Monoclonal Antibody Capture and Analysis Using Porous Membranes

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Monoclonal Antibody Capture and Analysis Using Porous Membranes Containing Immobilized Peptide Mimotopes Weijing Liu, Austin Landry Bennett, Wenjing Ning, Hui-Yin Tan, Joshua D Berwanger, Xiangqun Zeng, and Merlin L Bruening Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03183 • Publication Date (Web): 12 Sep 2018 Downloaded from http://pubs.acs.org on September 12, 2018

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Analytical Chemistry

Monoclonal Antibody Capture and Analysis Using Porous Membranes Containing Immobilized Peptide Mimotopes Weijing Liu1, Austin L. Bennett2, Wenjing Ning2, Hui-Yin Tan1, Joshua D. Berwanger1, Xiangqun Zeng3, and Merlin L. Bruening1,4* 1

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,

Indiana 46556, United States 2

Department of Chemistry, Michigan State University, East Lansing, Michigan 48824,

United States 3

Department of Chemistry, Oakland University, Rochester, Michigan 48309, United

States 4

Department of Chemical and Biomolecular Engineering, University of Notre Dame,

Notre Dame, Indiana 46556, United States *Corresponding author. [email protected] phone: +1 574-631-3024 Abstract Rapid, convenient methods for monoclonal antibody (mAb) isolation are critical for determining the concentrations of therapeutic mAbs in human serum. This work uses porous

nylon

membranes

modified

with

a

HER2

peptide

mimotope,

KGSGSGSQLGPYELWELSH (KH19), for rapid affinity capture of Herceptin, a mAb used to treat breast cancer. Covalent linking of KH19 to poly(acrylic acid)-containing films in porous nylon leads to a Herceptin binding capacity of 10 mg per mL of membrane and allows selective Herceptin capture from diluted (1:3) human serum in 5 min. Liquid chromatography/mass spectrometry demonstrates the high purity of eluted Herceptin. Moreover, the fluorescence intensity of the protein eluted from membranes

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increases linearly with the amount of Herceptin spiked in loading solutions containing diluted (1:3) human serum. These results demonstrate the promise of mimotope-modified membranes for Herceptin analysis that does not require secondary antibodies or derivatization with fluorescent labels. Thus, mimotope-containing membranes may form part of a simple benchtop analysis system for assessing the concentrations of therapeutic mAbs.

Monoclonal antibodies (mAbs) are the fastest growing class of pharmaceutical drugs.1-3 More than 40 antibody-related drugs are commercially available, in many cases with remarkable efficacy.1,4,5 However, antibody treatments are very expensive with a typical cost around $10,000 per month.6 Moreover, the levels of a therapeutic antibody in patient blood may vary 4-fold or more among patients, with higher concentrations potentially leading to side effects and lower levels resulting in ineffective treatment.7-9 A number of studies correlate serum antibody concentrations with patient response to mAb cancer therapies.7,10-16 Thus, methods to quickly and inexpensively determine serum mAb concentrations may help in choosing effective dosages and avoiding excessive use of expensive mAbs. A simple method for quantitation of mAbs could also facilitate determination of pharmacokinetic profiles during drug development. Current methods for monitoring specific antibody concentrations include optical immunosorbent assays,7,17-19 as well as immunosorbent assays that use detection with liquid chromatography/mass spectrometry (LC/MS),20 LC/MS-MS (after proteolysis),21,22 and monitoring of radioactively labeled antibodies.23 The MS methods often require


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expensive equipment, and standard ELISA with optical detection typically employs expensive secondary antibodies and protocols that include incubation times of 1.5 to >2 hours.24-28 This study develops mimotope-functionalized membranes that capture monoclonal antibodies in minutes. Subsequent rinsing and elution may enable antibody analysis through simple optical methods. The monoclonal antibody Herceptin binds to the human epidermal growth factor receptor 2 (HER2) on tumor cell surfaces and inhibits tumor metastasis as part of breast cancer therapies.29

