BPI Theater at the 2014 BIO Convention

When we launched the BioProcess Theater series at the Biotechnology Industry Organization’s International Convention in 2007, we hoped that our special programming would fill a need within that event’s exhibit hall. We wanted to bring into the hall the type of technical presentations that are not generally part of the main event’s more executive-level, business-focused programming.It has therefore been especially gratifying to see our attendance growing every year — such that standing-room-only is becoming more the rule than the exception. The “BPI Theater @ BIO” and a similar program that we began at Interphex earlier this year have become valued meeting places for bioprocessing professionals. We had wondered, though, how we might share these presentations with a larger audience. So when the number of requests for access to them began to overwhelm us, we decided to publish abstracts of each one and to provide our 30,000+ readers with the opportunity to listen to a number of them as podcasts. As with our Interphex theater summaries in BPI’s June 2014 issue, this special report summarizes each presentation and roundtable (in chronological order).The general program is arranged thematically each year, offering individual presentations and roundtable discussions on what we and our industry advisors determine are the year’s top subjects. Single-use technologies and cell therapies are topics of continuing interest. And over the past year a number of our authors have emphasized how automated single-use technologies will be essential to the commercial future of cell therapies.This is our second year of programming on antibody– drug conjugates (ADCs). Key issues toward success of those products include how to bring the small- and large-molecule components together within one facility as well how to manage these complex supply-chains.Other variations on familiar themes, reflected in the following summaries, include a heightened emphasis on single-use standardization and related sourcing concerns. Outsourcing relationships are being transformed through new strategies and technologies, and the biosimilar landscape is becoming more familiar territory.I hope you find these summaries useful and that you have time to listen to some of the recordings. Please let us know whether you are interested in speaking at one of our theaters in 2015 — and especially let us know which topics you would like us to offer as we continue to develop these programs for you.—S. Anne Montgomery, editor in chief

The audio from the following presentations is available in the BPI Theater section of our website.

Optimized Solutions for Better ADC Design Through SMARTag Technology
Gregory T. Bleck, global head of biologics R&D at Catalent Pharma Solutions
Bleck began by discussing the limitations of firstgeneration antibody–drug conjugate (ADC) technology:

  • Toxicity (aggregation and first-pass metabolism inliver, linker instability)

  • Variable potency (uncontrolled payload loading,pharmacokinetics, liability)

  • Chemistry, manufacturing, and controls (lot-to-lotreproducibility, complex manufacturing, challenginganalytics)

Then he described Specific Modifiable Aldehyde Recombinant Tag (SMARTag) technology and illustrated how it addresses those issues. To maximize ADC performance, developers can use this patented aldehyde-tagging method for site-specific, programmable drug placement on monoclonal antibody (MAb) carriers. ADC product consistency is improved for better regulatory compliance. A library of proprietary cytotoxin linkers and conjugation chemistry are designed to provide stability and optimize efficacy. An efficient and scalable manufacturing process allows for one-step in vivo tag generation that is compatible with any cell-based expression system and simplifies characterization analytics.

Catalent provides these solutions through a collaboration with Redwood Bioscience. In this partnership, the former provides biologic development expertise (with the GPEx expression system, state-of-the-art biomanufacturing and integrated analytical services) and the latter contributes advanced ADC technology as described above. Together, the companies aim to enable biologics innovators to more quickly develop “better ADCs.”

Bleck went on to present case-study data demonstrating potent in vitro cytotoxicity of SMARTag HER2 ADCs generated with Redwood’s linker library and different payloads and linkers. Specific conjugation sites were shown to affect in vivo efficacy. In a mouse PK study the ADC’s half-life was equivalent to that of the naked antibody. Both conjugation chemistry and linker composition also affected the ADC’s PK. In a rat toxicity study, the SMARTag HER2 ADC was tolerated even at the highest dose delivered, and it had a minimal effect on body weight.

One-Stop Shop for Antibody–Drug Conjugation Production
Andy Topping, PhD, director of development at Fujifilm Diosynth Biotechnologies

Topping began by defining his subject. An ADC is an antibody (or fragment thereof) linked by a stable chemical linker to a biologically active cytotoxic agent (typically an anticancer drug). Examples include gemtuzumab ozogamicin (Mylotarg), an anti-CD33 antibody conjugated to calicheamicin, which was withdrawn from the market in 2010; brentuximab vedotin (Adcetris), an anti-CD30 antibody conjugated by a cleavable linker to monoethyl auristatin (MMAE); and adotrastuzumab emtansine (Kadcyla), an anti-Her2 antibody conjugated by a noncleavable linker to cytotoxic DM-1 (a derivative of maytansine). With a market predicted to grow significantly, dozens of ADC molecules are now in development.

ADCs are complex products, Topping emphasized, first because MAbs alone are complicated molecules. Variants may be oxidized, deamidated, truncated, or differently glysolayted, and variation can destroy an antibody’s ability to function in vivo. Addition of a cytotoxic drug adds an additional layer of complexity. Most common conjugation methods do not target unique attachment sites on the MAb, so final ADCs are often a mixed population showing different drug-antibody ratio (DAR) values.

ADC manufacturing is challenging, beginning with an elaborate bioprocess to make the MAb. Conjugation adds new critical quality attributes (CQAs) for binding site, DAR, and reaction optimization. Formulation is difficult due to increased hydrophobicity and aggregation of the ADC. Analysis must address the MAb, the linker, the cytotoxic drug, and the conjugate.

Topping went on to describe his company’s alliance with Piramal Group to provide an integrated “one-stop shop” for ADC manufacturing. These products involve such differing sets of expertise that such partnerships are a common solution to the challenges described above. In this case, Fujifilm Diosynth’s experience in MAb production is combined with Piramal Healthcare’s experience in cytotoxics and conjugation. Together, the companies can simplify logistics (MAb produced at a Fujifilm Diosynth site and shipped to Piramal Healthcare UK, with program delivery between Piramal Healthcare UK and Fujifilm Diosynth UK) and provide single project-management contacts to their development clients.

