Article pubs.acs.org/ac
Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
Strategy for Generating Sequence-Defined Aptamer Reagent Sets for Detecting Protein Contaminants in Biotherapeutics James B. McGivney, IV,†,⊥ Andrew T. Csordas,‡,⊥ Faye M. Walker,‡ Elizabeth R. Bagley,§ Emily M. Gruber,‡ Peter L. Mage,§ Jose Casas-Finet,† Margaret A. Nakamoto,§ Michael Eisenstein,‡ Christopher J. Larkin,† Robert J. Strouse,† and H. Tom Soh*,§,∥ †
BioPharmaceutical Development, MedImmune, LLC, Gaithersburg, Maryland 20878, United States Institute for Collaborative Biotechnologies, University of California, Santa Barbara, California 93106, United States § Department of Electrical Engineering, Stanford University, Stanford, California 94305, United States ∥ Department of Radiology, Stanford University, Stanford, California 94305, United States ‡
S Supporting Information *
ABSTRACT: Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line. We identified a set of 32 DNA aptamers that achieve better sensitivity than a commercial pAb reagent set and can detect a comparable number of HCPs over a broad range of isoelectric points and sizes. Importantly, these aptamers detect multiple contaminants that are known to be responsible for therapeutic antibody degradation and toxicity in patients. Because HCP aptamer reagents are sequence-defined and chemically synthesized, we believe they may enable safer production of biologic drugs, and this strategy should be broadly applicable for the generation of HCP detection reagents for other cell lines.
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number of potentially contaminating species is likely to be considerably larger when different isoforms from splice variants and post-translational modifications are taken into account. Furthermore, the concentration of each protein species can vary widely,9 spanning four to five orders of magnitude.10 Finally, HCP detection must be achieved in the presence of a vast excess of the biologic drug. Polyclonal antibody (pAb)-based immunoassays are the current gold standard for detecting HCPs (as reviewed in Hogwood et al.11 and Zhu-Shimoni et al.12). Briefly, pAbs are generated in animals such as sheep, goats, or rabbits using host cell lysate as the immunogen. After administering serial doses of lysate to boost the animals’ immune response,13 the resulting pAbs are extracted and purified via affinity chromatography for use in enzyme-linked immunosorbent assays (ELISA) or Western blots.14 Unfortunately, pAb reagents suffer from a number of critical shortcomings. First, pAbs can only recognize
iologic drugs such as monoclonal antibody therapeutics are the fastest-growing category of drugs.1 Biologics are typically produced in mammalian host cells and therefore require a purification procedure that can efficiently eliminate host cell proteins (HCPs).2 Incomplete HCP removal can have serious consequences for drug safety and efficacy. For example, HCPs can elicit a dangerous immune response, and such problems have resulted in the suspension of clinical trials.3,4 HCPs can also introduce proteases and other contaminants that cause drug degradation5 or neutralization of activity via antitherapeutic antibodies.3 Accordingly, the US Food and Drug Administration (FDA) and other regulatory agencies require rigorous process control to monitor and control for the presence of HCPs and thereby ensure the purity of biologic drugs.6 Sensitive and reliable detection of HCPs is a highly challenging technical task.7 First and foremost, this is because of the very large number of potentially contaminating protein species. For example, the genome of Chinese hamster ovary (CHO) cells, a standard host for biologic drug production, contains more than 24 383 predicted genes.8 The actual © XXXX American Chemical Society
Received: November 18, 2017 Accepted: January 18, 2018
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DOI: 10.1021/acs.analchem.7b04775 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry HCPs that elicit a sufficient immune response,7,12,15 and several different animal species are sometimes used to improve antibody coverage.2 Second, pAbs may not exhibit sufficient affinity to achieve detection of low-abundance HCP contaminants in immunoassays.12 Finally, the composition of a pAb pool is inherently undefined,16 and thus, it is not possible to consistently obtain the same reagents.17,18 Thus, there remains an urgent need for more robust tools for HCP screening. Here, we describe a strategy for generating a defined set of nucleic acid aptamers for the reproducible and sensitive detection of a broad spectrum of CHO HCPs. Our aptamers overcome the shortcomings associated with pAb-based reagent sets. First, because they are discovered through in vitro screening rather than an in vivo biological process, they are capable of recognizing both immunogenic and nonimmunogenic proteins. Additionally, aptamers can be chemically synthesized in a highly reproducible fashion, which means that the exact same set of aptamers can be consistently produced.17 Finally, because the sequence of every aptamer in the reagent set is known, the composition of the aptamer reagent set can potentially be tuned to achieve maximum detection performance. We developed an HCP aptamer discovery strategy that enabled us to generate a set of 32 DNA aptamers that can detect a similar number of CHO protein species relative to a commercial pAb reagent. Importantly, these aptamers achieve a higher sensitivity (i.e., lower limit of detection) in detecting HCPs relative to the pAb reagents. We further show that our aptamer set can detect proteins spanning a wide range of isoelectric points and molecular weights, closely mirroring the natural distribution seen in the mammalian proteome. Finally, we show that our aptamers can detect common antibodybinding contaminants and enzymes that potentially degrade mAb drugs. Collectively, our data suggest that an aptamerbased reagent pool might be generally superior to pAbs as a means for sensitively and reproducibly detecting a broad and chemically diverse range of contaminant proteins.