Using phage display, Jiang et. al. isolated the peptide

QLGPYELWELSH as a HER2 epitope mimotope.28 Subsequently, Shang and coworkers added

a

7-amino

acid

linker

to

the

mimotope

to

create

the

peptide

CGSGSGSQLGPYELWELSH (CH19), in which the N-terminal cysteine enables immobilization on gold surfaces. They used this peptide in quartz crystal microbalance (QCM) immunosensors for Herceptin detection.30 However, the selectivity of such assays is not yet amenable to Herceptin quantitation in human serum. This work demonstrates rapid, selective capture of Herceptin in porous membranes coated with the immobilized peptide KGSGSGSQLGPYELWELSH (KH19). This peptide is similar to CH19 and contains the epitope for binding Herceptin, a 6-amino acid spacer, and a lysine residue for immobilizing the peptide to the surface through the formation of amide bonds.

Capture in porous membranes is particularly attractive

because convective flow and short radial diffusion distances in micron-sized pores31-39 allow protein binding during membrane residence times as low as 35 milli-seconds.40-42 In the present case, rapid mass transport to binding sites allows capture of Herceptin from 0.5 mL of human serum in as little as 5 min, and subsequent elution yields purified



Herceptin for direct analysis. Moreover, attachment of KH19 to polyelectrolyte multilayer films adsorbed in micropores yields a Herceptin binding capacity of ~10 mg per mL of membrane. Commercial Protein A/G columns and membranes also bind human IgG,43-45 but they are not selective for specific mAbs. The KH19-modified membranes specifically bind Herceptin from human serum diluted 1:3 with buffer, and gel electrophoresis and LC-MS show that mimotope-modified membranes exhibit low non-specific binding and effectively discriminate Herceptin from other antibodies. Measurements of native antibody fluorescence may allow quantitation of eluted antibodies within the concentration range found in patient plasma. Experimental Section Materials Hydroxylated nylon (LoProdyne LP, Pall, 1.2 µm pore size, 110 µm thick) and Aucoated Si wafers (200 nm of sputtered Au on 20 nm of Cr on Si (100) wafers) were cleaned in a UV/O3 chamber for 15 min prior to use. KGSGSGSQLGPYELWELSH (KH19) was synthesized by Selleck Chemicals with a purity over 95%. Poly(acrylic acid) (PAA, average molecular weight ~100,000 Da, 35% aqueous solution), polyethylenimine (PEI, branched, Mw=25,000 Da), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 3-mercaptopropionic acid (MPA, 99%), Tween-20 surfactant and human serum were used as received from Sigma Aldrich. Poly(vinyl) alcohol (PVA, 99–100 % hydrolyzed, approximate molecular weight 8600 Da) was purchased from Acros Organic. Coomassie protein assay reagent was obtained from Thermo Scientific, and Herceptin and Avastin in their therapeutic formulations were


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gifts from Dr. Mohammad Muhsin Chisti of Michigan State University. Buffers were prepared using analytical grade chemicals and deionized water (Milli-Q, 18.2 MΩ cm). Immobilization of KH19 in porous nylon membranes After exposure of nylon membranes to UV/O3 for 10 min, 20 mL of 0.01 M PAA (in 0.5 M NaCl, pH 3, PAA concentration is that of the repeating unit) was circulated through the membrane for 20 min, followed by passage of 20 mL of water through the membrane. Next, a solution containing 2 mg/mL of branched PEI (no NaCl, pH 3) was circulated through the membrane for 20 min, followed again by passage of 20 mL of water.

