector. The following voltages were used: ion source 1, 25.0 kV; ion source 2, 21.8 kV; lens, 9.5 kV; reflector, 26.3 kV; and detector, 1.6 kV. Data Processing. MALDI spectra were automatically processed in batches using Flexanalysis software version 2.4 as follows. First, the spectra were internally calibrated using the monoisotopic mass of the CNBr cleavage products of the stable isotope labeled peptide (either hGH N terminus 1580.837 or hGH C terminus 2377.157). Then, spectral information including intensity, area, signal-to-noise ratio, and resolution of the signal pairs at m/z 1573.8/1580.8 (hGH N terminus) and 2372.2/2378.2 (hGH C terminus) were automatically extracted. Spectra were excluded from quantitative analysis if the signal-to-noise ratio of the stable isotope labeled peptide did not meet the following criteria: m/z 1580.8 S/N g 500 or m/z 2378.2 S/N g 100. MALDI signal intensity ratios were calculated, and absolute amounts of detected peptide derived from growth hormone or its analogue were obtained by multiplication with the concentration of the stable isotope labeled peptide. RESULTS AND DISCUSSION Design and Performance of the Quantitative LC/MALDI Workflow. Proteomics and targeted protein quantitation studies normally employ trypsin to cleave proteins with high specificity into peptides that are subsequently detected and identified by mass spectrometry.19 However, there are a number of alternative enzymatic and chemical proteolysis agents that can also be used to generate specific peptide products if suitable cleavage sites are present. For example, for the effective proteolysis of heavily pegylated proteins with one or multiple pegylation sites, a small chemical agent is better suited than an enzyme, because access to cleavage sites will not be subject to steric hindrance. Cyanogen bromide cuts hGH into four fragments via cleavage of amide bonds C terminal to methionine residues20 and was chosen for this study. While two of the generated fragments are fairly large (>5 kDa) and hence not ideal for quantitation by MS, the N and C terminal peptides are of suitable mass and can be targeted by quantitative mass spectrometry. The MALDI spectrum of a CNBr digest of a hGH standard shows that this cleavage reaction is clean and no major side products are generated (Figure 2). The N and C terminal peptides are 14 and 21 amino acids in length, respectively (N terminus FPTIPLSRLFDNAM and C terminus DKVETFLRIVQCRSVEGSCGF; internal disulfide), and contain lysine or arginine residues that enhance their mass spectrometric ionization potential. In addition, upon CNBr cleavage, the methionine residue is converted into a homoserine lactone (HSL), which remains stable at low pH.21 This conversion neutralizes the negative charge of the peptide C terminal carboxylic acid thereby further enhancing the ionization potential in positive ion mode. Although HSLterminated peptides are reported to be in equilibrium with their respective homoserine hydrolysis products,22 CNBr conversions (19) (20) (21) (22)

Veenstra, T. D. J. Chromatogr., B 2007, 847, 3–11. Inglis, A. S.; Edman, P. Anal. Biochem. 1970, 37, 73–80. Kaiser, R.; Metzka, L. Anal. Biochem. 1999, 266, 1–8. Gross, E.; Hirs, C. H. W. In Methods in Enzymology; Academic Press: New York, 1967; Vol. 11, pp 238-255.

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Figure 2. MALDI-TOF mass spectrum of a CNBr digest of a hGH standard showing N terminal and C terminal peptides and the absence of major side products. The asterisk denotes the sodium adduct of the N terminal peptide and HSL is homoserine lactone.