Aptamer Enrichment. Prior to particle display, we performed two rounds of SELEX with biotinylated HCP (bHCP) bound to streptavidin-coated magnetic beads. Biotinylated HCP was prepared by covalently linking NHSPEG4-biotin (ThermoFisher Scientific) to 100 μg of HCP, following the manufacturer’s instructions. The bHCP was then conjugated to 1 μm streptavidin-coated magnetic beads (ThermoFisher Scientific) following the manufacturer’s protocol and stored at 4 °C. One nanomole of the ssDNA library was heated to 95 °C for 10 min, cooled on ice for 10 min, and then held at room temperature for 5 min. This ssDNA library was incubated with a final concentration of 10 μL of the stock concentration of HCP beads in a final volume of 100 μL of PBSMCT (1× DPBS, 1.5 mM MgCl2, 0.9 mM CaCl2, 0.01% Tween-20, pH 7.4). The ssDNA library and HCP−bead mixture was incubated for 1 h on a rotator at room temperature, after which the beads were washed 3 times for 3 min in PBSMCT. Enriched aptamers were eluted by heating the beads in 50 μL of water for 10 min at 95 °C. The aptamers were PCR amplified in preparation for the second round of selection. Amplification was performed with a phosphorylated reverse primer, after which Lambda exonuclease (New England Biolabs) was used to digest the phosphorylated strand of the dsDNA, yielding an ssDNA product for the next round of selection. In the second round, we combined 0.01 nmol of ssDNA with 10 μL of stock concentration bHCP-coated streptavidin beads in a 100 μL final reaction volume. This solution was incubated for 1 h at room temperature, after which the beads were washed and heat eluted as described above and then prepared for the first round of particle display. Aptamer Particle Preparation. The aptamer particles were made as previously described.19 Aptamer particles were made with 500 μL of MyOne 1 μm carboxylic acid magnetic particles (107 particles/μL; Life Technologies). The particles were washed with 500 μL of 10 mM NaOH and three times with 1 mL of nuclease-free water. The particles were then resuspended in a 150 μL solution containing 0.2 mM 5′-aminomodified forward primer (5′-amino-PEG18-CCC TGC GTT TAT CTG CTC TC-3′), 200 mM NaCl, 1 mM imidazole chloride, and 250 mM 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC) (Pierce Biotechnology) in 50% v/v dimethyl sulfoxide. Following resuspension, the particles were vortexed, sonicated, and placed on a rotator for overnight incubation at room temperature. To minimize nonspecific binding of HCPs, particle surfaces were passivated with PEG12 molecules. This was accomplished by incubation with 100 mM N-hydroxysuccinimide (NHS) and 250 mM EDC in a pH 4.7 100 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer at room temperature for 30 min. Next, 20 mM amino-PEG (Pierce Biotechnology) was conjugated to the particles for 1 h in MES buffer. Unbound amino-PEG12 particles were removed by 3 washes with pH 7.5, 250 mM Tris buffer containing 0.01% Tween-20. The FP conjugated particles were stored at 4 °C in 500 μL of the same buffer. To generate monoclonal aptamer particles, we carried out emulsion PCR19 with an aqueous phase of 1× GoTaq PCR Master Mix (Promega), 25 mM MgCl2, 3.5 mM of each dNTP (Promega), 40 nM FP, 3 μM reverse primer (RP), 0.25 U/μL of GoTaq Hot Start Polymerase (Promega), 1 pM template DNA, and 2 × 108 FP-coated particles, in a total volume of 1 mL. Before each experiment, an oil phase was made from 95% mineral oil, 4.5% Span 80, 0.45% Tween 80, and 0.05% Triton X-100 (each purchased from Sigma-Aldrich). Dropwise
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MATERIALS AND METHODS CHO HCP and anti-CHO HCP Polyclonal Antibody. A null CHO host cell line was grown using standard bioreactor conditions at the 50 L scale and harvested by continuous centrifugation. The resulting harvest was diafiltered into PBS, aliquoted, and stored at −80 °C. We also purchased a commercially available anti-CHO HCP polyclonal antibody reagent from Cygnus Technologies (3G-0016-AF). Aptamer Library and Amplification Conditions. The library, primers, and aptamers were purchased from Integrated DNA Technologies. The library sequence was 90 bases in length, containing two 20-base primer binding regions and a 50-base random region: 5′-CCCTGCGTTTATCTGCTCTC[50N]-GAACATTACATTGACGCAGG-3′. All individual aptamer sequences used for flow cytometry and affinity chromatography experiments were amino-modified, full-length 90-mer sequences. Thermal cycling conditions used for amplification of the aptamer candidate sequences in between rounds were as follows: 95 °C for 2 min, followed by repeated cycling at 93 °C for 30 s, 63 °C for 30 s, and 72 °C for 30 s. The number of cycles used for amplification between each round was determined by a pilot PCR reaction. The thermal cycling conditions used for amplification of aptamer candidate sequences in emulsion PCR were: 95 °C for 2 min, followed by 50 cycles of 93 °C for 15 s, 63 °C for 30 s, and 72 °C for 75 s. B
DOI: 10.1021/acs.analchem.7b04775 Anal. Chem. XXXX, XXX, XXX−XXX
