Methods and Materials
Thanks to vendors large and small — such as Invitrogen (www.invitrogen.com), ProteoChem (www.proteochem.com), Sigma Aldrich (www.sigmaaldrich.com), Soltec Ventures (www.soltecventures.com), and Thermo Scientific Pierce (www.piercenet.com) — bioconjugation chemistry is a field of many options. For example, amine coupling of lysine amino-acid residues typically involves amine-reactive succinimidyl esters. Sulfhydryl coupling of cysteine residues uses a sulfhydryl-reactive maleimide. Photochemically initiated free-radical reactions offer broader reactivity. Most processes couple small molecules to proteins or proteins to one another (e.g., antibodies and enzymes). Other payload molecules include oligosaccharides, nucleic acids, synthetic polymers (e.g., PEG), and carbon nanotubes — each according to its own application. Technical challenges come along with these introduced downstream operations after proteins are purified — most particularly in the heterogeneity that they introduce to the resulting product pool.
Process optimization for protein drug manufacturing involves three main considerations (4): product quality (purity, stability, and activity), process robustness, and cost. Conjugated biomolecules face further challenges. To simplify characterization and downstream processing as much as possible, for example, companies optimize the selectivity of their PEGylation reactions for a specific PEGylation degree and site. They want higher yields of the conjugate coinciding with lower yields of secondary products. Progression of the chemical reaction depends on a number of variables, and it’s hard to control reproducibly the exact attachment sites and number of attached molecules. It’s relatively easy to determine how many have stuck on — but identifying exactly where they’re attached is a real analytical puzzle. You may know which type of amino acids bind to your chosen linkers, but you don’t know which of those specifically actually caught them on the protein molecule.
Consultant and editorial advisor Sally Seaver says scientists can identify which possible sites on a known protein structure are actually conjugated, at least some of the time. “To me it is similar to the issue with knowing which sites are glycosylated or have other modifications and the rough percentage of times a given site is modified. We cannot say specifically, but we can determine the averages.”
Protective agents, different sizes and types of payload molecules, protein mutations, and specific solution pH conditions all provide options for fine-tuning the reaction parameters to produce more of a desired final structure and fewer unreacted or otherwise conjugated molecules. The final mixture of PEGylated conjugates can be separated by size-exclusion chromatography (SEC) to purify a desired structure. But scaling-up SEC is challenging because of relatively low throughput and high cost (4). At large scales, an additional purification step is often necessary, typically ion-exchange chromatography (IEC), although the chemical properties of PEG can lower both its capacity and resolution as well.
For ADCs, a cytotoxic agent is attached to a cleavable (e.g., peptide, disulfide) or noncleavable (e.g., thioether) linker molecule, which in turn binds to an immunoglobulin, Fab, or ScFV antibody fragment at a specific type of side chains (e.g., lysine or cysteine residues). Here too, attachment is not simply one-to-one; wherever the recognized amino acids are present on the protein surface, a linker may (or may not) bind — and wherever a linker binds, a payload molecule may (or may not) attach. Final ADC products are said to contain a defined average number of cytotoxic molecules per antibody.
Different linkers determine roughly where payload molecules will be attached, a key step in manufacturing. There are many options to choose from, depending on the properties of both antibody and payload. Generally, an ADC manufacturing process involves two chemical reactions (attaching the linkers, then the payloads) followed by purification to generate a final conjugate product pool. The “Conjugate Vaccines” box provides more detail. Such processes can be designed to be highly robust and scalable, if not entirely specific. These types of chemical reactions have been used and studied for many years and are quite well understood. Much of the separation required in purification is for removal of solvents and unbound and/or low–molecular-weight species. For quality-by-design (QbD) design space purposes, key quality attributes to be monitored include molecular size, charge, and sequence variants; drug/antibody ratios; product potency; and number of free, unconjugated drug and antibody molecules present.
Chemical linker bonds can release either by acidic/ reducing conditions or enzymatic cleavage when a conjugate is internalized in its target cell’s lysosome (5). Hydrazone bonds release under acidic conditions; disulfide bonds are subject to intracellular reduction. Peptides are conditionally stable and subject to rapid enzymatic cleavage by lysosomal proteases. Such methods are also useful for cleaving fusion proteins if their manufacturing processes require it.
