Biotech/biopharma startup companies are experiencing generally favorable funding environment for innovative drug development ideas thanks in part to the regulatory approval success of new drug products in recent years. In 2018, 63% of new molecular entities (NME) approved by FDA were initially developed by smaller bio/pharma companies, according to Switzerland-based venture capital firm HBM Partners (1). Today, it is these smaller, innovative companies that account for the majority of the early development of new therapeutics.

	Biotech startups are good environments for innovation and early drug development work for three key reasons. For one, startups have relatively low capital needs and overhead costs while they address key technical challenges. Second, founders typically have the luxury and the drive to constantly assess risks, which is crucial for any startup, and can make a thorough study of the best markets that their technology can best serve. Third, founders of startups have incentives in terms of real-world impact and financial return, so they are often most motivated to see their technology/innovation succeed and will push for that (2). 

	Because smaller companies are more flexible and agile, they can move faster and push projects forward more aggressively than larger pharmaceutical companies. The cost for developing a new drug and bringing it to market has been estimated at upwards of $2 billion, according to industry experts (3), and major pharmaceutical companies do not generally want to take on the burden of early innovation and failures.

	Instead, major pharma companies are looking at startup biotech firms-either by spinning out their own or collaborating with startups with interesting technology-to take on the work of early innovative drug development. This way, Big Pharma can minimize risks at the early stages of development and, if successful, have a pipeline of potential new, innovative drug products. This approach bodes well for biotech startups because, as part of an overall strategy to revive R&D and break stagnation, major pharma companies are actively seeking out startups and are investing in them (4). 

	Funding for biotech startups is favorable these days, not just from large pharma, but investment firms as well. Today, compared to two decades ago when the biotech bubble burst, venture capital (VC) firms show more willingness to return to the biotech space (5). A common model used today for starting up a biotech company is one where the VC creates the company by putting together a hand-picked executive team to develop an idea. The VC houses the team in its own offices and invests a substantial amount of money into the venture, which allows the VC to take a controlling ownership stake from the start.

	However, a VC-created startup is not the only model for getting a new biotech firm up and running. In addition to the larger investment houses, smaller firms have now also come into play and are looking to make small investments in promising new initiatives. Typically, a large VC does not waste time making small investments, which in the past was a challenge for biotech startups. Often, a startup had to work hard for major funding and could not begin work unless an influx of cash in the millions-of-dollars range was received from a VC. 

	Nowadays, the cost of starting up a new company has dropped significantly, supported by changes in infrastructure led by “a combination of open source software, modern web frameworks, SaaS [software as a service] developer tools, cloud hosting, and better distribution channels” (6). Instead, a biotech startup can run on a small infusion of cash sufficient to get it to the next funding point, such as validating the molecule through modeling processes or demonstrating efficacy in animal studies. This approach has opened up opportunity for smaller investment firms to fund early biotech companies. For example, for as little as $100,000-compared to a traditional startup investment of $10 million-biotech founders can work to prove that their innovative concept works; whereas in the past, they couldn’t move forward unless a large VC provided funding (6).

	Establishing a startup in biotechnology or biopharmaceutical drug development requires thought and strategy, as much as an adventurous spirit. Failure can hinge on many reasons, including a flawed financial strategy, inexperienced management, poor or lackluster science, bad timing, and/or not taking enough calculated risks (7).

	Industry experts recommend that entrepreneurs clearly identify the unmet market need that its technology can address. Founders must clearly state the problem that the startup can solve to the foundation of the company’s value to the marketplace.

	“It is important to solve a problem you are passionate about, but there must be a large enough market for this technology; in other words, make sure that there are enough people who care about this problem enough to pay for your solution. Going after a small or niche market is acceptable, too, but your sources of capital will be fewer, and you will need to clearly articulate how your company can even recover its costs. When looking for your market, ‘don’t be a hammer in search of a nail’; that is, be objective when identifying a need instead of trying to make your special interest into a market that might never exist,” wrote Adriana Tajonar from the California Institute for Quantitative Biosciences in a perspective piece published in Molecular Biology of the Cell (2).