resulting predominately in the HSL form have been reported.23 Indeed, under the conditions employed hereins the homoserineterminated N terminal hGH peptide could not be detected. While HSL formation applies to the N terminal CNBr peptide of hGH, the C terminus of the protein is not affected by it due to the absence of a terminal methionine. The stable isotope labeled peptides used as quantification standards mimic the N and C terminal sequences of the CNBr cleavage product of hGH, FPTIP(*L)SRLFDNAML (hGH, N terminus), and MDK(*V)ETFLRIVQCRSVEGSCGF (hGH, C terminus; internal disulfide). These peptides were designed to contain one additional amino acid such that the CNBr cleavage site is incorporated into the synthetic peptide sequence, and hence, the digestion step is included in the quantitative assessment. In this way, both the hGH-derived target peptides and the stable isotope labeled standard peptides that are used for mass spectrometric detection and quantitation are generated by CNBr. This concept is illustrated in Figure 3, which shows as an example the resolved isotope envelopes of the resulting hGH N terminal peptide and stable isotope labeled standard as detected by MALDI MS. Furthermore, both the target and the standard peptide contain the homoserine lactone generated by CNBr. Figure 3 also serves to demonstrate how MALDI spectra were used for signal comparison and peptide quantitation. The most intense signal of each isotopic envelope, in this case the monoisotopic signal, of the target peptide and the stable isotope labeled standard were integrated, signal area ratios calculated and multiplied by the concentration of stable isotope standard. This facilitated the determination of the absolute concentration of the hGH-derived target peptide and extrapolation to protein equivalents enabling the comparison of amounts of retained protein therapeutic between samples. This strategy to targeted quantitation of hGH was combined with the magnetic bead based sample preparation procedure of (23) Joppich-Kuhn, R.; Corkill, J. A.; Giese, R. W. Anal. Biochem. 1982, 119, 73–77. (24) Bugelski, P.; Treacy, G. Curr. Opin. Mol. Ther 2004, 6, 10–16. (25) Linnet, K.; Kondratovich, M. Clin. Chem. 2004, 50, 732–740.

Figure 3. Expanded region of a MALDI-TOF mass spectrum showing the isotopic envelopes of N terminal hGH native and stable isotope labeled peptide standard generated by CNBr. The monoisotopic signals (9) of both envelopes were integrated and used for signal comparisons.

the MS-based immogenicity assay, which is schematically illustrated in Figure 1. Briefly, sample overspiked with excess amounts of therapeutic protein and diluted in binding buffer is added to protein G magnetic beads. Following incubation and suitable wash steps to remove unbound serum components and non-ADA bound protein therapeutic, the retained components are eluted using a chaotropic agent. Known concentrations of stable isotope labeled peptide standards mimicking hGH sequences are then added to the eluate prior to acidification and CNBr proteolysis. Peptide digests are separated by capillary LC, and fractions containing the targeted peptides are collected onto MALDI plates. MALDI spectra containing the targeted hGH peptides and their stable isotope labeled standards are utilized for MALDI signal comparison and absolute quantification. The reproducibility of the LC/MALDI component of the workflow was assessed with nine human serum samples containing 2 µg/mL positive control anti-hGH antibody in addition to 150 µg/mL hGH. The resulting CNBr digests were pooled prior to repeat LC/MALDI analysis. The ratios of the integrated areas of the monoisotopic signal from native and standard N terminal hGH peptide were determined and their average calculated to be 0.424 + 0.012. The coefficient of variation (CV) was established to be 2.84%, which is comparable with conventional liquid chromatography electrospray ionization mass spectrometry based measurements. Effect of Excess Protein Spiking Concentration. The approach used in this method is based on incubating a large excess of protein therapeutic with a serum sample in order to saturate all available specific binding sites on antibodies that may be present in the patient sample generated in response to administration of the protein therapeutic. Experimentation established an optimum protein therapeutic spike concentration that would maximize the assay response while ensuring that the

Figure 4. Assay response for the C terminal hGH peptide originating from hGHA in response to varying concentrations of positive control antibody and hGHA spiked into human serum. The high washing efficiency is illustrated even when a large excess of protein therapeutic is present. A hGHA spike concentration of 150 µg/mL was selected as the optimum spiking concentration. (n ) 3; error bars positive standard deviation)