Topping went on to describe each company’s capabilities in detail:

  • cell-line development, MAb expression and platform technologies, and current good manufacturing practice (CGMP) manufacturing of bulk drug substance

  • an integrated development plan for MAb process development with same-day analytics for CQAs and standardized MAb analytics

  • flexible or platform MAb purification

  • rapid MAb assessment for ADC potential

  • ADC process development (e.g., partial reduction and/ or modification of MAb, linker and reductant equivalents and kinetics, conjugation, cosolvents, and stabilizing excipients)

  • ADC testing (identity, binding/charge profiles and peptide maps, purity, aggregates and fragments, drug-loading distribution, free drug and antibody, potency, DAR, antigen binding)

A single project team delivers representative material for early conjugation development (10–100s mg); MAb titer and quality optimization; representative material from laboratory-scale proving runs (>10 g); ADC development and optimization, with representative material supply (>10 g) quantities; MAb and ADC analytical characterization; management of raw-material supply chain for MAb, linker(s), drug(s); MAb and final ADC scale-up and manufacturing; and both non-GMP, pilot-scale (~350 g) and GMP materials (>1 kg); full process validation; and late-phase process characterization.

The Implementation of Single-Use Technology from Cell Line to Antibody–Drug Conjugate
Lee-Cheng Liu, chief executive officer at Eirgenix

Liu began by introducing his Taiwan-based company and its business strategy. Seeking to become a global biopharmaceutical player, Eirgenix wants to provide the highest reliability and quality with an effective cost structure for contract development, manufacturing, and product development:

  • Biosimilars, biobetters, and novel biologics (integrated mammalian and microbial cell line development with protein analysis, GMP process development, small-scale and large-scale API, and product characterization)

  • Antibody–drug conjugates.

Liu went on to describe his company’s partnership with Formosa Laboratories Inc., which brings expertise in highly potent APIs to their service offerings.

The main thrust of his talk focused on a facility expansion in which Eirgenix considered and chose to implement single-use technology. Space, budget, and timing constraints led the management to consider that option. Liu said his top reasons for choosing disposables over stainless steel included increased flexibility/capacity and improved efficiency (decreased turn-around and handling times, reduced maintenance and labor/headcount), without needing to expand the facility’s existing clean-in-place and steam-in-place (CIP/SIP) systems.

“Choosing a disposable system is like building a partnership with the vendor,” Liu explained. Users need to bridge their internal expertise with that of the supplier. Considerations include bag materials, increasing demand on the vendor’s quality system (e.g., plastic supplier auditing, material test methods and characterization), technical service and support, and life-cycle monitoring and verification.

When evaluating available bioreactor systems, Eirgenix leadership looked at their fitness for the available space; software for control, batch management, and connection to the larger facility; technical aspects; agitation and aeration design concepts; mixing times and kLa; spargers and gas aeration controls; temperature control; bags and other consumables; integrity testikng; leachables and extractables; materials, validation documents, and film suppliers; reference information; and service and support. They also took into consideration the impacts on their facility’s downstream capacity. Eventually, the company chose new 50-L, 200-L, and 1,000-L Sartorius Stedim Biotech BIOSTAT STR bioreactors.

The expansion project began in December 2013, and the final systems were delivered in early April of 2014. Installation, installation and operational qualification (IQ/OQ) were complete in May and performance qualification (PQ) at the end of June. The system was expected to be ready for production in early July.

Sourcing ADC Development and Manufacturing Services for a More Efficient Supply Chain
Jason C. Brady, PhD, director of business development for conjugates and cytotoxics at Lonza Custom Manufacturing

Brady began with an ADC market overview before focusing on ADC supply chain challenges such as vendor management and partnerships that involve multiple quality systems, contracts, and geographical locations. All the necessary specialized services — biologics, chemistry, analytics — are seldom available from a single contract manufacturing organization (CMO). With limited specialized manufacturing capacity available in the marketplace, this can become a problem for ADC developers. And starting up from scratch can involve significant capital expenditures and employee training, raw materials, timelines, and risk of batch failures.

Lonza offers full development, GMP manufacturing, and analytical services for monoclonal antibodies and recombinant proteins, cytotoxic payloads, and conjugation. Brady emphasized his company’s commitment to safety and quality. “Lonza is a recognized industry leader in HPAPI containment to ensure product and worker safety. Our internal toxicologists work with clients to help classify development toxins and breakdown products into Lonza’s banding system.”

Since 2006, Lonza has invested in a new ADC production facility in Visp, Switzerland. Large-scale CGMP production started in mid-2008. The site passed an FDA preapproval inspection in October 2012 and had been expanded by May 2014. Single–use technology is being introduced late this year. A dedicated conjugates team of more than 135 people are employed in R&D, quality control (QC), project management, and manufacturing.

To illustrate his company’s work in the ADC area, Brady offered two case studies. The first, involving a small biopharmaceutical client, produced a MAb using GS technology at Lonza’s biologics facility in Slough, UK, and used that to make the final conjugated product at its Visp site. With 400 grams of MAb input and a 4.0 DAR, the overall process yield for this three-batch campaign was 95% with a 380-g API output. The second case study involved a midsized biotechnology company and a GMP toxin/linker and ADC made at Lonza Visp. This project is still in progress.

Finally, Brady explained how his company’s approach can simplify the ADC supply chain. Teams are trained in aseptic biologics handling as well as high-potency chemistry (all in a GMP environment). The company provides full or complementary support of MAb development “from gene to GMP” including its GS expression technology for license. Lonza’s analytical expertise covers biomolecules, ADCs, free toxins, cleaning validation, interbatch changeovers, and maximum allowable carryover calculations. Its existing global network of biologic manufacturing facilities complements the high-potency chemistry (both linker/toxin production and conjugation) centralized at the Visp location. That includes back integration into chemical raw materials at a site in Nansha, China. Project management and QC harmonization places all manufacturing steps under Lonza quality systems.

Roundtable-300x168.jpgRoundtable: The History, the Challenges, the Promises of Antibody–Drug Conjugates

Moderator: Morris Rosenberg, founder and president of Rosenberg Consultants


David Rabuka, PhD, founder, president, and chief scientific officer for Redwood Bioscience

Cynthia Wooge, PhD, manager of global strategic marketing at SAFC

Jason Brady, PhD, director of business development for conjugates and cytotoxics at Lonza Custom Manufacturing

David Rabuka addressed the future direction of ADC development. He began by highlighting the complexity of ADC molecules and the processes involved in developing and manufacturing them. Cytotoxic molecules, he pointed out, are often water insoluble. Linkers must allow for release of the drug payload once it has reached its target, but not before. The mechanism of target delivery is important: The ADC binds to a tumor-associated antigen and gets internalized and eventually trafficked to the lysosome, where the protein is degraded and its payload released for action.