	Executives in the startup trenches have a first-hand look at the challenges of building a biopharma company. For example, when building a biotech/biopharma company from the ground up, key goals that founders should pursue include getting to cash-flow positive as quickly as possible to limit dilution; attracting motivated, entrepreneurial and experienced team members; creating shareholder value; and applying a business model that enables continuous growth, but is simple to execute, according to Lindsay A. Rosenwald, MD, chairman, president, and CEO of Fortress Biotech, Inc. 

	Fortress Biotech, a rapidly growing New York-based biopharmaceutical company specializing in the identification, in-licensing, development, and marketing of FDA-approved pharmaceuticals and high-potential clinical-stage assets, has more than 25 programs in clinical and preclinical development with product candidates spanning six therapeutic areas, including oncology, rare diseases, and gene therapy. The company has established partnerships with academic research institutions and biopharmaceutical companies. 

	Evelina Vajeso, CEO of Ilya Pharma, an Uppsala, Sweden-based clinical-stage biopharmaceutical company, says building up data step by step, reducing risks, and planning out the development strategy for a drug candidate are also important.

	Ilya Pharma targets unmet clinical needs such as the treatment of post-surgical wounds in special populations, problematic diabetic wounds, and indications in the gastrointestinal tract, such as inflammatory bowel disease. The company’s lead candidate, ILP100, EudraCT No. 2019000680-24, is currently in its first in-human trial, and a Phase II program is planned to start in 2020 under investigational new drug status. 

	Rosenwald recommends using business development as R&D (don’t try to invent anything); putting parameters around the types of product candidates to acquire (e.g., clinical-stage product candidates that have excellent human data, unmet medical need, and a clear path to market); and building relationships with the best research organizations. 

	Success also hinges on keeping in line with certain best practices, such as maintaining excellent relationships with key opinion leaders (KOLs) and listening to advice from quality consultants and advisors. “KOLs (particularly those well-versed in the field of medicine that a startup’s product candidate is meant to serve) will help you avoid poor choices when it comes to drug development. Another best practice is to work with world-class consultants and advisors to make sure your development programs are formulated with the highest chances for clinical success. And last but not least, hire experienced and motivated employees to facilitate efficient and successful execution of your programs,” advises Rosenwald.

	Vajeso also points out that, even though many may say something will not work, experience has taught her otherwise. For her, a good practice to follow is surrounding oneself with people who have an open mindset and are eager to learn and develop.

	Ilya’s lead candidate, ILP100, is in the cell- and gene-therapy space, and Vajseo says she is surprised at how fast the project is growing. “We are currently running a first-in-human study but really mostly working with the Phase II programs for new trials to start next year. In developing a new and innovative compound, it is crucial to work with the best senior experts with an open mind and who have seen multiple complex compounds before. Do not waste time with generic advice,” Vajeso says. 

	Experts also advise avoiding major pitfalls early on, such as taking on too much risk at the start. “My advice is to create a durable business model, grow slowly, forge partnerships, and spread the risk,” Rosenwald cautions. “Another pitfall to steer clear of is sticking with losing product candidates for too long. Cut them early!” 

	In Fortress Biotech’s case, the company had to overcome a challenge related to its drug candidate for amyloidosis. Rosenwald explains that the company had to deal with market confusion over Fortress Biotech’s drug candidate and a competitor’s product that was further along in clinical trials. Though Fortress Biotech believed its candidate demonstrated superiority over the competitor’s product, the confusion over the two drugs made it difficult for the company to raise the necessary development capital. “We recognized that we had to pivot and were fortunately able to forge a partnership with a large pharmaceutical company in a $500-million deal to develop the drug candidate,” Rosenwald explains.

	Quality is an integral aspect in drug development, not just quality of the product, but quality of the environment, processes, and company mindset. In Vajeso’s opinion, enforcing quality is about “always keeping the science and data in mind.” Beyond that, having good people with key expertise goes a long way in implementing the necessary quality aspects in drug development.  