A second layer of PAA was deposited on top of branched PEI using the

conditions for adsorption of the first layer. Subsequent immobilization of the KH19 peptide to the (PAA/PEI/PAA)-modified membranes followed a literature procedure.42 Five mL of 0.1 M NHS, 0.1 M EDC was circulated through the membrane for 1 h, followed by passage of 20 mL of water. Finally, a pH ~9 solution containing 1 mg/mL of KH19 was circulated through membranes for 1 h, followed by passage of 20 mL of water or a 10 µM NaOH (pH=9) solution. In all modification procedures, the flow rate through the membrane was 1 mL/min, and the membrane surface area was 3.1 cm2 (exposed membrane diameter of 2 cm). Membrane modification used single membranes and the holder shown in Figure S1 of the supporting information. Adsorption of Herceptin and Avastin in KH19 modified nylon membranes Single membranes (exposed diameter of 2 cm, membrane volume of 0.035 cm3) modified with KH19 were washed with 20 mL of 20 mM phosphate buffer (pH=7.4) containing 150 mM NaCl. A solution containing 0.1 mg/mL of Herceptin or Avastin in the same buffer solution was passed through the membrane, and effluent aliquots were



collected at a flow rate ~0.5 mL/min. The Herceptin and Avastin concentrations in the effluent aliquots were determined using Coomassie protein assay reagent with calibration curves prepared using 0, 0.02, 0.04, 0.06, 0.08, and 0.1 mg/mL mAb concentrations.46 Using the concentrations determined for effluent aliquots, we plotted breakthrough curves that show the effluent concentration versus the total loading-solution volume passed through the membrane. The adsorption isotherm of Herceptin was determined from binding capacities at different feed concentrations (0.01, 0.02, 0.05, 0.1, and 0.2 mg/mL) in 20 mM phosphatebuffered saline (300 mM NaCl, pH 7.4). At a flow rate of 1 mL/min, 9 mL of Herceptin solution was passed through KH19 modified membranes (3.1 cm2 external area), followed by 10 mL of a phosphate buffer (500 mM NaCl, pH 7.4) wash at flow rate of 1 mL/min. The Herceptin feed, effluent, and wash solutions were collected and analyzed using fluorescent spectroscopy (Synergy H1 Microplate Reader) to determine the Herceptin binding capacity. Using a calibration curve based on standards in loading buffer, samples were analyzed with fluorescence spectroscopy (emission wavelength of 270 nm and determination of the maximum intensity around 330 nm). Capture of Herceptin from human serum for gel electrophoresis Human serum diluted 1:3 in phosphate buffer was spiked with Herceptin to a concentration of 100 µg/mL. One mL of the spiked, diluted serum was passed through a KH19-modified membrane (external area of 3.1 cm2) in 10 min, followed by washing with 10 mL of phosphate buffer containing 150 mM NaCl, washing with 10 mL of phosphate buffer containing 500 mM NaCl, and elution with 4 200-µL aliquots of 2% sodium dodecylsulfate (SDS) containing 100 mM dithiothreitol (DTT). Thirty µL of


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eluate aliquots were loaded on 4-20% gradient SDS-polyacrylamide gel electrophoresis (PAGE) gels.

Mass spectrometry analysis of eluates from KH19-modified membranes For the eluates mentioned in the prior subsection, SDS was removed with a Pierce Detergent Removal Spin Column (Thermo) according to the manufacturer’s instructions. For antibody reduction, 10 µL of 0.1 M TCEP in 10% HOAc was added to 100 µL of the eluate aliquot, and the mixture was incubated at 75 oC for 15 min. LC-MS analysis of the reduced sample employed an Agilent Poroshell C3 column to separate heavy and light chains during a 20 min gradient from 5-50% B on an UltiMate 3000 HPLC system (Thermo) flowing at 0.4 mL/min. Solvent A was 0.1% formic acid (FA) in H2O, and solvent B was 0.1% FA in ACN. MS was performed on a micrOTOF-Q II instrument (Bruker). The second 100 µL of an eluate aliquot was reduced and alkylated prior to insolution digestion. (Section S5 in the supporting information describes the detailed procedures.) The digests were then desalted with a C18 spin column and dried with a SpeedVac before reconstitution in 0.1% FA for LC-MS/MS analysis. Nano-Ultra High Performance Liquid Chromatography MS/MS was performed to identify nonspecific binding species. Injections (2 µL) corresponding to ~300 ng of insolution digested tryptic protein (reconstituted in 0.1% FA) were loaded onto a 100 mm x 75 µm C18-BEH column (Waters Billerica, MA) and separated over a 90 min gradient from 5-35%B on a nano-Acquity system (Waters) flowing at 500 nL/min. Solution A was