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colonies were sent to GeneWiz for Sanger sequencing. We obtained 119 sequences and used Geneious software for analysis. Microarray Analysis. Selected sequences with deleted primer regions were in situ synthesized onto an array (MYcroarray, Ann Arbor, MI). The 50-mer sequences from the HTS data were ranked based on copy number, and the top 6000 sequences were synthesized in triplicate on a single slide at known locations. The slide was blocked with 0.5% fetal bovine serum (Fisher) in PBSMCT at room temperature for 4.5 h and then washed in 25 mL of PBSMCT for 1 min. The slide was then incubated with 5 μg/mL of bHCP in PBSMCT containing 100 μg/mL salmon sperm DNA (Invitrogen) and 10 μg/mL acetylated bovine serum albumin (acBSA, Promega) for 7.5 h at room temperature. The slide was washed again with 25 mL of PBSMCT for 1 min. Next, the slide was labeled with 10 nM streptavidin−Alexa Fluor 647 conjugate (Thermo Fisher Scientific) for 40 min at room temperature. The slide was then washed with PBSMCT for 5 min, PBSMC for 5 min, and then with water.20 The slide was dried using an array spinner (ArrayIt), then scanned with a GenePix 4400A microarray scanner (Molecular Devices) at 2.5 μm resolution. The fluorescence intensity of all features on the slide was quantitated; the median fluorescence intensity from each triplicate of individual aptamers was averaged, and the resulting fluorescence intensities were plotted. Aptamer families were determined by using the Tree Building function in Geneious using the neighbor-joining method. Affinity Chromatography. Two prepacked 5 mL HiTrap NHS-activated HP Sepharose columns (GE) were conjugated with either anti-CHO HCP pAbs or the selected 90-mer aptamers according to the manufacturer’s instructions, unless otherwise stated. The aptamers were conjugated to the column in a 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3 buffer, while the antiCHO HCP pAbs were conjugated to the column in 1× PBS. The pAb column was prepared such that it would have the same number of binding sites as the aptamer column. The average molecular weight of the 90-mer aptamers was 28 kDa, whereas the average molecular weight of pAb IgG is 150 kDa. However, there are two binding sites for each IgG molecule, making the effective molecular weight 75 kDa per binding site. Therefore, the molecular weight ratio between the pAb and aptamers per binding site was 2.68, and we aimed to conjugate 2.68 times as much pAb by mass as aptamer on each column. We determined the amount of 3′-end amino-modified aptamer and pAb conjugated to each column by using the Qubit DNA assay and Qubit Protein Assay (Thermo Fisher Scientific), respectively. We loaded 17.0 mg of DNA aptamers onto the column and determined that 2.9 mg were actually conjugated to the column. We used 8.7 mg of pAb solution for conjugation and determined that 7.1 mg were conjugated. By mass, this resulted in a pAb to aptamer ratio of 2.45, reasonably close to the value of 2.68 that we sought to achieve. Chromatography was performed according to the manufacturer’s protocol. Flow rates were manually adjusted as necessary with a Harvard syringe pump. Each column was equilibrated with 10 column volumes (CVs) of PBSMCT during the aptamer experiment, and with 10 CVs of PBS for the pAb experiment, prior to loading. HCPs were diluted to 0.15 mg/ mL in 45 mL of PBSMCT or PBS, and this HCP mixture was loaded onto the column at a flow rate of 1 mL/min. This flowthrough was retained for additional cycles of affinity chromatography. The column was then washed with 5 CVs
addition of 1 mL of the aqueous phase into 7 mL of the oil phase in a DT-20 tube (IKA) attached to an Ultra-Turrax Device was carried out to generate the water-in-oil emulsions. The dropwise addition took place over 30 s, as the solution was continuously stirred at 620 rpm for a total of 5 min. Following this step, we took approximately 80, 100 μL aliquots of the emulsion solution and added this to a 96-well PCR plate for 50 cycles of amplification. The individual reactions were then collected by centrifugation at 500g for 2 min into an emulsion collection tray (Life Technologies). Emulsions were broken by addition of 10 mL of 2-butanol to the collection tray. This solution was transferred to a 50 mL tube, vortexed for 30 s, and then pelleted by centrifugation for 5 min at 3000g. Following removal of the oil phase, the particles were transferred to a new 1.5 mL tube in 600 μL of pH 7.5 emulsion breaking (EB) buffer (10 mM Tris, 100 mM NaCl, 1% Triton X-100, 1% SDS, and 1 mM EDTA). The particles in the new tube were vortexed for 30 s and centrifuged at 21 000g for 90 s, followed by removal of the supernatant. Particles were washed one more time in EB buffer and then resuspended in 100 μL of 10 mM Tris, pH 7.5 buffer. To generate ssDNA particles, the Tris buffer was removed from the beads by magnetic separation, and the beads were incubated with 100 μL of 100 mM NaOH for 20 min on a rotator. This NaOH incubation was repeated