The chemical reactions of bioconjugation occur in biological buffer solutions that can support bacterial growth, so aseptic conditions and clean utilities are needed (5). And ADC manufacturing environments must allow for safe manipulation of highly toxic drugs. Biological facilities are primarily designed to reduce microbial contamination because most proteins present no significant health risk to workers or the exterior environment. But cytotoxic drugs are among the most potent chemicals used in therapeutic applications, with occupational exposure levels designated in the 10–9 gram/m3 range (5). Workers (and the outside environment) must be protected from them just as much as the products must be protected from contamination (3).
Multiproduct facilities introduce further concerns. Companies that “campaign” their manufacturing cannot exceed the maximum allowable carryover (MAC) of residual product between different campaigns. Analytical methods based on high-performance liquid chromatography (HPLC) or measurement of total organic carbon (TOC) content aren’t necessarily sensitive enough to make these assurances during product changeovers (5). In many cases, enzyme-linked immunosorbent assays (ELISAs) or mass spectrometric (MS) techniques are necessary to detect residual drug in the ng/mL range. Alternatively, dedicating a facility to a single product’s manufacture increases costs, which are likely to be passed on to patients.
Several biopharmaceutical companies have active ADC development programs: e.g., ImmunoGen (www.immunogen.com), Genentech (www. gene.com), Pfizer (www.pfizer.com), and Seattle Genetics (www.seagen. com). Some contract biomanufacturers present themselves as especially capable in this area: e.g., CellMosaic (www.cellmosaic.com), Diosynth Biotechnology ( et=”new” href=”http://www.diosynthbiotechnology.com”>www.diosynthbiotechnology.com), Fleet Bioprocessing (www.fleetbioprocessing.co.uk), Goodwin Biotechology (www.goodwinbio.com), Lonza (www.lonza.com), and SAFC Pharma (www.safcglobal.com). Conjugate vaccines are the business of giants such as Pfizer and Sanofi Pasteur (www.sanofipasteur.com) as well as innovative newcomers such as Nabi Biopharmaceuticals (www.nabi.com), which recently engaged Diosynth RTP to manufacture its antismoking nicotine conjugate vaccine product. When it comes to protein conjugates, all these players face similar challenges in manufacturing, product characterization, and testing.
Depending on the linkage chemistry, molecular attachment sites, and synthetic methods involved in bioconjugate processes, significant regulatory issues (e.g., chemistry, manufacturing, and controls) can arise during product development. With newer product forms such as ADCs, manufacturers are asking how to determine the appropriate assays for characterization and quality assurance and how to identify critical quality attributes (CQAs) for antibodies, linkers, and small drug molecules as well as the combined final products. The answers to most of those questions are product specific.
Like fusion proteins, conjugate products are more subject to heterogeneity than are strictly protein drugs, which already present great challenges with multimers, glycosylation variants, and other product variants. Linker molecules often attach fairly randomly — not one at a time, but usually a few to several at various points across the surface of the protein — in a process that cannot be precisely controlled. Few antibodies get fully loaded on all possible linkage sites. Product characterization issues thus are introduced, including the number of payload molecules present/ needed, the structure and folding of the conjugate molecule, and so on. The presence of small molecules makes additional assays necessary beyond those typical for an antibody: e.g., for determining the amount of free, unbound cytotoxic agents. A mass distribution profile is a good test of process consistency. Ultraviolet (UV)– based assays measure average loading, with MS providing orthogonal verification. That also provides information on the relative amounts of antibodies loaded with one, two, three, four, or more individual small molecules (as well as on empty/ unconjugated antibodies), but it doesn’t show where they’re attached. It can, however, detect free crosslinkers, free linker–drug combinations, and antibodies with linkers but no drug attached.
In process development, single- and dual-point experiments help companies identify key operating parameters and their ranges, define broader testing ranges for those parameters, and determine acceptable ranges. Design of experiment (DoE) studies help determine significant factors and criteria, identify interactions among them, assess process variability, build predictive models and design robust processes, and determine operating ranges for manufacturing with predictive outcomes.
ADC product characterization is a two-step process, with tests performed — some for the antibody, some for the payload — both before and after the crosslinking reactions. Infrared (IR), nuclear magnetic resonance (NMR), and MS methods are used for product identification before conjugation; then UV absorption and immunosorbent assays can confirm identity of the conjugated molecule. Small molecules have many issues of their own, most unfamiliar to biologics companies. These include residual solvents, melting points, and heavy metals.