0.1% FA in H2O, and solution B was 0.1% FA in ACN. MS/MS was performed on a QExactiveTM Hybrid Quadrupole-OrbitrapTM instrument (Thermo, San Jose, CA) running a top-20 data-dependent method, where a single MS at a resolution of 70,000 was acquired, and the top-20 precursors were selected for fragmentation. Raw LC-MS/MS files were processed by MaxQuant version 1.5.6.0. MS/MS spectra were searched against the Human serum proteome (618 proteins). The database also included common contaminants and Herceptin sequences. MaxQuant analysis parameters included a precursor mass tolerance of 20 ppm for the initial search, a precursor mass tolerance of 6 ppm for the main search, and an FTMS MS/MS match tolerance of 20 ppm. We set trypsin as the specific enzyme. Variable modifications included methionine oxidation (M), deamidation (NQ), and Gln->pyro-Glu, while the fixed modification was carbamidomethyl on cysteine. The minimal peptide length was set to 6 amino acids, the maximum peptide mass was 10,000 Da, and the maximum number of missed cleavages was 10. Quantitation of Herceptin in human serum Human serum diluted 1:3 in 20 mM phosphate buffer (pH=7.4, 150 mM NaCl) was spiked with Herceptin to concentrations of 0, 10, 30, 60, 80, 120 µg/mL. Spiked diluted serum (0.5 mL) containing different amounts of Herceptin was passed through a stack of 3 KH19-modified membranes (the top surface area of each membrane was 0.78 cm2, and the total membrane volume was 0.026 mL). The membrane was rinsed with 10 mL of phosphate buffer containing 150 mM NaCl, 0.05% Tween-20 and 0.5 % PVA, followed by 10 mL of the same buffer with 500 mM NaCl. Herceptin was recovered in 2 % SDS containing 100 mM DTT in 600 µL aliquots. The flow rates were 0.1 mL/min for


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loading and elution and 1 mL/min for washing. Eluate fractions were analyzed using fluorescence spectroscopy with excitation at 280 nm and emission scanned from 310 – 400 nm. The amount of Herceptin in the first 1.2 mL of eluates was determined using a calibration curve obtained with Herceptin standards in 2 % SDS and 100 mM DTT.

Results and Discussion This section first describes immobilization of the KH19 mimotope in PAAmodified porous nylon. Subsequently, we examine breakthrough curves for Herceptin binding to these membranes to determine the binding capacity and adsorption isotherm, and also show selective binding of Herceptin over another mAb, Avastin. Capture of Herceptin from human serum and LC-MS/MS analyses of the membrane eluate further show the high selectivity of these membranes for Herceptin. Finally, elution of bound Herceptin and determination of the eluate fluorescence yield linear calibration curves that demonstrate the potential of mimotope-modified membranes for label-free Herceptin analysis. Quantitation

of

KH19

immobilization

in

PAA/PEI/PAA-modified

nylon

membranes. Immobilization of the KH19 peptide in porous nylon membranes includes adsorption of a PAA/PEI/PAA film in the pores (and on the exterior surfaces) followed by activation of the adsorbed PAA using EDC/NHS chemistry and reaction with the terminal lysine group of KH19 (Figure 1). Previously we used a similar procedure to anchor



Nylon Pore

COOH COOH

1. PAA 2. PEI 3. PAA

KGSGSGSQLGPYELWELSH COOH

1. NHS/EDC 2. KGSGSGSQL GPYELWELSH

COOH

Page 10 of 24

KGSGSGSQLGPYELWELSH

COOH

COOH

COOH PAA: poly(acrylic acid) PEI: branched polyethylenimine

KGSGSGSQLGPYELWELSH

NHS: N-Hydroxysuccinimide EDC: 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

Figure 1. Scheme of the modification of porous nylon with a PAA/PEI/PAA-KH19 film.