background signal was stable; i.e., the washing conditions were stringent enough to remove excess and nonspecifically retained protein therapeutic. As an example, the result of such an experiment is shown for hGHA for which the positive control antibody exhibits a reduced affinity compared to hGH. The experiment included human serum samples that were incubated with increasing levels of protein therapeutic spike ranging from 1 to 500 µg/mL in addition to no (0 µg/mL), medium (10 µg/ mL), and high (100 µg/mL) levels of positive control anti-hGH antibody. Figure 4 shows the assay responses for measuring the C terminal CNBr fragment of hGHA from three separate bead preparations and MS experiments performed on different days. As expected, the mean assay response increases with increasing concentration of the protein therapeutic and, for the high positive control antibody, spike reaching a plateau at concentrations above 100 µg/mL. The assay response from samples without any positive control antibody present remains consistently low, even at very high hGHA concentrations demonstrating the efficiency of the washing procedure. While the washing conditions of sufficient stringency are required to remove excess, unbound protein therapeutics, the removal of specifically bound protein from ADAs with very low affinity and high dissociation rate is a possible consequence. Hence, we presume that this method is unlikely to capture low levels of low-affinity ADAs. Here, a hGHA spike concentration of 150 µg/mL was selected as the optimum spiking concentration. For each protein therapeutic of interest, this optimum spike concentration needs to be established separately, and for hGH, it was also determined to be 150 µg/mL. Expectedly, lower levels of anti-drug antibody can be saturated with lower protein therapeutic spike concentration (Figure 4); however, the use of a high spike concentration is particularly important in order to ensure that high antibody concentrations can also be adequately measured in the same experimental setup. Analytical Chemistry, Vol. 80, No. 18, September 15, 2008

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Figure 5. Representative calibration line for the determination of positive control antibody in cynomolgus serum spiked with 150 µg/ mL hGH. The graph shows the detection of N terminal hGH fragment. The error bars represent ( standard deviation (n ) 3).

This mass spectrometric immunogenicity assay enables assessment of ADA responses in sera that potentially still contain a large concentration of the protein therapeutic, i.e., up to several tens of microgram per milliliter or higher. For the analysis of preclinical or clinical samples, the implication is that following the addition of an excess protein therapeutic concentration such as 150 µg/mL the final levels in the assayed serum can be 150 up to ∼250 µg/mL depending whether the sera were taken at baseline, during the dosing period, or after washout. This range of protein therapeutic levels will saturate possible ADA binding sites while maintaining a stable low background signal. This provides a suitable means of assessing a large range of ADA concentrations in the presence or absence of excess protein therapeutic concentration. Calibration Curve for Detection of Positive Control Antibody. Preclinical immunogenicity studies are not regarded as highly predictive for clinical studies;24 nonetheless, they are performed as they may provide information on potential impact to humans and assist with the interpretation of toxicology end points. We investigated the performance and relative assay sensitivity of this method in cynomolgus serum by diluting the positive control antibody in negative cynomolgus serum across 2 orders of concentration range to 0, 50, 100, 250, 500, 1000, 2000, and 5000 ng/mL. These samples, additionally spiked with 150 µg/ mL hGH, were analyzed in triplicate. The calibration curve was

constructed by plotting the mean assay response, i.e., the amount of detected target peptide, against the concentration of positive control antibody. Due to the underlying antibody kinetics, which drives the sigmoidal shape of the calibration curve, a weighted (1/y2) unconstrained sigmoidal curve fit was used (Figure 5)). The quality of the curve fit was assessed by generating backcalculated values for the calibrants, and for the presented case, these ranged from 95.4 to 103.8% of the expected value of the prepared dilutions of the positive control antibody (Table 1. The CV values determined from the samples containing positive control antibody were found to be below 20% with an average of 14%. On a general note, the sensitivity of this immunogenicity assay observed during assay characterization strongly depends on the affinity of the positive control antibody for the protein therapeutic whose immunogenicity is assessed. Thus, different apparent assay sensitivities will be noticeable depending on the positive control antibody that is employed. However, the methodology is capable of quantifying subfemtomole amounts of peptide originating from protein therapeutic bound to specific IgG antibody, and in the example presented in Figure 5, the relative assay sensitivity was ∼50 ng/mL positive control antibody. Negative Cut-Point Determination. This methodology is intended for utilization as a screening tool, and for this a negative assay cut point can be established above which a sample is deemed to be positive. In order to determine the cut point, an adequate number of negative sera, i.e., not containing any ADA (or positive control antibody), should be analyzed. To this end, the negative cut point of the hGH assay in baseline cynomolgus serum was determined using the N terminal hGH fragment after analysis of 24 normal serum samples spiked with 150 µg/mL hGH assayed on three different days. A distribution of the individual negative assay responses of one of the analyzed batches is illustrated in Figure 6, and no outliers could be identified. All samples of this batch gave similar responses, and the mean result was found at 0.51 ± 0.16 fmol [N terminal hGH peptide] with a CV of 31%. The other two batches (data not shown) gave mean responses of 0.49 ± 0.11 or 0.43 ± 0.08 fmol [N terminal hGH peptide] with their CV being 23 or 17%, respectively. The negative cut point was determined statistically for each analysis run using the formula: Cut point ) mean negative +1.645 × SD based on the 95% upper confidence limit.7,25 Consequently, a false positive rate for the screening assay of ∼5% will be expected. The assay cut points were found to be 0.77, 0.68, and 0.56 fmol [N terminal