Categorizing the future into “four buckets,” Rabuka characterized them as such: site specificity, programmable payload placement, cost of goods, and manufacturing robustness and reproducibility. As he described them, they are interdependent and interconnected. His company is focusing on site specificity as a solution for the next generation of ADCs. “Another future approach to ADC is thinking about new payloads. You need to have the appropriate potency. These molecules need to be very potent, very toxic. So there’s a lot of interest and excitement about different mechanisms for modes of action.”

Cynthia Wooge discussed the benefits of using a contract manufacturing organization (CMO) for ADCs, which she described as the final realization of Paul Ehrlich’s “magic bullet.” The primary reason for making the CMO choice is that ADCs are complex molecules that require expertise in handling biologics, linkers, and highly potent active pharmaceutical ingredients (HPAPIs). They also require access to specialized containment that protects not only the product from the environment, but also the personnel handling it from the HPAPIs. Because conjugates have both biological and chemical attributes, they require very sophisticated analytical techniques.

The associated supply chain, Wooge explained, is complicated in and of itself. First, the antibody is manufactured. Often the client supplies the MAb or at least a process for making it. The linker and payload come from a different source. Formulation, testing, and characterization require different expertise. And the regulatory package in support of such products will be a challenge in itself. Thus, many companies — from startups to “big pharma” enterprises — use CMOs for their ADC needs.

Wooge next elaborated on her company’s offerings in the areas she’d defined. She detailed the ADC development process from technology transfer planning to analytical method development and validation, defining chemistries, GMP manufacturing, and quality assurance and quality control (QA/QC). She highlighted the importance of “front-loading the analytical” aspects. “We’ve seen a shift toward more complicated or sophisticated analytical techniques, those that increase the speed at which we can process samples, those that give us additional detailed information, and those that for the most part require more high-end analytical instrumentation and knowledge in terms of running those instruments.”

Finally she pointed to the advantages in using a CMO for ADC development and manufacturing of having access to that company’s experience with a number of different products, different technologies, and different scales. CMOs have significant experience across a range of platforms, which enables them to leverage platform approaches to quickly develop and get their clients’ products into clinical testing and onto the market.

Jason Brady concluded the roundtable discussion by offering up a brief summary of ADC manufacturing supply chain trends for consideration. He believes that the possibility of expedited drug development programs could change manufacturing strategies. The product class could expand beyond existing indications to other cancer types, nononcology indications, and even earlier lines of therapy. Site-specific conjugation, novel linker technologies, and optimized payloads are all in development already.

Brady highlighted manufacturing challenges, echoing those described by others preceding him. ADC products are extremely potent, thus requring small doses and hence small manufacturing batches. He pointed to targeted niche indications and small patient populations. That’s good because ADCs will be more expensive to produce than standard chemotherapies or even “naked” biologics. So Brady says that developers must improve the therapeutic windows and efficacy over time, identifying candidates for accelerated approval, and using platform technologies wherever possible. Novel linker payloads are becoming more common, however.

A perennial question will be when/whether to invest in dedicated equipment. Initial small-batch campaigns help companies make decisions on when/how to invest. And CMOs offer flexibility: multiproduct facilities that can accommodate small campaigns and novel payloads while remaining equipped for validation and commercial launching to bridge late clinical supply and commercial readiness.

Solutions to all those challenges were discussed. Single-use technology is already well accepted for making preclinical toxicology testing and early clinical supplies. Makers of niche products may implement “mid-scale” single-use or hybrid stainless/disposable set-ups for supplying phase 2a–2b and pivotal trial supplies. The trick is to minimize capital expenditures during that early, high-risk period of development and minimize costs associated with each product candidate. Companies seek faster change-overs and limited cleaning and QC times.

Plant scheduling becomes critical as development goes along. Manufacturers must align their campaigns using similar payload technologies in consecutive slots whenever and wherever possible. CMOs manage facility use with client timelines. Manufacturing processes are industrialized after product launch and before expansion of indications, but before or during phase 3 if a product’s initial indication involves a large patient population. When facility expansion is necessary, companies should expect ~12–16 months lead time. And finally, an experienced, collaborative regulatory affairs team will be critical to ADC success.


An Integrated Approach to Cell Line and Process Development of Therapeutic Antibodies: A Case Study in Development of a Biosimilar
Victor A. Vinci, chief scientific officer and vice president at Cook Pharmica

After introducing his company (a wholly owned subsidiary of Cook Medical group) and its mission (to provide GMP process, analytical, and formulation development, drug substance and drug product manufacturing, and fill–finish), Vinci launched into a discussion of the MAb “molecule to market” life cycle. He spoke of criticality assessments, preliminary analytical control strategies, product and process understanding, risk management, process characterization, product and process mapping, design space, control strategies, and links between critical process parameters (CPPs) and critical quality attributes (CQAs). He also provided a detailed case study based on rapid development of a biosimilar trastuzumab (a model molecule). In collaboration with Selexis (using a client-owned Selexis cell line), Cook created a scalable process using a technology platform approach for commercial-ready titers with minimal development.

Before early development, in-depth molecule characterization supports a criticality risk assessment of product quality attributes and provides insight into protein stability. Based on a criticality risk assessment, a testing plan based on criticality of product attributes is established (with one or more analytical methods associated with each CQA). A key strategy for success and quick turnaround is an established development platform. It allows for significantly reduced parameter screening to develop a process for a class of molecules that meets the target product profile (TPP) and provides assurance of quality. A flexible technology platform enables rapid development for early phase clinical manufacturing. Risk-driven process characterization helps establish design space(s) during late-phase clinical manufacturing. Increased product and process knowledge provides a strong foundation.

The Importance of Media Selection and Scale-Down Models for High-Titer Expression in CHO Cells
Simon Boa, PhD, MBA, director of the Merck Biodevelopment Center

Companies want to optimize expression levels and yields for their cell lines, but most people don’t look at the media and feed stage carefully enough. You can increase your titer three- to five-fold by trying different media, which makes a monumental difference at the commercial stage.

Boa recommended four approaches: You can use the template that arrives and not change anything; you can start from scratch and design your own media; you can optimize by starting with one media and change amino acids or make additions as you go; or you can feed a commercially available media of high quality.