once, and then the beads were washed twice with 100 μL of pH 7.5 Tris buffer and finally resuspended in 100 μL of pH 7.5 Tris buffer and stored at 4 °C. HCP Aptamer Screening with Particle Display. The R1 particle display pool was generated by incubating monoclonal aptamer particles with 250 ng/mL of bHCP for approximately 2 h at room temperature on a rotator. Following incubation, unbound bHCP was removed, and the beads were incubated with a 1:500 dilution of a stock solution of streptavidin− phycoerytherin (SA-PE, Thermo Fisher Scientific) for 15 min on a rotator at room temperature. Unbound SA-PE was removed, and the beads were resuspended in PBSMCT. Fluorescence-activated cell sorting (FACS) was carried out using a FACS Aria I (BD) system. Gates were set for sorting based on a negative control reaction containing only FP-coated beads. During sorting, all particles with more fluorescence than the negative control reaction were collected. We intentionally gated broadly to capture as many aptamer candidates as possible because individual HCPs span a wide range of concentrations. After obtaining the R1 pool, we amplified these aptamer candidates and carried out emulsion PCR again in preparation for the next round of screening. For R2, the bHCP concentration was reduced to 150 ng/mL. The final R3 pool was obtained in the same fashion but with 40 ng/mL of bHCP. Analysis of the pools at a constant concentration of 150 ng/mL was carried out with a BD Accuri flow cytometer. High-Throughput Sequencing of the R1 Pool. Candidate aptamer sequencing was carried out by Genewiz. Library preparation was carried out with the Illumina TruSeq Kit. Samples were sequenced on an Illumina MiSeq system in a 2 × 76 paired-end format. We obtained a total of 3.67 × 106 merged reads, with 218 557 unique sequences containing primer-binding regions. These sequences were ranked based on the number of copies of each sequence in the output of the run. Cloning and Sequencing of the R3 Pool. We cloned the R3 particle display pool with a TOPO TA cloning kit (Invitrogen). Chemically competent Escherichia coli were transformed with the aptamer candidates using a plasmid with a kanamycin resistance marker. The resulting bacterial C
DOI: 10.1021/acs.analchem.7b04775 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry of the appropriate binding buffer at a flow rate of 2 mL/min. Bound HCPs were eluted in 3 CVs of 2 M NaCl, 10 mM Tris, pH 7.5 for the aptamer column or 100 mM sodium citrate, 0.5 M NaCl, pH 3.0 for the anti-CHO HCP pAb column. The pAb column was eluted directly into 15 mL of 1 M Tris, pH 7.5 to neutralize the protein solution coming off of the column. After elution, each column was washed with 2 CVs of the appropriate binding buffer at 5 mL/min. The retained HCP flow-through was then passed back over the corresponding column for an additional round of binding, washing, elution, and column equilibration. This process was carried out a total of five times. The eluates from each affinity chromatography column were then concentrated using 3 kDa MWCO Amicon columns (Millipore) for further analysis. Mass Spectrometry Analysis. MS and associated sample preparation was carried out by MS Bioworks. A total of 20 μg of protein from the aptamer column sample, the pAb column sample, and the load sample were run in separate lanes on a 4− 12% Bis-Tris gradient SDS-PAGE gel with MOPS buffer. The gel was Coomassie-stained, and each lane was cut into 40 equally sized segments. Gel pieces were processed using a robot (ProGest, DigiLab) with the following protocol: wash with 25 mM ammonium bicarbonate followed by acetonitrile → reduce with 10 mM dithiothreitol (DTT) at 60 °C followed by alkylation with 50 mM iodoacetamide at room temperature → digest with trypsin (Promega) at 37 °C for 4 h → quench with formic acid. The supernatant was analyzed directly without further processing. The gel digests were analyzed by nanoLC/ MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive MS instrument. Peptides were loaded on a trapping column and eluted over a 75-μm analytical column at 350 nL/min; both columns were packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed in the Orbitrap at 70 000 fwhm resolution and 17 500 fwhm resolution, respectively. The 15 most abundant ions were selected for MS/MS. Data were searched using a local copy of Mascot with the following parameters: enzyme: trypsin; database: UniProt Cricetulus griseus (Chinese hamster; www. uniprot.org; Proteome ID UP000001075): forward and reverse appended with common contaminants; fixed modification: carbamidomethyl (C); variable modifications: oxidation (M), acetyl (protein N-term), pyro-Glu (N-term Q), deamidation (NQ); mass values: monoisotopic; peptide mass tolerance: 10 ppm; fragment mass tolerance: 0.02 Da. At least two unique peptides were required per protein to achieve a positive identification. Isoelectric point data were plotted for all proteins where available using the expasy.org “Compute pI/Mw Tool.” The protein titin is extremely large (MW = 4007 