Purity of antibodies and component molecules is measured before conjugation by analytical HPLC (primarily) — and final product purity after with methods that measure conjugated and free-drug content (including derivatives), residual solvents and water, optical rotation, melting point, and even chiral HPLC if stereo isomers are present. Although product identity testing traditionally involves charge-based assays, these may not work after conjugation, especially when lysine linkage is used. Binding assays, peptide mapping, and sequencing may be used instead. The need for one or more assays depends on product CQAs and whether other products are manufactured at the same facility. Although not as robust or reliable and more difficult to create than are binding ELISAs, cell-based potency assays are needed to demonstrate potency from lot to lot. Identity tests are needed for both the antibody and linker/drug combination — anything that might vary. But only the binding assay will show antibody performance before and after conjugation (to measure efficacy before the in vivo testing). Such assays are used in testing MAb products whether or not they are conjugated.
Final-product purity is tested using sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), although cysteine-linked ADCs can cause problems for that method. Other options include capillary gel electrophoresis (CGE), SEC-HPLC, and charge-based analytical methods. Aggregates and fragments are of particular issue at the final product stage. Potency is tested using cytotoxicity and binding assays to demonstrate that conjugation does not affect an antibody’s binding ability. Here is where drug/antibody ratio, drug loading distribution, and measured free drug and antibody come into play. Different conjugation technologies often involve different assay requirements and possibilities.
For therapeutic MAbs, a single potency assay (typically cell based or method-of-action) is usually sufficient for lot-release and stability testing. But for ADCs, two are preferred. A cell-potency assay demonstrates the mechanism of action: target binding, cell internalization, drug release, and cell death. Although an ELISA shows only target binding (which is part of product characterization and comparability testing for process changes), its redundancy of data is necessary. However, the chemical process of conjugation is usually straightforward. Good reference standards and archived samples of every lot will help companies demonstrate the robustness of their processes.
At a recent CMC Forum on ADCs, quality consultant Nadine Ritter explained that their QC testing requirements (e.g., identity, purity, impurities, activity, concentration, and stability) are the same as for other biopharmaceutical products according to the current International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) (6,7). However, she cautioned, this is a new class of drugs with multiple different CMC elements — so their CMC regulation is still evolving. “Key concerns for QA/QC departments include physiochemical and functional assays as well as adequately specific identity assays for both the MAb moiety and drug moiety. This could be especially of concern for multiproduct facilities, where specific identity of each component of the ADC might be necessary to distinguish it from other, similar products (e.g. MAbs) produced in the same place. Also, regulators may have more detailed questions regarding product heterogeneity (product-related substances) before and after conjugation. Additionally, there may be unique issues related to ADC immunogenicity studies beyond what is currently expected for unconjugated protein therapeutics.”
Manufacturing Conjugate Vaccines
Conjugate vaccine production is a stepwise process:
Protein production: culture of host organism followed by collecting, washing, homogenizing, recovering, and purifying protein
Polysaccharide production: fermentation of donor organisms followed by cell disruption, polysaccharide purification, and trimming by hydrolysis (acid, periodate)
Derivativization: makes both components reactive
Conjugation: bringing components together
Purification: removing unwanted compounds
Finish and fill: Concentration to final strength, dialysis into appropriate solute(s), and sterile fill as a liquid (then possibly lyophilized)
Preparation and purification of the protein is a familiar procedure to most BPI readers; preparing the bacterial polysaccharide components and adding/removing reagents is less so. Many reagents are available for derivatizing and conjugating vaccines. Purified bulk saccharides are trimmed and sorted into size classes to meet product specifications. Size-exclusion chromatography is commonly used, followed by concentration of the dilute results by ultrafiltration. In ultrafiltration sizing, a saccharide mixture is filtered through two or more different membranes: e.g., a 50-kD rated membrane to exclude large contaminants, polysaccharide aggregates, and large polysaccharide molecules, followed by a 10-kD membrane to remove small molecules, salts, and solvents. The oligosaccharides that remain trapped between those two membranes are those in a narrowed size range.