Merck tested a number of conditions. For example, the company tested MAb production using different combinations of media and feed and saw a great diversity of results from one cell line depending on the combination. Boa’s advice is not to look only at media A with feed A. Some cell lines and cell types respond better to new combinations of media and feed, so one combination isn’t perfect for all cell lines. The right combinations can provide a 300% increase in cell density and fivefold increase in product yield (titer). You should also look at different media’s availability in relationship to the location of your processing center and choose a medium that both works the best and is readily available. Regulatory concerns make it difficult to adjust your medium and feed later on in drug development.

Genome Engineering and SURECHO-Mplus Libraries for Optimal Transgene Expression and Recombinant Protein Production
Igor Fisch, PhD, chief executive officer and chairman of Selexis

Selexis has been investing in innovation. The company was founded in 2001 and is based in Switzerland. It has recently developed a way to create a fertile environment for transcription. The platform is controlled by three elements: Chinese hamster ovary (CHO) cell lines, CHO cell libraries, and CHO genomes.

Selexis identified 1,600 genomic elements located in noncoding regions of the CHO genome that play important roles in increasing expression and stability. When starting with a protein to be expressed by CHO cells, the company can identify a team of “silent” proteins to be coexpressed with the protein of interest, improving protein development and leading to an increase in production. The platform does not change the gene of interest but rather the genes around it, which can help it to express itself better. Use of SURE CHO-Mplus libraries can improve production levels of proteins including monoclonal antibodies, enzymes, structural proteins, and fusion proteins.

One case study began with low production in a CHO line. The company used its CHO libraries and found genes that expressed the wanted molecule better by slowing down the ribosomes so that the protein had time to fold properly. In another case, a client had low productivity and high downstream costs for product recovery. By running the libraries, Selexis was able to find a pool of cells that reversed the situation and rescued the project.

Navigating the Biosimilars Landscape: Dispelling the Myths
Sarfaraz K. Niazi, PhD, chairman and chief executive officer of Therapeutic Proteins InternationalNiazi-e1410461787925-300x229.jpg

Why aren’t follow-on biologics called bioidentical instead of biosimilar? Because of the nature of biology, such molecules are never identical to each other. For example, a native molecule such as insulin that is made by the body is variable: The molecule can be different when expressed in the morning from the version expressed in the afternoon.

Chemical drugs are identical, defined molecules. But biological drugs are huge molecules (3-D proteins that fold in particular ways) that even involve a “fourth” dimension based on the environment (media) around them. Variability does not necessarily make them unsafe or undesirable; variability is intrinsic to all biologicals, and that is why they are called similar. As long as a biosimilar is safe and effective, then it is acceptable even if the structure is a little different from the original molecule. However, the manufacturing process must be designed to reduce variability.

Niazi discussed issues specific to the FDA’s ability to approve biosimilars. Their production is increasingly based on single-use technologies. He commented that one advantage with such technologies is that processing will exert less stress on proteins than older methods did. It is a misconception that single-use technologies always raise development costs, minimizing eventual savings for patients. New, smaller, more nimble companies that can keep production costs down will be able to produce lower-cost biosimilars.

Masking and Demasking of Endotoxin (LPS) in Biopharmaceutical Formulations
Wolfgang Mutter, PhD, general manager of Hyglos

Hyglos has invented a new way to detect endotoxins for improving biosimilar development and production. An endotoxin is a breakdown product from bacteria, and such products are everywhere. Normally, endotoxins are not a problem, but when they get into a patient’s bloodstream, they can trigger fever and sepsis. Detection depends on molecular structure.

During processing, endotoxins dissolve and are then “masked” and undetectable. After testing for endotoxins, you remove what you detect. But masked endotoxins can remain; no test can measure them. Masking can have a combination of causes including time, temperature, buffer, and bacterial species. The longer the time you wait to test for endotoxins, the more will have dissolved. More will dissolve at higher temperatures and more alkaline pH levels, so you can reduce masking by keeping temperatures low and using lower-pH buffers. Endotoxins from different bacterial species will dissolve at different rates. The challenge is how to convert an endotoxin from its undetectable structure back to a detectable structure.

Hyglos has filed a patent on its process for adding particular reagents to solution that will demask endotoxins by causing them to return to a structure that can be detected. Depending on your formulation, you would add a different concentration of the reagents. You can purchase a kit from Hyglos containing the reagents. During questioning, Dr. Mutter responded that some drugs are not gaining approval from the FDA because the endotoxin testing is not good enough. The FDA is convinced that Hyglos’s protocol for demasking endotoxins works.


Developing Successful Strategiesfor Commercial Manufacture of Cell Therapies: Case Studieson MSCsand IPSCs
Thomas Fellner, PhD, MBA, head of business development in cell therapy development services at Lonza Walkersville, Inc.

After introducing his company and its process development and manufacturing services, Fellner focused on two main topics: technical and economical challenges of induced pluripotent stem cell (iPSC) based therapies; and strategies for commercial manufacturing of mesenchymal stem cell (MSC) based therapies.

Induced pluripotent stem cells are imbued with the ability to self-renew and the propensity to generate any cell type. “Before either autologous or allogeneic iPSC-based therapies can become technically and economically viable,” Fellner explained, “several hurdles need to be overcome.” Besides (and because of) technical issues of scale, regulatory compliance, and so on, development of iPSCs is a costly endeavor.

Fellner went into more technical detail in his discussion of MSCs. After describing the scaling necessary for cell expansion — with 100 million to a billion cells per dose and millions of potential patients with conditions such as critical limb ischemia, Crohn’s disease, chronic obstructive pulmonary disease (COPD), and congestive heart failure — he showed how his company expands MSCs. Lonza uses microcarrier-based suspension culture rather than the tray systems common at preclinical stages and claims significant cost savings as a result. Fellner showed how it is possible to get a trillion MSCs out of a single bioreactor.

Integrating Technologies to Seamlessly Transition Cell Therapy Products from Clinical Research to Commercial-Scale Manufacturing
Clive Glover, PhD, product manager for cell therapy technologies at GE Healthcare

Industrializing autologous cell immunotherapies requires radically new equipment that is different from that used in a research lab. The three challenges for commercial production are to develop automated systems, minimize the process footprint, and ensure the chain of custody.

A primary source of expense is the cleanroom space needed at commercial scale. To treat 400 patients, you need 1,100 ft2; to treat 4,000 patients, you need 4,000 ft2; and for 20,000 patients, you need 15,000 ft2. To make the footprint as small as possible, use a single, repeatable unit that can handle parallel processes (multiple samples). You can start stacking and use all three dimensions.