Table 1. Individual Assay Responses from a Positive Antibody Control Calibration Using hGH (Detection of N Terminal Peptide) in Cynomolgus Seruma assay response (fmol) positive control antibody (µg/mL)

1

2

3

mean

SD

% CV

5 2 1 0.5 0.25 0.1 0.05 0

48.1 20.7 6.58 3.08 1.56 0.97 0.80 0.68

47.3 14.3 7.05 2.37 1.26 0.75 0.71 0.54

50.8 15.7 4.95 3.18 1.20 0.76 0.62 0.39

48.7 16.9 6.20 2.87 1.34 0.83 0.71 0.54

1.8 3.4 1.1 0.4 0.2 0.1 0.1 0.1

3.8 19.9 17.8 15.4 14.2 15.4 13.3 26.6

a

Basic statistics and back-calculated values are presented.

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back-calculated positive control antibody (µg/mL)

% target

4.98 2.04 0.96 0.52 0.24 0.10 0.05

100.4 97.9 103.8 95.4 102.7 99.3 99.1

Figure 6. Representative negative cut-point determination of an antihGH immunogenicity assay in cynomolgus serum. The dashed line illustrates the calculated negative cut point.

hGH peptide]. These negative cut points, which illustrate assay sensitivity, are employed during subsequent sample analysis, but a unique negative cut point may need to be determined using samples obtained from representative subject populations for each study. Study samples with responses above the assay cut point will then be considered positive and recommended for confirmatory analysis. Case Study: ADA Determination in Cynomolgus Sera. In order to demonstrate the utility of this magnetic bead based LC/ MALDI approach, the ADA responses detectable in cynomolgus sera following a subcutaneous administration regimen of a pegylated growth hormone analogue were analyzed. In this pilot investigation aimed at demonstrating the feasibility of this assay, we determined ADA responses against hGH. A number of selected sera were used that were available from baseline, day 45 and 87 during the dosing period, and in some cases from day 56 of the recovery phase where the protein therapeutic was cleared from systemic circulation. As described above, the samples were spiked with 150 µg/mL hGH and incubated at +4 °C for 16 h to allow saturation of possible ADA binding sites.

Representative MS data from different sampling time points illustrating no, medium, and high ADA responses are displayed in Figure 7. In the presented examples, the relative quantitation of the ADA reactivity was based on the detection of the N terminal CNBr fragment of hGH and the corresponding spiked stable isotope labeled standard peptide at m/z 1580.9. For instance, Figure 7A demonstrates samples that did not exhibit any ADA responses in any of the analyzed time points. However, the presence of ADA directed against hGH of varying amplitude resulted in intense MALDI signals that could be observed in other samples taken during the dosing time points (Figure 7B, C); but assay responses below the negative cut point were observed in samples from the recovery phase. For practical reasons, it is desirable to convert the assay response from an amount of detected target peptide (in fmol) into a biologically more relevant unit. Herein we propose to use “hGH binding capacity” expressed as a concentration (in ng/mL). The values are derived by first extrapolating the molar amount of detected target peptide into mass of intact protein from which the peptide originated using the molecular weight of the protein. For this calculation to be valid, a complete yield of target peptide from the precursor protein has to be achieved. Alternatively, as in this case, the conversion of an extended stable isotope labeled peptide incorporating the cleavage site compensates for possible lower conversion efficiencies, where standard peptide and protein are converted with equal relative yields. Further incorporation of the used sample volume enables expressing the assay response as hGH binding capacity (in ng/mL), which refers to the total detected binding independent of the ADA affinities. The assay responses from all baseline samples were used to calculate the negative cut point for this small feasibility study set, which was determined to be 26.7 ng/mL hGH binding capacity (Figure 8; horizontal red line). Responses above this cut point were considered positive for ADA. Sera from the sampling time point in the recovery phase were not available in all cases, but a number of different trends in ADA