aminobutyl nitrilotriacetate in nylon membranes.42 FTIR spectra (supporting information Figure S2) confirm the activation procedure and the peptide immobilization on Au surfaces. To determine the amount of KH19 binding in PAA/PEI/PAA-modified nylon membranes, we compared the fluorescence intensity of KH19 loading and effluent solutions (see supporting information Figure S3). The activated PAA/PEI/PAA-modified nylon membrane captures almost half of the KH19 peptides in the loading solution to give 25±4 mg of peptide per mL of membrane. The molecular mass of the KH19 peptide is 2032 g/mole, so if every immobilized peptide bound one antibody (molecular mass of 150 kDa), the membrane would capture ~1800 mg of antibody per mL of membrane. Such a high binding capacity is not possible because the density of pure protein is around 1000 mg/mL, and the membrane porosity is only 50%. Thus, most mimotopes will not bind an antibody due to steric constraints.

However, the KH19 immobilization is

sufficient to potentially bind nearly all of the mAb in patient sera (see below). Herceptin

and

Avastin

binding

in

KH19-PAA/PEI/PAA-modified

membranes.


nylon

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After determining the amount of KH19 immobilization in PAA/PEI/PAAmodified membranes, we examined Herceptin binding to these membranes using breakthrough curves. Figure 2 shows that the modified membranes initially capture essentially all of the Herceptin, but as the membrane begins to saturate the mAb “breaks through”. Integration of the difference between the feed concentration and the effluent concentration in Figure 2 gives the binding capacity.

The breakthrough curve is

relatively sharp as the binding capacity at 10% breakthrough is approximately 50% of the equilibrium binding capacity, which is ~10 mg/mL based on the entire breakthrough curve. Figure 2 also shows a control experiment that examines the capture of the monoclonal antibody Avastin. Both Avastin and Herceptin are humanized antibodies and have essentially the same constant regions.

However, because KH19 binds to the

complementarity determining region of Herceptin, membranes modified with this mimotope should not bind Avastin. The Avastin breakthrough curve shows negligible binding, indicating both low dead volume in the system and minimal non-specific binding of this antibody.



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Figure 2. Breakthrough curves for Herceptin and Avastin adsorption in membranes modified with (PAA/PEI/PAA)-KH19 films. Loading solutions contained 0.1 mg/mL of either antibody in phosphate buffer, and the outer surface area of the membrane was 3.14 cm2 (diameter of 2 cm).

Herceptin adsorption isotherm To assess the affinity of the KH19-modified membranes for Herceptin, we determined binding capacities at a range of Herceptin concentrations. As Figure 3 shows, the concentration-dependent binding capacities fit reasonably well to a Langmuir adsorption isotherm. Eq. 1 describes this isotherm where ‫ ݍ‬is the observed binding capacity at a given mAb concentration, ‫ݍ‬଴ is the number of available binding sites, and ‫ܭ‬௔ is the association constant. ‫ݍ = ݍ‬଴

௄ೌ [ு௘௥௖௘௣௧௜௡]

(1)

ଵା௄ೌ [ு௘௥௖௘௣௧௜௡]

Fitting of the data in Figure 3 yields values of 2.9 x 106 M-1 and 11.8 mg of Herceptin per mL of membrane for ‫ܭ‬௔ and ‫ݍ‬଴ , respectively. This value of ‫ܭ‬௔ corresponds to a dissociation constant, ‫ܭ‬ௗ , of 345 nM between Herceptin and the immobilized mimotope, which is higher than the 5 nM ‫ܭ‬ௗ reported in cell-based Herceptin-HER2 affinity assays.47 The relatively low affinity of the modified membrane for Herceptin may stem from either steric constraints on binding to the immobilized mimotopes or a lower affinity for the mimotope than native antigens. However, these affinities still allow Herceptin capture at µM concentrations, and nM affinities may make elution difficult.