GE is used to building infrastructure and designing software. Its software tracks patient samples and ensures that the right facilities, equipment, and raw materials are all available when needed. During the presentation’s Q&A session, Glover discussed processing at small rather than large scale, avoiding stacking as a solution in earthquake-prone areas (or using antivibration beds instead), and the fact that you don’t need an incubator with this system because each unit comes with complete gas heating and full environmental control.

Best Practices in Supply Chain Logistics: How Blood Centers Participate in Collection, Storage, and Delivery of Cell Therapies
Lisa Shaffer, senior vice president of Blood Centers of America

One major resource for logistical distribution of cell therapies is the existing network of blood, plasma, and tissue-collection centers. Shaffer highlighted the capabilities of Blood Centers of America (BCA), which has been working with cell therapy companies since 2006. Started 28 years ago with four blood centers, BCA has grown to 55 across the United States and in Canada. Some centers have multiple collection sites in multiple states — overall, blood is collected, processed, and/or tested in over 400 sites.

The blood is tracked from start to finish, from the needle in a donors’ arm to the actual transfusion of product or outdate. Every unit is considered a drug and given a lot number. Whole blood is usually not transfused, but instead broken up into cells, platelets, and plasma.

BCA follows an approved healthy donor protocol for which every donor must be consented and qualified. Blood centers are always in need of donors because blood remains alive for only 42 days. BCA also recruits donors for companies for preclinical studies and helps find patients with certain blood characteristics for clinical trials.

The company is currently contracted to provide five biotech companies with phenotyped red blood cells. It is also collecting (by consent) cord and birth tissues (with nine cord banks) and is experienced in disease and genomic testing (with a large database on typed donors). In the future, BCG hopes to store products for companies locally and to assist companies through its existing shipping network. The company’s goal is to get more involved in processing and manipulation of cells.

During Q&A, Shaffer was asked how far along BCA is in working with cell therapy companies. She responded that in these early stages, BCA is working with companies to develop protocols and quality standards.

Cell Therapy Commercialization Through Automated Processing
Robert Speziale, vice president, business development, Invetech (substituting for Richart Grant, executive vice president, life sciences, Invetech)

Invetech is a >30-year-old company that has been working in cell therapy for 10 years. Speziale began by commenting that the trouble with research isn’t failure, but rather finding affordable success. He suggested “profit by design”: translating great science into good manufacturing practices by considering safety and efficacy, reproducibility, scalability, and affordability. Invetech uses engineering to turn the science into commercial success. Back in 1980, recombinant proteins were challenging to work with: Raw materials were expensive and fragile, companies didn’t have the technology to work with them, regulatory guidance was lacking, and the manufacturing process was variable and unpredictable. Now in 2014, we are hearing the same challenges described about cell therapies. Many difficulties can be overcome with automation. It can close the process to keep a totally aseptic environment, optimize manufacturing resources, support running parallel processes, sustain process integrity (chain of command maintained, products segregated, errors minimized), and improve data integrity by computer-tracking all steps in a process.


Roundtable: Advances in Novel, and Single-Use Technologies in Protein Purification — the Future of Chromatography

Moderator: Eric S. Langer, managing partner at BioPlan Associates, Inc.


Chris Forespring, senior manager of downstream production at Medimmune/Astra Zeneca

Francis Torres, principal in bioprocess manufacturing and technology at Merck

Paul Jorjorian, senior scientific manager for purification at Gallus BioPharmaceuticals

Duncan Low, executive scientific director at Amgen

The speakers began with short introductory statements. Moderator Eric Langer, who publishes an annual survey of the industry’s developments and directions, noted that once challenges are solved in downstream operations, the industry will take a huge leap in growth. Regarding whether the industry will stay with protein A or move to alternatives, he reported that ~45% of survey respondents are looking for alternatives, but only 1.6% are actually trying them out.

With 17 years of experience in process development, downstream processing, and manufacturing design, Francis Torres has evaluated different chromatography systems including protein A and alternatives. He identified different process development needs (number of samples, volume, and speed), clinical space (flexibility and speed), manufacturing space (reliability, low cost operations, productivity, and increased capacity), and customers and investors (productivity and meeting demand for patients). Torres is looking ahead to sterile chromatography, resin reuse, membrane chromatography, reusable prepacked columns, and increased availability of single-use technologies. Another area of interest is crystallization without any chromatography.

With 12 years of bioprocess development, engineering, and industry experience, Chris Forespring specializes in evaluating and implementing new technologies. He said manufacturers are interested in the three “Cs”: cost, consistency, and capacity. Processes need to be simple and robust. A barrier to convincing companies to use novel processes is that it is hard to guarantee consistent success. You can overcome this barrier if a product or process can fill a need or solve a problem, improve throughput, help ergonomically (e.g., by expanding operator space and/or increasing flexibility), and demonstrate robustness. MedImmune tests new technologies at pilot scale to provide proof of concept, sometimes in partnership with a supplier. As a technology shows promise, the company demonstrates scale-up and reproducibility and evaluates the reliability of supplies.

Paul Jorjorian’s team specializes in development and scale-up of GMP protein processes. Gallus BioPharmaceuticals is focused on increasing efficiency and reducing costs. Jorjorian gave two case studies showing cost-effective alternatives to protein A. In the first study, a small biotech company was working on a shoestring budget and substituted Repligen’s CaptivA resin for protein A at a fraction of the cost. In the second case, Gallus collaborated with Natrix Separations (Canada) to use a hydrogel chromatography technology. Although it was not quite as good as protein A in removing impurities, it was a lower-cost and effective option.

Duncan Low, who has been with Amgen for 11 years, leads the Raw Materials Global Network Material Sciences team and is an active member of the PDA’s task force on single-use and ISP’s executive committee for process analytical technology (PAT). Low focused on materials selection and design. He emphasized the importance of paying attention to compatibility of materials and functional specifications. Novel technologies must be robust to have value. Verification must be performed throughout a process through preuse inspection, in-process monitoring, and postproduction testing. A quality audit can be complemented through a technical diligence visit to a supplier’s facility. Change control must be clearly defined and communicated along the entire supply chain. Understanding variability across the supply chain and throughout the product life cycle is essential. Complaints from patients must be dealt with expediently by tracking down the reasons, whether they are attributable to materials (supplier issues) or procedures.