kDa) and was therefore excluded from plots for conciseness. Individual Aptamer-Induced HCP Precipitation and Identification. The HCPs bound to individual aptamers were identified using magnetic bead-based precipitation. Magnetic beads (streptavidin MyOne C1 Dynabeads; Invitrogen) were washed 3 times with 2× binding buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, 0.01% Tween-20, pH 7.5). Two hundred microliters of beads were then added to 200 μL of each individual 3′-biotinylated aptamer (final NaCl concentration = 1 M) and gently mixed for 15 min at room temperature. The beads were then washed 3 times with 1× binding buffer followed by 2 washes with PBSMCT and 3 washes with PBSMCT + 0.1 mg/mL salmon sperm DNA (PBSMCT + ssDNA). The beads were resuspended in
PBSMCT + ssDNA + 0.05 mg/mL CHO HCP and incubated overnight at 4 °C. The beads were then washed 3 times with PBSMCT + ssDNA and prepared for SDS-PAGE and MS as described above, except with the individual aptamer gel lanes cut into 10 equally sized slices. Flow Cytometry-Based Detection of Host Cell Proteins. To determine the detection limit of the 32 90-mer aptamers and the pAb affinity reagents, we conjugated them to 1 μm carboxylic acid magnetic beads and carried out flow cytometry to measure signal change as a function of bHCP concentration. The 3′-end amino-modified aptamers were conjugated to the beads using the same protocol used to conjugate FP to the magnetic beads for emulsion PCR. The pAbs were conjugated to the beads following the manufacturer’s recommendations. Following conjugation, binding assays were carried out with biotinylated HCP (bHCP) in PBSMCT for the aptamers and in PBST (1x PBS + 0.01% Tween-20) buffer for the pAb. Binding assays were carried out by varying the concentration of bHCP from 0 to 10 000 ng/mL in a constant background of 0.5 mg/mL of human IgG (SigmaAldrich). One microliter of a 1:200 dilution of the stock solution of beads (∼107 beads/μL) was added to a 100 μL final volume containing bHCP and IgG and incubated for 2 h at room temperature on a rotator. Each sample was washed once with PBSMCT for the aptamer experiments and once with PBST for the pAb experiments and then incubated with a 1:500 dilution of streptavidin−Alexa Fluor-647 conjugate for 15 min on a rotator at room temperature. Following removal of the unbound fluorophore solution, the beads were resuspended in 150 μL of the appropriate binding buffer, and the fluorescence associated with the beads in each sample was measured on a BD FACSVerse. Median values for each sample were recorded. We used the average of three samples at each concentration to generate a plot of fluorescence signal intensity as a function of bHCP concentration. The detection limit for the pAbs and aptamers was obtained by finding the sample concentration associated with a signal of three times the standard deviation of the negative control reaction (with no bHCP added) based on a linear regression of the negative control and the three lowest HCP concentrations.
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RESULTS AND DISCUSSION HCP Aptamer Discovery Procedure. HCP aptamer discovery is carried out in three steps: selection, highthroughput sequencing (HTS), and aptamer array analysis (Figure 1). The selection step involves multiple rounds of particle display screening, in which we employ emulsion PCR to convert a library of DNA sequences into a pool of monoclonal aptamer particles that each display many copies of a given sequence, as previously described by our group.19 These particles are then analyzed via FACS to isolate aptamers that bind to labeled HCP. Given the limited throughput of the FACS instrument (7 × 107 particles/h), we reduced the size of our initial random library of 6 × 1014 DNA sequences by performing two rounds of “pre-enrichment” with conventional SELEX (see Methods). This allowed us to reduce the size of the library to a scale that could be efficiently sorted without converging the library to a point where sequence diversity is compromised. We began the particle display screening process by converting this pre-enriched pool into aptamer particles through emulsion PCR (Figure 1, step 1). These aptamer particles were then incubated with labeled HCP (step 2). We D
DOI: 10.1021/acs.analchem.7b04775 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 1. HCP aptamer screening and characterization procedure. A pre-enriched library of random DNA sequences is converted to aptamer particles (step 1), which are incubated with biotinylated HCP (step 2). Unbound HCP is removed (step 3), and the HCP-bound particles are labeled with a streptavidin-conjugated fluorophore (step 4). FACS is used to isolate HCP-binding aptamers (step 5), which are then sequenced (step 6) and used to synthesize an array (step 7) for confirmation of HCP binding. The best aptamer candidates from the array are then compared to pAbs by using affinity chromatography followed by mass spectrometry to characterize the proteins bound by each affinity reagent set (step 8).