	For Rosenwald, enforcing quality into the core of a company depends on having a dedicated management team, and having external advisors who are experienced in the areas you are working in to help grow, support, and maintain a culture of quality.

 (HBM Partners, Zug, Switzerland, January 2019).
	2. A. Tajonar, “How to Start a Biotech Company,” 

25 (21) 3280–3283 (2014).]
	3. J.A. DiMasia, et al., 

47, 20–33 (2016).
	4. JLL, “Big Pharma Poised for Disruption in 2019 While Mid-Tier Companies Double Down on Innovation,” Press Release, Jan. 17, 2019.
	5. B. Booth, “

The Creation of Biotech Startups: Evolution Not Revolution

How Biotech Startup Funding Will Change in the Next 10 Years


	Vol. 32, No. 12
	December 2019
	Pages: 12–14

	When referring to this article, please cite it as F. Mirasol, “Building a Biopharma Company,” 

Articles

Cover Story

The innovative spirit of biotech startups is a driving force behind the development of new therapeutic products, but building a successful biopharmaceutical company from the ground up has its risks and challenges.

Building a Biopharma Company

The number of product approvals for cell and gene therapies is rising steadily. Earlier this year, FDA issued a statement revealing that there are already more than 800 active cell-based or directly administered gene therapy investigational new drug applications (INDs) currently on file, and that by 2020, they expect to be receiving more than 200 INDs per year (1). Based on this forecast, the industry expects cell and gene therapy products to surge in the market and remain a prominent part of the pharmaceutical industry for years to come (2).

	FDA has developed four distinct pathways to speed the progress of promising new therapies through clinical trials, and if results merit, on to approval. Advanced therapies may be eligible for one or more of these designated pathways if they treat a serious condition, address unmet medical needs, or obtain early clinical results that show significantly improved outcomes compared to current therapies. In 2018, FDA’s Center for Drug Evaluation and Research (CDER) issued a record-breaking 59 new drug approvals (3). Of these new approvals, 32% were authorized as first-in-class, and 58% were approved to treat rare or “orphan” diseases. 

There are three cell therapy products that have been approved thus far: Provenge (sipuleucel-T) from Dendreon Pharmaceuticals, Kymriah (tisagenlecleucel) from Novartis, and Yescarta (axicabtagene ciloleucel) from Kite Pharma, a Gilead Sciences company. These are likely to be the tip of the iceberg. Because many new cell and gene therapies are targeting unmet medical needs, orphan diseases, or both, they are much more likely to request (and receive) accelerated approval. The Alliance for Regenerative Medicine reports that as of the end of 2018, 263 cellular therapies and 362 gene-modified cellular therapies were being evaluated in clinical trials (see 

) (4). The majority of these clinical trials are for cancer indications, with chimeric antigen receptor (CAR)-T cell therapies alone accounting for 181 Phase I–III clinical trials (5). 

	While a robust pipeline is certainly a strong indicator of future therapeutic success, the rapid increase in the number of cell-based therapies headed toward commercialization is placing growing demands on cell-therapy starting material suppliers. Unlike other biological drug products such as therapeutic proteins, monoclonal antibodies, or vaccines, drugs based on living cells are by nature uniquely vulnerable to supply chain complications and setbacks. Apheresis center capacity, donor network access, sample handling expertise, and quality control systems all contribute to the ability to achieve reproducible starting material quality and efficacy.  

	The raw materials for allogeneic cell therapies cannot be manufactured on demand, but instead, rely on a network of volunteer donors. This creates variability in cell therapy starting material from the outset. Small donor networks are vulnerable to issues, such as unexpected drops in the number of donations, uneven reliability, and inadequate diversity to meet the specific needs of their clients. Sourcing cells from a large, diverse donor network avoids many of these complications, but cell-therapy manufacturing and supply companies must still deal with the inherent variability of living cells and tissues. 

	Many different factors can introduce variability into the total yield of white blood cells (WBC) and other therapeutic cells during apheresis collection. Disease, for example, impacts cellular functional capabilities and overall cell health. Cells collected from patients with illness display significant physiological differences compared to those collected from healthy donors. Cell subset composition, cell phenotype expression, and the presence of inflammatory or stress-related factors can all impact target cell health, function, yield, and downstream therapeutic potency.  