Figure 7. Determination of ADA to hGH in cynomolgus sera: Raw MALDI spectra from baseline sera, two time points during the dosing period, and the recovery phase illustrating the detection of the hGH N terminal CNBr fragment and the corresponding stable isotope labeled standard peptide. Selected examples show (A) no, (B) medium, and (C) high responses. Analytical Chemistry, Vol. 80, No. 18, September 15, 2008

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Figure 8. Overview of assay results from a feasibility study for determination of ADA to hGH in cynomolgus sera. Baseline responses were used to calculate the cut point of 26.7 ng/mL hGH binding capacity. Sera from the sampling time point in the recovery phase were not available in all cases.

response could be noted with relative signal intensity spanning more than 2 orders of magnitude (Figure 8). Some animals did not exhibit any measurable ADA levels at any of the analyzed time points (n ) 5). In contrast, ADA levels were transient and increased from baseline and became measurable in other animals, but returned below the negative cut-point value during the recovery phase if a sample was available for analysis (n ) 6). In one case (animal 4), the ADA response seemed to be persistent and was still measurable in the recovery phase. This selected data set demonstrates how this methodology can be utilized in the assessment of ADA responses and their dynamics in the presence of high levels of protein therapeutic. CONCLUDING REMARKS This study illustrates how a bead-based sample fractionation strategy hyphenated with mass spectrometry can be applied to monitor ADA responses to therapeutic proteins in the presence of high circulating concentrations of the therapeutic protein. This

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method measures total IgG responses and is intended to supplement existing methodologies for immunogenicity testing. The analysis of samples from a preclinical immunogenicity investigation demonstrated feasibility and proof of concept of this methodology. The workflow is scalable in terms of sample/injection volume and throughput and can be further developed or modified to fit existing analytical instrumentation, e.g., for bead processing and mass spectrometry. To date we have evaluated this assay with protein G based capture, realizing this limits the detection to IgG responses as other immunoglobulin subclasses do not bind to protein G.26 Furthermore, while IgGs from many species including human, monkey, rabbit, and goat bind strongly to protein G, IgGs from certain other species such as rat are only weakly or moderately bound by protein G. It is conceivable, however, that the format of this assay can be adapted to incorporate specific capture and enrichment of other immunoglobulin subclasses, particularly including early IgM-based antibody responses, for example, by utilizing antihuman IgM antibodies coupled to magnetic beads potentially allowing immunoglobulin isotyping. Similar approaches can be developed using protein A capture, antibodies directed against certain other immunoglobulin subclasses, or mixed capture phases. The adaptation of those strategies will benefit from the fact that the sample processing after bead handling and the MS component of the workflow remain unchanged. We also envisage this approach to be transferable to other protein therapeutics and also other matrixes and species. Distinct advantages are the minimal requirements for immunoaffinity capture reagents and that existing mass spectrometric approaches for the assessment of pharmacokinetic profiles of therapeutic proteins can be adapted to measure ADA response. ACKNOWLEDGMENT The authors are very grateful to Deborah Finco-Kent for valuable discussions concerning this method. The authors also thank Debbie Carter and Marina Lumbreras for providing Biacore data.

Received for review March 14, 2008. Accepted July 2, 2008. AC8005439 (26) Fahnestock, S. R. Trends Biotechnol. 1987, 5, 79–83.

Assessing Immunogenicity in the Presence of Excess Protein

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