Upon derivativization, the unreacting species can be flushed through a 10-kD membrane and treated oligosaccharides diafiltered into a solution for conjugation. A similar process is used to remove unwanted reagents from the protein solution. After the components have reacted together, conjugates can once again be passed over a small-porosity ultrafiltration membrane to remove unreacted chemicals. Final concentration/diafiltration takes the product to its final formulation strength in a carrier fluid before formulation with whatever adjuvants and preservatives are required.
Some conjugate vaccines are multivalent: made up of multiple saccharide serotypes. Each serotype is prepared and conjugated separately, then they’re mixed together in a final formulation. Wyeth’s Prevnar pediatric pneumonia vaccine is one example that uses 13 different polysaccharides. This further complicates manufacturing because each is produced and controlled separately. Complexity is a barrier to cost-effective transfer of conjugate vaccines to developing areas of the world.
Companies such as Pall Life Sciences sell equipment that may help mitigate such costs. Flat-sheet ultrafiltration membrane cassettes, for example, are typically used with dedicated holders and systems for each stage of use. It can be expensive to purchase and maintain multiple systems. But single-use technologies allow reagent preparation, sterile filtration, and compounding with less capital investment. Although larger-scale UF systems are not currently available as disposables, those with plastic liners let companies produce even multivalent vaccines with only a single set of stainless steel tanks.
Stability testing is vitally important for conjugated products of any kind. How long will the molecular linkages remain over time spent in formulation — and at what temperatures? At one recent meeting, a scientist working in this field called these “the mother of all stability studies.” Shelf-life monitoring for ADCs could cover up to 20 years of storage due to the potency and cytotoxicity of the payload molecules — and the dangers they could propose should their linkages fail over time.
In some biotechnological/biological products, potency is dependent upon the conjugation of the active ingredient(s) to a second moiety or binding to an adjuvant. Dissociation of the active ingredient(s) from the carrier used in conjugates or adjuvants should be examined in real-time/real-temperature studies (including conditions encountered during shipment). The assessment of the stability of such products may be difficult since, in some cases, in vitro tests for biological activity and physicochemical characterisation are impractical or provide inaccurate results. Appropriate strategies (e.g., testing the product prior to conjugation/binding, assessing the release of the active compound from the second moiety, in vivo assays) or the use of an appropriate surrogate test should be considered to overcome the inadequacies of in vitro testing. (8)
Pharmacokinetics (PK) testing is of particular interest with protein–drug conjugates, especially for antibodies carrying cytotoxic cancer drugs (9). Genentech has such a product in phase 3 clinical trials; it’s based on the Herceptin antibody. When the company began developing conjugate products, its analytical group realized it would need a PK laboratory that combines two types of analysis that have previously existed in more or less separate worlds: liquid chromatography with mass spectrometry (LC-MS) and immunoassays (9). Enzyme-linked immunosorbent assays (ELISAs) are the mainstay for studying MAbs, and MS offers great utility in the analysis of small molecules. Combine the two types of products, and you’ll need both types of analysis.
In the body of a preclinical or clinical subject, enzymes can cleave the linker that holds a small molecule to its antibody host. They may also be separated by chemical reduction. Metabolic and catabolic processes can act on the conjugate drug before it reaches its tumor target. This causes great safety concerns when the payload molecule is cytotoxic, as in cancer therapeutics. If it falls off, where will it go? First in preclinical animal studies, and later in clinical trials, product analysts must test several different types of matrices for presence of free drug molecules: e.g., plasma, serum, and various tissues. That presents a problem. Such matrices are more complex than even the cell culture supernatants that challenge process analysts.
The selectivity and sensitivity of affinity LC-MS makes it an attractive choice for the task of characterizing both intact conjugates and fragments thereof. Genentech’s PK laboratory also uses it to optimize/troubleshoot immunoassays: e.g., screening affinity antibodies for selectivity and identifying custom ELISA reagents.
“The technology for PK studies of proteins and chemical entities has been around for decades,” Sally Seaver poins out. “And some [unconjugated] proteins need PK studies that detect their active region versus the rest of the protein because protein cleavage occurs naturally at a high rate.”