 The collection of healthy donor cells is also subject to considerable variability, and a well-established donor center will be aware of the many factors that impact WBC counts (6). Data generated illustrates the variability present in apheresis collections from normal healthy donors (

). For example, WBC yields vary significantly with donor body mass index (BMI) and advanced age. Factors such as age, gender, BMI, ethnicity, current medications, and medical history have all been documented to result in differences in WBC profiles (7, 8). Stressful personal issues and lifestyle habits such as smoking, drinking to excess, poor sleep habits, and poor nutrition all impact WBC counts. Good donor communication can give cell collection center staff valuable knowledge about which factors known to affect target cell collection may be present, as well as the opportunity to modify cell collection protocols appropriately. 

	Apheresis and healthcare experts should understand donor variability issues and be trained to optimally match the unique physiological characteristics of each donor with the project needs of the client. They also need the training and expertise to operate and maintain apheresis instrumentation and to decide which collection methods best suit the donor and client. Differences in staff training and apheresis equipment and supplies can also introduce variability. Standardizing equipment and training between the manufacturers’ various collection sites can alleviate starting material variability and downstream processing risk. 

	Along with donor-related variability, collection methods and post-collection sample handling also contribute to starting material variability. It is all but inevitable that a small percentage of cells will be lost to purification or target cell modification steps, which is why it’s best to develop a process that is as integrated as possible. Automation and closed-loop systems can cut down on process related cell loss.

	Flawed sample handling during cell collection and transfer to the manufacturing facility, however, carries a greater risk than minor process losses. Contamination risk, whether from outside sources or intrinsic contamination from other cell types, is an ongoing concern for cell therapy developers because it can result in serious medical and financial consequences. Risks of this type can be avoided through stringent oversight of good manufacturing practices (GMP)-compliant cleanrooms and isolator hoods as well as through the use of closed culturing systems and automated, inline quality attribute monitoring.

	Contamination isn’t the only risk to starting material quality. Cells are capable of responding to their environment, and exposing cells to physiological stress factors can lead to apoptosis and loss of target cell function and potency.

	For years, the mesenchymal stem cell (MSC) therapy field struggled with intermittent loss-of-function issues, despite promising clinical trial results for treating a host of different diseases. Several research groups reported that the cells were vulnerable to loss of viability and function following cryopreservation and suggested that it might only be possible to use MSCs that were freshly isolated (9). Because this would present obvious logistical problems for a commercialized therapy, some intense investigation followed. It was eventually determined that MSCs are particularly susceptible to heat stress during cell thawing. Post-thaw recovery of MSCs in culture for a 24-hour period allows these cells to fully regain their functional potency (10).

	Cell therapy source material losses resulting from sub-optimal cell preservation and storage techniques can result in bottlenecks that slow the entire manufacturing process. Biopreservation and cryopreservation cocktails, as well as cell freezing and thawing methods, can have an enormous impact on product quality and efficacy. Similarly, shipping and storage logistics can introduce risk to these vulnerable products in the form of unforeseen delays or inadequate temperature regulation, which can affect product stability, again leading to loss of viability and functionality. Because cryopreservation of therapeutic cells is central to their successful commercialization, cryopreservation and shipping logistics need to be optimized as early as possible during process development.

	Cell sourcing and variability are two of the most important challenges facing the fledgling cell therapy industry. Inconsistent access or inconsistent starting material quality can leave manufacturers shorthanded, increasing the risk of batch failure or missed production deadlines. With increased pressure on hospitals and other apheresis centers to procure greater quantities of high-quality target cells, these centers are scrambling to increase yields without sacrificing quality.

	While ideally all donors would be able to donate higher blood volumes rich in the target cell population, realistically, donors with these characteristics are not always readily available. Manufacturers need to incorporate collection methods that help counter issues such as low volume yields or the presence of contaminating cell types. 