The panel discussed the use of affinity tags, the price of protein A, continuous processing, antibody purification, acceptance of novel approaches, comparative benefits of new and retrofitted facilities, the industry’s willingness to adopt alternative technology, supplier and customer integration, supplier partnerships, and the overall goal of safe, effective, and affordable medicines.

Implementation and Use of Single-Use Products: A CMO’s Perspective
Michiel Ultee, chief scientific officer at Gallus BioPharmaceuticals

Gallus is an experienced and “pure-play CMO” that works only on its clients’ products. It embraces single-use technology for the ability to work on many kinds of projects with a quick changeover between products, the quick set-up and installation, the variety and flexibility in design, the portability of equipment, the lower capital and utility costs, and the reduced carbon footprint. Ultee showed examples of single-use equipment. The upstream application consisted of small bioreactors (48 on a benchtop unit), which can test many variables in a short time using either wave or rocker designs. In a scaling study, he reported consistency between small and large sizes of bioreactors. The small ones can be representative of the large ones.

Gallus likes the convenience of using buffers in bags. For the chromatography step, he said that a company can use membrane chromatography or columns. Some membranes are made to work with higher salt concentrations, so you can streamline the process by not having to perform a dilution. One caveat is to make sure that your product doesn’t stick to the membrane. If you use columns, compare prepacked columns and standard ones — the trade-off is the time and expense to clean, repack, and test columns compared with the price of prepacked columns. CMOs have embraced single-use technologies because of cost, turnaround time, and flexibility.

Lowering Operational Costs with New Protein A Solutions for Single-Use Facilities
Aaron Gilden (general manager and sales director), stepping in for Kiran Chodavarapu, PhD, business development manager, Grace Discovery Sciences

Five key downstream challenges are the cost of chromatography media, the lack of disposable options, the expense of membranes, the need for ongoing cleaning and validation steps, and the efficiency of operations. Using protein A can require much labor and be time-consuming, but it also comes in cost-effective, disposable, prepacked columns.

Gilden presented two case studies. In one, a 48-L stainless steel column was replaced with a 48-L single-use column. The stainless steel column cost $920,000 to use, and the single-use column cost $330,000, representing ~$600,000 savings. Although greater speed wasn’t a goal (197 versus 250 hours), the savings in column cost and the short running time gave the company a 57% total cost saving. In the second case study, a CMO wanted to reduce labor and in-suite time. It was using a 13-L column in a four-cycle run. The company replaced it with a 24-L disposable column. It operated for two cycles, was cleaned in place, and then run for two more cycles. That cost $290,000 and took 145 hours to use the 13-L column compared with $190,000 and 27 hours to use the 24-L column. Purification time was reduced by 81%, and the cost of operations dropped ~40%.

During questions, Gilden said that his company’s largest column is 45 cm but that a 60-cm column is being developed. The resins have a silica backbone, Columns are sold prepacked and can handle sodium hydroxide for eight hours and 100 cycles.

SU Development and Use of Single-Use Vials for Sterile Processing
Jean-Pascal Zambaux, president and chief executive officer at Disposable Lab

Disposable Lab is a four-year-old company based in France, with a single-use fill-and-finish system. The patented technology is being used in Europe, the United States, and Japan. The enclosed system prevents environmental contamination and protects operators who work with toxic products. The system (which is not yet automated) uses gravity rather than a pump for vial filling. A modular unit can be shipped and installed anywhere in the world. This system can be used for both small and large filling needs (1,000 to 10,000 vials per batch with fills from 0.2 mL to 100 L). For small batches, disposable kits are available. The system can fill, stopper, and cap syringes and weigh vials. Disposable Lab will deliver a prefilled, decontaminated isolator with the specified vials in baskets and already sterilized. Customers can then connect the isolator to their own equipment and start filling vials. The vials are validated and tested for particulates and sterility according to USP guidelines.

How to Go into Commercial Scale with Single-Use
Ian Sellick, director of marketing at Pall Life Sciences

Single-use is becoming the industry standard at the clinical scale, but for commercial-scale manufacturing, most companies are still using stainless steel. Sellick noted that within five years, 46% of manufacturers are expected to have a single-use commercial facility. The industry is also moving toward automated processes with complete data logging.

To transition to a truly automated, single-use, large-scale commercial operation, a company must remove operator intervention, make it as foolproof as possible when it must occur, make the single-use set up easy to assemble (e.g., using color-coded tagging, placing simple pictograms next to operation), and have the ability to completely automate the process (including batch recording of every step and electronic sensors, detectors, and gauges). Tubing and connectors are now being developed in larger sizes (1 in.) at the commercial scale. Process control and reproducibility can be improved using “friendly” software. Companies should look into available downstream applications (disposable chromatography is being developed along with automated systems to run the columns) and use disposable filling for larger applications.

An alternative to a separate single-use system can be a multiplex system with a single controller. The benefits are the control of key parameters and critical process steps, consistency of product quality, reduction in equipment and labor costs, reduction in operator errors, and batch reporting. Single-use technologies might make some cell therapies commercially viable. A question was asked about advances with centrifugation, and the answer was that two single-use versions are now available.

Regulatory Compliance in Single-Use Biomanufacturing Facilities Requires a Collaborative Approach with Your Supply Chain
Todd Kapp, market development manager, Life Sciences Group, Parker domnick hunter; member of the Bioprocess Systems Alliance (BPSA) Board of Directors

Todd Kapp’s six main topics covered the history of single-use and collaboration acceptance, the goal of collaboration, expectations from both the user and the supplier, an overview of the supply chain, guidelines and standards, and experiences of Parker domnick hunter. Single-use supplies were first available in the 1960s as bags for collecting and storing blood. In the 1990s, single-use bioreactors were first being used. The Bioprocess Systems Alliance (BPSA) was formed in 2005 by suppliers to develop guidelines for the industry. The supply chain begins with plastic resins, which are converted into films, tubes, connectors, and membranes and then assembled into filters, tubing manifolds, and bioprocess containers or bags. These products must be sterilized, have a shelf life that includes functionality and packaging integrity, and perform as expected. A key point emphasized was that “the process is the product.” The ultimate goal of collaboration is patient safety and wellness. It is critical for both users and suppliers to communicate their expectations to each other and to identify critical quality attributes.