Regulatory and Facility Challenges
Being composed of both small-molecule components and proteins, many conjugate products face unique regulatory challenges. Often the nature of the linked molecules makes the difference. Because of their cytotoxic components, for example, ADCs are subject to review in the United States by the FDA Center for Drug Evaluation and Research (CDER), whereas most other conjugate products are covered by the Center for Biologics Evaluation and Research (CBER). ADCs are both drug and biologic molecules. Reviewers may change throughout product development — as different sorts of expertise become necessary. But clinical approaches to oncology products in general are becoming more harmonized across the divisions — just as regulations are harmonizing internationally — so thi
s may not be as confusing as it sounds. Even so, companies accustomed to the biologics license application (BLA) face an inevitable learning curve when it comes to the new drug application (NDA) of CDER. But it is worth noting that the Genentech/Roche filing of T-DM1 was a BLA. Regardless of the regulatory pathway, conjugate products need individually appropriate characterization, comparability, release, and stability assays.
Conjugate production requires a CGMP aseptic biological manufacturing environment that, in the case of ADCs, protects personnel from cytotoxic chemicals in the occupational exposure range of 40 ng/ m3 of room air (5). Quality control workers cannot touch or inhale ADCs during product testing. Single-use technology is useful for conjugate manufacturing facilities; otherwise all equipment and process contact surfaces require steam-in-place (SIP) and clean-in-place (CIP) capability to remove even minute traces of residual drug between batches and during product change-over. Cross-contamination is prevented by cleanrooms with dedicated utilities. The “CMO Perspective” box provides further detail.
Operational and system redundancies provide for safety as well as efficiency. Raw materials costs for ADC and other conjugate-product manufacturing are higher than for either classical drug or biologics production (5). Thus, batch failures are more costly for companies involved in this market. This justifies spending money on automation and redundancies to prevent such losses. “Execution of this multifaceted design concept could be achieved by Lonza,” wrote the company’s senior director of ADC and biochemical technology in 2009, “because of the high level of synergy between its biopharmaceutical and small-molecule business units” at a facility in Visp, Switzerland (5).
TECHNICAL CHALLENGES OF ANTIBODY DRUG CONJUGATES: A CMO PERSPECTIVE
CMOs face several challenges in both the traditional non–high-potency and high-potency conjugation fields. For a start, there needs to be an ability to work effectively with both small and large molecules in development, manufacturing, and quality control. In addition, CMOs often need to perform and be competent in several different traditional biopharmaceutical and biologics seperation techniques: e.g., size-exclusion chromatography, ion-exchange chromatography, ultrafiltration, and membrane filtration.
In some cases, a protein and small molecule may need to be chemically modified before conjugation, so the ability to perform that task adequately is a prerequisite. In addition, removal of remaining amounts of nonconjugated small molecules is often necessary. A conjugation reaction may produce undesired impurities that then need to be removed using traditional protein purification techniques.
QC analytical testing with both protein assays and small molecule testing is critical for both conjugated and nonconjugated forms.
Most drugs used in antibody–drug conjugates are highly potent, so facilities making them must include specialized engineering controls and safe handling procedures. Numerous design, anticontamination, and decontamination considerations come into play.
Engineering Controls: Use of an isolator barrier enclosure allows for the safe handling, weighing, and dissolution of highly potent compound powders. Surrogate powder testing is used to qualify hood effectiveness. Before “going live,” an isolator or glove-box system is tested using a nonpotency solution (usually naproxen sodium). In addition, manufacturing cleanrooms are designed to contain potent compounds in the event of accidental release or spills. The system includes single-pass air (both supply and return air HEPA-filtered). Cleanroom pressure is positive with respect to the atmosphere but negative in air pressure relative to the adjacent airlocks, thus mitigating the risk of contamination. Employees exiting the cleanroom do so through a decontamination misting shower located in a personnel exit air lock.
Equipment used in these cleanroom is low-energy and non–aerosol-forming to prevent the risk of generating an aerosol form of either a highly potent compound or a drug conjugate into the air. Only slow-moving pumps are used, and no high-speed mixing or centrifuges are allowed. Everything is tightly contained in the closed environment, with compounds kept preferably in liquid form to prevent airborne distribution. A number of smaller details also need to be considered when dealing with high-potency compounds. For example, a cleanroom design with coved corners makes cleaning easier.