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12

Binding Capacity (mg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10 8 6 4 2 0 0

0.2

0.4 0.6 0.8 1 Feed Concentration (µM)

1.2

1.4

Figure 3. Equilibrium Herceptin binding capacities as a function of Herceptin concentration for KH19-modified membranes. The line shows the best fit Langmuir isotherm and error bars represent standard deviations of the binding capacities obtained with three replicate membranes. Capture of Herceptin from human serum. KH19-modified membranes successfully isolate Herceptin from human serum diluted 1:3 with phosphate buffer. Figure 4 shows the SDS-PAGE analysis of four consecutive eluate aliquots (lanes 6 to 9) from a membrane loaded with diluted human serum containing 100 µg/mL of Herceptin. Notably, the dominant bands in the eluate stem from the heavy and light chains of Herceptin, and nearly all of the eluted mAb appears in the first two eluate aliquots.



Figure 4. SDS-PAGE analysis showing selective capture of Herceptin from human serum diluted 1:3 with phosphate buffer; Lanes 1 and 4: Molecular weight markers; Lanes 2 and 3: Herceptin-spiked serum before and after passing through the membrane, respectively (the spiked 1:3 diluted serum was further diluted 1:9 prior to loading 30 µL of the diluate for SDS-PAGE). Lane 5: 3 µg of Herceptin standard; Lanes 6 to 9: 30 µL from 4 consecutive eluate aliquots (2% SDS and 100 mM DTT, each aliquot had a total volume of 0.2 mL) from a KH19-modified membrane loaded with one mL of diluted human serum spiked to a final concentration of 100 µg/mL of Herceptin. Later eluates (lanes 8 and 9) contain minimal protein.

Figure 5. LC-MS analysis confirms the identity of the IgG eluted from the KH19modified membrane as Herceptin. (A) UV chromatogram (orange plot) of eluted protein showing the light chain and heavy chain of Herceptin; (B,C) Charge envelopes of light and heavy chains, respectively; (D, E) Deconvoluted mass spectra of Herceptin light (LC) and heavy (HC) chains, respectively.


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To verify that the KH19-modified membrane binds Herceptin and not other IgG antibodies in human serum, we reduced the eluted antibody and performed LC-MS analysis. The liquid chromatogram (UV detection, Figure 5(A)) of the eluate shows dominant peaks for the Herceptin light and heavy chains. Mass spectra verify that the strong bands in the chromatogram stem from the light and heavy chains of Herceptin and not other antibodies (Figures 5(B) and (C)). The deconvoluted average mass of the envelope in Figure 5(B) is 23443 Da (Figure 5(D)), which is equal to the average mass of Herceptin light chain (LC). The deconvoluted spectrum of Figure 5(C) give peaks at 50601 Da, 50763 Da and 50925 Da (Figure 5(E)) which are the average masses of the Herceptin heavy chain (HC) plus G0F, G1F and G2F glycosylation units, respectively. G0F, G1F, and G2F denote glycosylation with a polysaccharide containing 4 Nacetylglucosamine units, 1 fucose unit, and three mannose units and addition of 0, 1, and 2 galactose units, respectively. The deconvoluted peaks are separated by 162 Da which is the mass of a galactose unit.

Identification of proteins that show nonspecific binding to modified membranes Although the mass spectra in Figure 5 suggest highly pure Herceptin in the eluate, SDS-PAGE revealed some weak bands for proteins other than the heavy and light chains (see Figure 4, lane 6). Identification of the non-specifically adsorbed proteins may aid in future studies aimed at reducing the amounts of these proteins in the eluate. To identify such proteins, we digested the eluted antibodies and accompanying impurities and performed LC-MS/MS analysis. The process included removal of surfactant from the eluate, alkylation, digestion, and desalting followed by LC-MS/MS analysis (see the