A supplier must have robust quality claims that can be consistently supported by testing data, understand the global regulatory requirements and track updated standards, demonstrate strong internal quality requirements, and respond rapidly questions and concerns. Because there is variation in raw materials and consumables, users should have a policy in place for accepting or rejecting raw materials, a system for evaluating suppliers, agreed upon specifications for the materials purchased, transparency, and a change-control process. Companies that use single-use technologies no longer have total control over their processing. They are like parachutists, and suppliers are the parachutes.

The Future of Biomanufacturing: Faster, Better, Cheaper
Nihir Parikh, business development leader for Enterprise Solutions ASIA (substituting for Parrish Galliher, CTO, upstream, GE Healthcare)

Large biopharmaceutical facilities are giving way to smaller, more efficient facilities. Considerations for design of new facilities include bringing the entire facility together as a single solution with a standard platform. Nahar noted that the money lies in making medicines, not facilities, so facilities can be made simpler. With companies establishing multiple facilities around the world, it is essential to have the right people in the right place with cost-effective timelines.

One technological solution is a FlexFactory approach, in which an entire process (from seed to bulk filling) is in one building. This is a flexible, multiproduct facility enabled by single-use technology and centralized automation that can operate any equipment within the facility. Product management including documentation and staff development and training are customized to clients’ needs.

Parikh believes that the industry is moving toward personalized medicines. He says that after two generations — from big facilities to single-use bioreactor facilities — the industry is now investigating “wearable devices.” In 20 years, a hand-held device might make one dose at a time when a patient needs it.


Preparing for Preapproval Inspection (PAI): How toTransition Operations from Early Stage to Phase 3 and Commercial Production
Patricio Massera, general manager of the Copenhagen facility for CMC Biologics

CMC Biologics was founded in 2001 by six people in Copenhagen. The company now has three sites (Copenhagen, Seattle, and Berkeley) and 400 employees. Massera shared their experience in moving from clinical manufacturing to commercial production, necessitating changes in the company culture, systems, implementation, and facilities.

First, they determined what was needed to meet FDA guidelines and regulations. They used gap assessments to look at their systems. Some gaps were fixed by small procedural changes whereas others required systems upgrades. For example, the company implemented a master control system. It established leaders in areas being upgraded to oversee employee training, and upgrades ran parallel to routine work. Manufacturing and quality teams were the most affected. Teams were restructured according to particular tasks and projects, new objectives were set, and roles and responsibilities were clearly defined. The company’s multipurpose facilities include both mammalian and microbial lines and use both stainless-steel and single-use technologies; projects are segregated. To prevent unnecessary human traffic from suite to suite, the company generated a unidirectional flow by attaching an exterior corridor. In that case, an engineering solution (remodeling the facility) was preferable to a procedural solution. Water distribution was upgraded, and warehouse capacity was increased.

Assessment through validation took 18 months, and in another six months the company started using the new system. In 2013 the facility was approved in Denmark. CMC Biologics is expecting to receive FDA approval by next year.

Product Development Partnership: AERAS as a Model
Peter Alexander, senior director of technical operations at AERAS

Founded in 2003, AERAS is a small but fully integrated biotech organization (funded by the Bill and Melinda Gates Foundation) focusing on diseases of global impact. It functions as a product-development partner linking funders, academics, manufactures, pharma partners, government agencies, and clinical sites. Its mission is to develop tuberculosis (TB) vaccines. As a product-development partner, it helps develop its partners’ vaccine products by working as a CMO. AERAS has facilities in Maryland, Beijing, and South Africa.

In the past 200 years, more than a billion people have died from TB. In 2011 alone, there were almost 9 million new cases and 1.4 million deaths worldwide; in 2012, there were 10,000 cases in the United States. Over one third of the world’s population is infected with TB. A major complication is the tendency for coinfection, especially with AIDS. Drug-resistant strains of TB are becoming more prevalent. The only vaccine presently in use was developed 90 years ago and can be used only in pediatrics.

The AERAS partnering model is to stimulate product development by supplying grants to drive discovery and preclinical development through clinical trials, cost sharing, and late-stage development and market launch through commercial partners. Contract manufacturing includes preclinical development through clinical trials management, process development, and cell banking. GMP manufacturing includes purification and formulation, fill, and finish. AERAS does not use single-use technologies.

Developing a Robust and Scalable CGMP Protein Manufacture Process
Daniel Smith, PhD, chief scientific officer of Cobra Biologics, and Julian Hanak, commercial director of Cobra Biologics

Julian Hanak spoke about setting up a manufacturing process. In addition to considering titer, expression stability, expression levels, and cell line growth, a company must characterize its product to select the clones that are best for productivity and potency.

Cobra’s expression system is the MaxXpress (maximum protein expression) platform with ubiquitous chromatin opening elements (UCOEs) from ubiquitously expressed genes that are never turned off. Inserted transgenes are sometimes silenced by chromatin, so UCOEs keep that from happening. Expression is enhanced from 6% to 80%, with more stable expression in more clones. Cobra looks at thousands of clones robotically to pick the best, most productive cells.

In one example, the company compared selection processes of robots and scientists; a robot could deal with 366 clones, whereas a scientist dealt with 110. Both found high-producing clones, but the robot found more of the best producing clones. Candidate clones are evaluated using a microbioreactor robot: 24 individual fermentations are performed simultaneously. The analytical step is automated to screen a large number of clones. Different fermentations provide different profiles and can be optimized at the small scale.

The real key is to look at potency and CQAs early on. This is particularly important in biosimilar development. Cobra’s automated platform for cell-line development maximizes the chance of finding high-producing, stable clones. It can make the correct product at a very small scale in disposable bioreactors and go to full-scale production using disposable 250-L to 1,000-L bioreactors under CGMP conditions.

The Future of Aseptic Filling: Automated Filling Robots Coming to a Clean Room Near You
Gene Yoshioka, director of manufacturing at Avid Bioservices

Avid is a CMO specializing in biologic development and commercial manufacturing since 2005. The typical filling line is built for high throughput rather than for minimizing product loss, but Avid sees every drop of product as valuable. Throughout different product phases, it is desirable to be able to use the same system to fill different types of containers (vials, syringes, bags, or cartridges) so as not to have to requalify a filling line.

Avid is excited to introduce robotics into the pharmaceutical industry. Robotics can be programmed with a certain amount of intelligence that can react to situations. Avid worked with Automated Systems of Tacoma in 2012 to design a robotic filling system with certain features: It can function intelligently (adjusting for required filling volumes and reacting to environmental events), fill different types of containers, occupy a small footprint, minimize product loss, and be used from pre-clinical through commercial stages (managing batch sizes from small to large).