Safe Handling Procedures: In addition to cleanroom garments, sometimes operators wear Saranex chemical suits (Dow Chemical Company, www.dow.com) and supplied breathing-air hoods. They use wireless radios for communication and follow effective, process-specific decontamination procedures to dissolve highly potent compounds with organic solvents or mild caustic solutions before regular equipment cleaning. Finally, all disposables and process waste streams are collected and sent for incineration offsite, with the waste being drum-sealed and logged.
Other Challenges: Highly potent compounds require product contact surface residue testing with detection to nanogram levels. Cleaning verification includes swabbing and testing by high-pressure liquid chromatography (HPLC) or total organic carbon (TOC) analysis.
When working with highly potent compounds, one additional challenge is cost-related. The starting materials (antibodies and drug linkers) are expensive relative to what is typically used in traditional protein isolation/purification. Essentially, we are starting with purified molecules, and volumes are relatively small. This adds to the overall product cost, which can sometimes be a major supply chain consideration for drug development in such a competitive market.
Finally, when we deal with highly potent compounds, safety certification by a third party is important: e.g., Safebridge Consultants, Inc. (www.safebridge.com). Certification verifies that a company’s safety and industrial hygiene programs for working with highly potent compounds are effective. This adds a further step to the overall process, but it is entirely necessary to ensure that a company’s high-potency programs are both effective and safe.
Kent C. Robertson is a manufacturing manager at SAFC, 3360 South 2nd Street, St. Louis, MO 63318; 1-314-286-8071, fax 1-314-289-6027; firstname.lastname@example.org.
In the United States, generally speaking, ADCs are regulated as drugs through 21 CFR 201, 210, 211, 312, and 314 with NDAs. For their antibody components, 21 CFR 600 and some biologic guidance documents also apply. Because conjugates are not considered to be combination products, 21 CFR 300.50 does not apply. Through the efforts of ICH, the regulatory approaches of Europe, Japan, and North America are coming into alignment, with the rest of the world tending to follow suit. Australia, for example, generally follows Europe’s lead. A common technical document (CTD) will be accepted for review by the FDA, European Medicines Agency (EMA), or Japanese regulators. However, the more complex is the product involved, the wiser it is for the company to take advantage of opportunities for pre-IND and other meetings with reviewers beforehand — no matter which market it has in mind and which regulatory agency is invol
Some developers lament that ADCs aren’t regulated more as antibodies — like other conjugate products — given the complexity of the comparability steps that the BLA requires for process changes (compared with the NDA). In Canada, they are in fact regulated solely as biologics. At the ADCs conference mentioned above, Chana Fuchs of CDER told audience members to be guided by what they hope to achieve with any given test or process step. Because of the wide variation in types of conjugates, the answers to most questions will be product specific. This is a product class based on a technological method rather than a particular type of molecule. Health Canada’s Anthony Ridgway said at the ADC meeting earlier this year that the most important thing is to get the right group of people together working on it. Regulators want companies to begin discussions early with their advisors and reviewers — and continue those meetings right through development. As in computing, where you “save early, save often,” in conjugate drug development perhaps you should “consult early and consult often.”
Ask the Experts
As in any other biotechnology endeavor, the technical challenges of protein conjugates inevitably translate to business concerns. In many cases, the best advice is for a company to focus on the part of the process that its staff knows well — its core competency — and leave the rest up to people with the necessary expertise. This may include contract development/manufacturing, it may involve consultants or licensing partners, and it may even lead to mergers or acquisitions.
In an interview with BioProcess International last year, consultant and editorial advisor Sally Seaver put it this way in the context of advice to small, entrepreneurial companies: “A smaller company needs to understand exactly what its strengths are. I had one client that wanted to know what the latest was in cell culture techniques for monoclonal antibodies. They were conjugating, and they had their tox molecule and knew all about it. So I said, ‘Why would you spend time producing the MAb? Why not contract that out? What you need to spend time on is handling the addition of the toxic agent, how you assay for it, and how you optimize that process.’ The thing is to know your niche.”
Online Supporting Material
Find more information — including a discussion of crosslinking reagents, an interview excerpt with Pfizer’s associate director of oncology, and a list of further reading materials — on the BPI website at www.bioprocessintl.com/bpiextra.
Cheryl Scott is senior technical editor of BioProcess International, 1574 Coburg Road #242, Eugene, OR 97401; 1-646-957-8879; email@example.com.
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