Figure 2. Enrichment of HCP-binding aptamers. (A) We incubated our initial pre-enriched aptamer pool as well as the pools from R1−R3 of particle display screening with 150 ng/mL bHCP and then analyzed each pool via FACS. The percentage of HCP-binding aptamer particles (HCP gate) consistently increased from round to round. (B) We generated a custom array comprising the 6000 most highly represented sequences in the R1 pool and performed a binding assay in which we incubated the array with bHCP followed by streptavidin−Alexa Fluor 647. We measured relative binding based on fluorescence intensity and plotted the mean feature fluorescence intensity for each of the 6000 aptamer candidates, which are ranked by sequence copy number. The red line indicates the 20 000 fluorescence unit threshold used to select high-affinity aptamers for further characterization.
chose biotinylated CHO HCP (bHCP) because ∼70% of biologics are produced in CHO cells.21 After removing unbound bHCP (step 3), we then labeled the bHCP-bound aptamer particles with a streptavidin−phycoerythrin (SA−PE; step 4) conjugate and sorted the particles via FACS (step 5). This allowed us to collect individual aptamer particles that exhibit high levels of fluorescence, indicating strong binding to CHO bHCP. We established a reference gate by running unlabeled beads, which are coated only with the forward primer (FP) used for emulsion PCR, through the FACS instrument. We then sorted all particles with fluorescence above that of the reference gate to capture aptamers that recognize both highand low-abundance proteins. The isolated aptamer particles were then subjected to further rounds of screening, in which we used decreasing concentrations of bHCP to gradually increase the stringency (see Methods). We characterized the output of our selection process by using HTS to survey the contents of the resulting aptamer pool (step 6). We identified the most highly represented sequences and incorporated these into a custom aptamer array, which allowed us to assess the binding characteristics of our pool (step 7). Finally, we used MS to characterize the proteins recognized by our selected aptamers (step 8). We observed increased enrichment of HCP aptamers over the course of successive rounds of screening (Figure 2A). This was determined by measuring the percentage of aptamer particles that bound to a fixed concentration of fluorescently labeled CHO HCP in each round. We incubated beads from the pre-enriched pool or aptamer particles from each particle
display round (R1−R3) with 150 ng/mL bHCP. In the preenriched pool, only 0.2% of the particles resided in the sort gate. This population slightly increased in the R1 pool (0.5%), while the R2 and R3 pools had a relatively large fraction of particles within the sort gate (7.5 and 13.0%, respectively). This increase in the sorted population indicates successful enrichment for HCP-binding aptamers from round to round. In targeting a heterogeneous mixture such as HCP, we wanted to capture the broadest diversity of aptamer sequences possible, allowing us to identify the greatest possible number of individual HCPs. We therefore needed to ensure that the aptamer pool had not converged to an overly narrow population of sequences. We sequenced individual clones from the R3 pool and found the pool had converged to a point that might limit the spectrum of targets recognized. We therefore focused on the R1 aptamer pool, which exhibited a slight increase in the sorted population relative to the preenriched pool, but was unlikely to have undergone extensive convergence. To ensure that we were not unknowingly enriching for sequences that bind to SA-PE, we performed a flow cytometry analysis of the round 3 pool after incubating with SA-PE in the absence of HCP, and our results confirmed that no such selection for off-target binding was occurring (Supporting Information, Figure S1). We subsequently used an aptamer array to survey the HCP binding characteristics of our aptamer pool.22 We generated an in situ-synthesized custom array comprising the 6000 most highly represented sequences from the R1 pool by copy number. This is the maximum number of sequences that could E
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Analytical Chemistry be synthesized in this particular array format, allowing for each sequence to be represented in triplicate for a total of ∼18 000 aptamer features (see Methods). After incubating the aptamer array with 5 μg/mL bHCP and washing, we added streptavidin−Alexa Fluor 647 to label aptamer-bound HCPs. We measured the fluorescence of each aptamer feature, averaging the fluorescence for each set of triplicates. The fluorescence intensity ranged from 7168 to 47 550 fluorescence units (FU), with an average signal of 13 326 FU (Figure 2B). This level of variability was to be expected, given that this pool had undergone only two rounds of SELEX and one round of particle display screening. As a threshold for defining high aptamer affinity for CHO HCP, we established a cutoff of 20 000 FU (approximately 2 times the average signal intensity). Based on this threshold, 321 of the 6000 aptamers exhibited high affinity for CHO HCP. To simplify additional characterization of these CHO HCP binding aptamers, we grouped the 321 aptamers into 32 sequence families using sequence homology analysis (see Methods) and selected the aptamer with the highest fluorescence intensity from each family. We then evaluated the performance of the resulting 32 aptamer set. Flow Cytometry Binding Assay with Aptamer and pAb Affinity Reagents. We first compared the sensitivity of this set of 32 aptamers with a commercially available anti-CHO HCP pAb reagent in a flow cytometry-based assay. The 32 aptamers were mixed in equimolar amounts and then covalently conjugated to activated carboxylic acid-coated magnetic beads. A second set of carboxylic acid magnetic beads was modified with covalently linked anti-CHO HCP pAbs. We incubated each set of beads with various concentrations of bHCP in the presence of 0.5 mg/mL of human IgG to reproduce the antibody concentrations that are typically present in a biologic drug preparation. Finally, we measured the level of bHCP binding by labeling with Alexa Fluor 647-conjugated streptavidin and analyzing the fluorescence of individual beads. The 32-aptamer set showed a better limit of detection (LOD) than the pAb reagent (Figure 3). We determined the