A questioner asked, “Do you monitor for viable and nonviable particulates?” Avid does have both passive and active monitoring to detect for microbial growth.

Roundtable: CMO/CDMOs — How Technologies Are Changing Outsourcing Strategies, Services Offered, and Risks Taken

Moderator: Susan Dexter, principal consultant at Latham Biopharm Group


Economic Trends: Rob Silber, director of supply chain/business contracts at Boehringer Ingelheim

Technology Advancements: Andrew Sandford, vice president of business development at Catalent Pharma Solutions

Facility Designs: Bill Reed, PhD, senior vice president of operations at Kalon Biotherapeutics

Service Offerings: Mark Bamforth, president and chief executive officer at Gallus BioPharmaceuticals

Moderator Susan Dexter has been working with CMOs since 1986 and believes that people are now much better informed than in previous years about how to select a CMO.

Rob Silber manages third-party contracts and supply chains. Started in 1885 in Ingelheim, Germany, his company now has 47,000 employees and produces medicines for both humans and animals. Silber said that companies outsource to enter new markets quickly, reduce risk in developing new products, reduce cost of building and maintaining facilities, create more capacity and flexibility, and bring in technology that they do not have in-house. BI uses both stainless steel and single-use technologies: single use during phases 1 and 2 (when producing a small amount of product) and stainless steel for large-scale manufacturing. BI’s facilities are identical to each other, making process transfer extremely efficient; knowledgeable staff go with the project from one plant to another. CMOs must keep up with the technology that their customers want. Customers want robust processes, high-quality systems, and low regulatory risk.

Mark Bamforth has 22 years of experience in the United Kingdom and the United States. Gallus uses single-use technologies in commercial manufacturing. Selecting a CMO partner is a big decision for small companies, so they should consider regulatory compliance, both short- and long-term needs, technology fit, experience with complex molecules, ability to meet timelines, location, culture fit (including ability to communicate with one another other), and one-stop-shop approaches rather than specific process expertise.

Andrew Sanford is a microbiologist with experience in gene expression. Catalent is a leading supplier of technologies for drug delivery systems and biologics.

Instead of changing existing processes, the company looks at lowering the cost of goods and accelerating development timelines. The FDA supports innovation that drives down costs and timelines but maintains quality. Personalized medicine will be the wave of the future, including phenotypical medicines produced individually for patients. Using small-scale technologies such as miniature bioreactors is a faster way to produce testable material.

Bill Reed has spent 25 years as a manufacturer. He led one of the largest vaccine platforms in the world and oversaw providing the H1N1 vaccine during the pandemic. Located on the campus of Texas A&M, his company focuses on at cell culture and viral process development and vaccine manufacturing. Through a partnership with the university, it contributes to curricula, provides lectures, supports interns with training on industrial equipment, and recruits from the best of the graduates. The facility has capacity for 20 completely closed mobile clean rooms with integrated disposables. Segregation is both structural and procedural to ensure integrity of individual projects. Reed gave a virtual tour of the facility and explained its set-up, how sterile conditions are maintained, how the design keeps things flexible, and the ease with which workers can do their jobs.

Dexter then asked a series of questions for discussion. Gallus focuses on product development in one plant and on commercial manufacturing at the other plant. It is able to move from one to the other by sharing people and having common practices and equipment. BI regularly transfers projects from Germany to the United States or China; multilingual employees travel with the projects. Global quality systems meet local requirements. Catalent is making the right investments where the market is most seeking innovation.

All panelists agreed that single-use technologies are here to stay, particularly during process development, and are also becoming more common at commercial scales. Companies are looking at the risks and benefits of using new technologies including the ability to get FDA approval.

The Client Experience: The Key to a CDMO’s Sustainable Future
Safa’a Al-Rais, senior director of venture management at Therapure Biopharma

Therapure is located near Toronto, ON, in Canada. Al-Rais says the secret to its success is a process of “painting pictures” in planning that evolves with clients. By “telling stories,” the company shows clients and staff how to achieve their vision and objectives. In the industry’s highly competitive markets, clients use CDMOs that are experts in particular fields. Elements of a client-centric organization include sharing clients’ vision and focusing on their needs, aligning those with Therapure’s vision, helping clients identify needs and objectives, adopting innovative ideas, suggesting process improvements based on expertise,

emphasizing a team culture, adopting the client’s passion for its project, and valuing patient safety above all.

GMP Considerations for Bioprocess Design: A CMO Perspective
Les Tillack, chief executive officer at PharmaSynth Pty Ltd.

PharmaSynth is one of Australia’s most experienced CMOs, producing vaccines, immunotherapies, gene therapies, recombinant proteins, and synthetic molecules for 20 years. Tillack shared lessons learned in designing new bioprocesses. He advised finding the simplest expression system (bacteria, yeast, and mammalian cells) that will work for your product and aiming for the highest yield. Look for a protein that is expressed in a robust form and is not hard to keep intact.

Tillack suggests that when going from the laboratory to GMP manufacture, companies should document the generation of the cell line so that it can be validated. He cautioned against using ampicillin as the selective agent in a bacterial expression system because it can’t be used in a GMP facility. “Keep some of the original cell line aside for subcultures,” he said. “When selecting media and growth conditions, make sure that your raw materials are suitable for a GMP facility. Make sure that the media you plan to use can be used in the country where your facility is located, and pay attention to sterilization and quarantine requirements.”

Cost should be considered at early stages: If you use an expensive resin at the development phase, it will be too expensive to scale up. For purification, Tillack advises using the minimum number of steps possible because “every time you add a chromatography step, you will lose 20% of your protein.” Initial analytical work can show whether the extra steps are increasing purity or not. “Don’t use novel purification systems,” said Tillack, “because regulatory agencies might not accept them, and they can be cost prohibitive. Design processes to use a minimum of polymers. Minimize formation of protein dimers because they are hard to get rid of. It is easier to manufacture products without protein tags because they have to be removed. In analytical work, plan ahead for testing your product’s efficacy, potency, and purity.”

Alison Center is editorial assistant ([email protected]); Cheryl Scott is senior technical editor ([email protected]), and S. Anne Montgomery is editor in chief ([email protected]) of BioProcess International, PO Box 70, Dexter, OR 97431. Recordings of many of these full presentations are available online at http://bit.ly/BPI-BIO2014.

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