Page 16 of 24

experimental section). The peptides identified in LC-MS/MS give 100% amino acid coverage of the Herceptin light chain and 78.4% coverage of heavy chain. Table 1 lists other identified proteins that have an Andromeda score above 70.48 The molecular weights of some of these non-specific binding proteins (albumin, Histidine-rich glycoprotein, Clusterin and Vitronectin) are in the 50-70 kDa range and likely correspond to the bands above 50 kDa in the SDS-PAGE analysis of the eluate (Figure 4, lane 6). Even though LC with UV detection could not detect these nonspecific-binding proteins before digestion (see Figure 5A) due to their low abundance, LC-MS/MS can identify their tryptic peptides after digestion. MaxQuant label free quantification analysis indicates that 70% of the normalized intensity of peptide ions stems from Herceptin. This is a remarkable purity considering that the 1 mL of diluted serum contains approximately 20-30 mg of total protein and only 100 µg of Herceptin. In summary, SDS-PAGE and LC show that KH19-containing membranes specifically capture Herceptin. This is a distinct advantage over affinity chromatography with protein A columns that bind to the Fc region of all IgG antibodies.

Table 1. Proteins identified from LC-MS/MS analysis of the eluate from a membrane loaded with 100 µg/mL of Herceptin in human serum diluted 1:3 with phosphate buffered saline. The table also gives the protein molecular weights (MW), sequence coverages and Andromeda scores from LC-MS/MS. Proteins

Sequence coverage/%

MW/kDa

Score

Herceptin Lc

100

23.4

249

Herceptin Hc

78.4

49.2

323

Albumin

70.3

69.5

175


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Apolipoprotein E

63.1

36.2

147

Complement C3

48.6

187.2

321

Immunoglobulin heavy constant mu

41.3

49.4

75

Histidine-rich glycoprotein

40.4

59.6

179

Clusterin

30.5

52.5

80

Vitronectin

27.4

54.3

79

Quantitation of Herceptin in human serum After capture of Herceptin from serum, elution may enable simple native fluorescence analysis of this mAb if it is sufficiently pure. Thus, we determined the fluorescence of the eluate from membranes loaded with different concentrations of Herceptin and estimated the amount of protein in the eluate using a calibration curve based on Herceptin standards prepared in the elution buffer. In this case, we employed stacks of 3 membranes to ensure capture of nearly all of the antibody. For the spiked 1:3 diluted serum, we examined Herceptin concentrations from 10-120 µg/mL. In a prior study, the concentration range of Herceptin in patients’ undiluted plasma was 20 to 440 µg/mL.25 Notably, Figure 6 shows that the total protein recovered from membranes loaded with spiked diluted serum varied linearly with the spiked Herceptin concentration. Washes containing 0.05 % Tween-20 and 0.5 % PVA help to remove contaminant proteins (Coomassie stained gel; data not shown). However, the non-zero intercept in Figure 6 suggests some non-specific adsorption of contaminant proteins in serum, even with stringent washes. Nevertheless, the curve demonstrates the feasibility of using the native fluorescence of eluted Herceptin to determine its concentration. In contrast to ELISA, this method requires no secondary antibody and no oxidation of dyes.



Loaded Herceptin (µg/ml) 0

Total Protein Detected (µg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20

40

60

80

100

120

70 60 50 40 30

y = 0.83 x + 8.8 R² = 0.97

20 10 0 0

10

20

30

40

50

60

Loaded Herceptin (µg)

Figure 6. Total protein detected in the eluate from membranes loaded with different amounts of Herceptin spiked in 0.5 mL of diluted serum.

The total protein was

determined using the eluate fluorescence compared to the fluorescence of Herceptin standards prepared in the elution buffer.

Error bars show standard deviations of

experiments with three replicate membranes.

Conclusions Covalent linking of lysine-terminated peptide mimotopes to adsorbed PAA provides a simple method for creating membranes with high affinity for specific mAbs.

The

membranes show low non-specific adsorption and high selectivity for Herceptin in the presence of other mAbs or other antibodies in human serum. Herceptin capture from 0.5 mL of solution can occur in 5 min or less, but rinsing and elution require longer times. Capture and elution followed by fluorescence analysis allows determination of Herceptin concentrations over the expected range in diluted serum without the use of secondary antibodies. Future studies with spin membranes or membranes in 96-well plates may afford convenient mAb analyses in

Monoclonal Antibody Capture and Analysis Using Porous Membranes

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