LOD based on the concentration of bHCP that produced a signal equivalent to three times the standard deviation of measurements obtained from a negative control reaction containing no bHCP. We tested both affinity reagents over a bHCP range from 0 to 10 000 ng/mL in the presence of 0.5 mg/mL human IgG and found a LOD of 16 and 78 ng/mL bHCP for the aptamer set and pAb, respectively. This is a critical benchmark, as 100 ng/mL is the industry-accepted threshold for HCP contamination in a biopharmaceutical.15 Mass Spectrometry Characterization of HCPs Bound to Aptamer and pAb Affinity Reagents. To assess the diversity of targets that can potentially be detected by the two reagent sets, we used MS to quantitate and identify the proteins captured by pAb and aptamer columns over the course of affinity chromatography with NHS-activated sepharose highperformance columns. After conjugating the 32 aptamers or anti-CHO pAbs to columns, we performed binding by loading 45 mL of HCP at 0.15 mg/mL over each column at a flow rate of 1 mL/hour. After washing, we eluted the bound HCP from each sample and concentrated the proteins with 3 kDa molecular weight cut off (MWCO) spin filters. HCP samples were then separated using SDS-PAGE and segmented into 40 equally sized gel slices. After extracting the proteins from each slice and digesting with trypsin, the resulting peptides were analyzed via MS (see Methods). These data are provided in the Supporting Information, Table S1. Positive identification was obtained when at least two unique peptides from each CHO HCP were found in the UniProt CHO genome database.8 In addition, we performed 32 independent experiments to reveal the identities of the various proteins bound to each individual aptamer (Supporting Information, Figure S2 and Table S2). This quantitative analysis showed that our set of 32 aptamers bound nearly as many proteins as the commercially available pAbs (Figure 4A). In comparison with a total of 1818 HCPs in the load sample, we identified 842 from the aptamer column eluate (37.9% of load) and 892 from the pAb column eluate (44.8% of load) with 490 proteins present in all three HCP subsets. We also found 70 proteins that were uniquely identified in the pAb sample and more than twice as many proteins (146) that were unique to the aptamer sample and could not be detected in the load sample, likely due to their low concentration. We directly compared the biochemical and physical characteristics of the HCPs isolated from the aptamer and pAb columns by plotting molecular weight as a function of isoelectric point (pI) for each protein (Figures 4B−D and Figures S3 and S4). We observed substantial overlap between the aptamer and pAb HCPs over a wide range of molecular weights and pIs. The average molecular weight of the bound CHO HCPs was approximately ∼55 kDa for both the aptamers and the pAbs. Likewise, the median molecular weight for both HCP subsets was approximately 40 kDa, which is close to the value reported for higher eukaryotes (375 residues).23−25 Thus, the aptamers and pAbs seem to recognize equivalent subsets of proteins in terms of size. Critically, our aptamers recognized a number of HCPs that are known to affect the therapeutic efficacy or safety of biologic drugs. These included three high-risk HCPs that have been shown to cause enzymatic degradation of monoclonal antibodies: matrix metalloproteinase-19, serine protease HTRA1, and protein disulfide-isomerase A6.26 Proteolytic enzymes can fragment IgG, and contamination with the matrix metalloproteinase-19 and serine protease HTRA1 enzymes could
Figure 3. Assessing the sensitivity of HCP aptamers versus pAbs. We compared bHCP binding at a range of concentrations from 0 to 10 000 ng/mL for our 32 aptamer set (orange) and a commercial pAb reagent set (blue) using a flow cytometry assay. These measurements were performed in the presence of 0.5 mg/mL IgG, which is a typical concentration for biologic drug preparations. The inset shows a similar LOD for both sets of affinity reagents. Error bars represent the standard deviations of the mean from triplicate samples. F
DOI: 10.1021/acs.analchem.7b04775 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
damaging effect on the disulfide bonds that hold mAbs together.26 Our aptamers also detected other proteins that are known to consistently interact with mAbs. Zhang et al.9 evaluated a protein A eluate pool of HCPs from nine unique mAbs and found that clusterin was in all pools examined, and in seven of nine mAbs evaluated, clusterin was the most abundant HCP identified. Our aptamer set was easily able to detect clusterin, which was the 94th most abundant protein out of 842 aptamer column proteins. This ranking was based on a list of largest to smallest normalized spectral abundance factors (NSAF). The second most abundant protein identified by Zhang et al. was actin, which is the most abundant protein in the majority of eukaryotic cells and is involved in more protein−protein interactions than any other protein.28 We also detected actin in our aptamer affinity chromatography experiments, and it ranked as the 61st most abundant protein as measured by NSAF. Finally, Zhang et al. found a small set of proteins that were initially abundant in the cell culture fluid and tended to be associated with mAbs. Because it has been noted that mAbs tend to bind to highly abundant proteins, it was encouraging that our aptamers were able to identify 9 out of the 10 most abundant HCPs in our load sample. These results demonstrate that even a small set of DNA aptamers is sufficient for the broad recognition of HCPs that interact with mAbs.
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CONCLUSION We demonstrated the generation of an aptamer reagent set that matches or exceeds the HCP detection performance of pAbs and overcomes fundamental limitations of such reagent sets. Our high-throughput FACS-based screening strategy yielded 32 aptamers that exhibit superior sensitivity to a commercially available pAb HCP detection reagent set with the capacity to detect a roughly equivalent number of proteins with a broad distribution of pI and molecular weights. With just 32 aptamers, we were able to detect 842 different HCPs, including 345 not recognized by the pAb reagent set. Given that each reagent set recognizes sizable populations of HCPs that are not detected by the other, one could consider combining aptamers with pAbs to broaden the coverage of HCPs and potentially increase the detection sensitivity. However, this would also eliminate a key asset of our aptamer reagent set: the fact that it is entirely sequence-defined, which means that it can be relied on to deliver reproducible performance and can consistently be manufactured in a simple and low-cost fashion. We also demonstrated that a number of the proteins recognized by our aptamers are known common contaminants in biologic drug preparations as well as enzymes that can potentially degrade mAbs. Although we demonstrated that our aptamer set can achieve excellent HCP detection sensitivity against a high background of IgG (Figure 3), we acknowledge the potential for crossreactivity between our aptamers and antibody-based drugs. We are in the process of assessing the extent of such cross-reactivity and strategies for mitigating it; for example, making use of “sandwich assays” or additives such as dextran sulfate and salmon-sperm DNA to improve specificity. Importantly, individual aptamers with measurable affinity for IgG can readily be eliminated or replaced in the final reagent set. It should also be noted that there are certain high-risk HCPs that were not identified by the current reagent set, such as cathepsin B26 and phospholipase B-like 2.29 It is possible that some HCPs may not have been efficiently biotinylated in the current approach
Figure 4. Quantitative analysis of HCP detection with aptamers and pAbs. (A) A Venn diagram shows the overlap between HCPs bound by the aptamer or pAb column relative to the load sample. (B−D) Scatterplots showing molecular weight vs pI for proteins in (B) the aptamer and pAb samples, (C) the load and aptamer samples, and (D) the pAb and load samples. Black dots indicate proteins identified in both subsets.
potentially cause degradation of mAb-based drugs.5 The third high-risk HCP, protein disulfide-isomerase A6, can catalyze the disruption of disulfide bonds, as has been shown with insulin,27 and there is reason to believe that it could exert an equally G
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(for example, due to a lack of exposed lysine side-chains), and the use of alternative biotinylation regimens (e.g., targeting sulfhydryl or carboxyl groups) could help overcome this. However, it should be relatively straightforward to generate additional aptamers that target specific contaminants with known biological/clinical impact as a supplement to our 32aptamer set, and we believe that this capacity to customize the reagents in a controlled fashion is a powerful feature of our approach. More generally, this represents only a first effort to generate an aptamer-based HCP reagent set. In future work, we will be pursuing additional strategies to further refine the screening process, and it should be straightforward to assemble curated reagent sets that greatly exceed this performance, or which have been tailored for non-CHO bioproduction cell lines such as HEK293. Indeed, given the fact that aptamers can be transmitted as sequence information, it is entirely feasible that such assembly could be a community-driven effort. In conclusion, we believe our aptamer-based HCP detection toolkit should offer a superior and more economical strategy for improving the safety of biologic drug preparations.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b04775. Figures showing aptamer pool and fluorophore label results with no target present, unique CHO HCPs bound to individual aptamers and pI values for all CHO proteins plotted versus molecular weight and unique CHO HCPs bound by aptamers and the anti-HCP pAb plotted versus pI; Tables showing mass spectrometry data for HCPs bound to aptamers and the anti-HCP pAb and individual HCPs identified by pull-down experiments (PDF)
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Article
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
H. Tom Soh: 0000-0001-9443-857X Author Contributions
⊥ J.B.M. and A.T.C. contributed equally to this work. H.T.S. and J.B.M. conceived the project. H.T.S., J.B.M., and A.T.C. designed the experiments. J.B.M., A.T.C., F.M.W., and E.R.B. carried out experiments. H.T.S., J.B.M., A.T.C., F.M.W., E.R.B., C.J.L., R.J.S., P.L.M., J. C.-F., and M.A.N. analyzed the data. H.T.S., J.B.M., A.T.C., F.M.W., M.A.N., and M.E. wrote the manuscript.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by DARPA (Grant N66001-14-24055) and the Garland Initiative. The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. H
DOI: 10.1021/acs.analchem.7b04775 Anal. Chem. XXXX, XXX, XXX−XXX