	Cell collection efficiency refers to the ratio of the target cell population to contaminating cell types present in the apheresis product as well as the ratio of total yield of target cells collected to those calculated to be present in the donor’s circulation. Because contaminating cell types can slow or complicate downstream isolation protocols, yield and purity should be balanced with the goal of consistently delivering maximum therapeutic efficacy to the patient.

	The best way to protect cellular starting material quality is to have strong quality management systems in place from start to finish (11). Real-time monitoring and quality control checkpoints using validated, physiologically relevant functional potency assays are a necessary part of the process. Cell collection and processing centers need to be tightly coordinated to prevent loss of efficacy due to raw material transfer logistics. 

	As the industry evolves, steps are being taken to improve access to donor materials and ensure their consistent quality. Cell collection centers and their pharmaceutical manufacturing partners need to integrate and coordinate their efforts early in the development process. Establishing and maintaining large, diverse donor networks involves proactive outreach to potential donors by experienced, highly trained staff. Training should include expertise in donor selection and an understanding of the impact of demographics and lifestyle characteristics on cell yield and purity. It should also cover collection methods, instrumentation, data collection, and biological sample handling and storage. 

	Cell and gene therapy companies are still newcomers to the medical field. As new products start emerging onto the scale-up and marketing stage, manufacturing and supply companies are actively building the expertise and infrastructure necessary for commercial success. 

	Issues such as starting material sourcing and quality validation will require the integration of a number of different strategies. One of the most important strategies manufacturers need to implement is investing significant resources early on for intensive product testing and characterization, including characterization of apheresis materials that are the critical foundation for the product. 

	Discerning which quality attributes are the most critical and developing quality control assays that accurately assess these attributes throughout process development and manufacturing are crucial to commercial success. In some ways this is an ongoing process; scale-up may necessitate manufacturing changes that impact which quality attributes are the most critical to track, or whether further critical quality attribute (CQA) assays need to be appended to the quality control systems. This is where exhaustive product characterization pays off. Understanding the optimal operating limits of critical reagents and methods can speed process adaptation and product re-validation.

	Cell-therapy starting material sourcing is a tremendous challenge, but one that can be mitigated through a variety of integrated efforts. Hospitals and apheresis clinics are set up to handle patients; they are generally not prepared to accommodate increased demand for cell therapy starting materials. While one cannot always avoid a hospital setting for the collection of patient donor materials, perhaps a new paradigm is necessary to transition away from hospitals as the main resource for healthy donor materials, and toward apheresis suppliers whose business model is specifically geared toward supporting cell and gene therapy development. 

	Some larger-scale apheresis material collection centers are already incorporating the preparation of GMP-compliant materials on site, integrating cell collection with GMP-compliant preliminary processing (12). This provides the benefit of regulatory compliance and assured quality from the outset, while also avoiding the need to transport collected apheresis products to an off-site facility for preliminary characterization, processing, and target cell isolation procedures. 

	The development and standardization of suitable sample handling protocols based on tissue and cell type is another critical support issue for both cell-therapy starting materials and final product. Automation, process integration, closed-system design, and compliance with current GMP can all be used to safeguard cell-therapy product quality. 

	Regulatory guidelines issued by FDA and other regulatory agencies are still evolving to suitably accommodate advanced medicinal products such as cellular therapies. Feedback from newly approved products has led to an increased emphasis on commercial preparedness on the part of cell therapy companies seeking expedited approval in order to assure patients are protected from supply chain shortages (13). As the cell and gene therapy industry matures, manufacturing and supply companies are putting in place the physical infrastructure and expertise needed to minimize variability and expand starting-material sourcing capabilities. Standardizing quality control systems and nomenclature, strengthening raw material and reagent characterization, and optimizing sample handling methods will significantly reduce risk for the industry. Close cooperation between research and development labs, regulatory agencies, apheresis supply centers, and manufacturing facilities will drive successful commercialization of these much-needed therapies.

	1. FDA, “Statement from FDA Commissioner Scott Gottlieb, MD, and Peter Marks, MD, PhD, Director of the Center for Biologics Evaluation and Research on New Policies to Advance Development of Safe and Effective Cell and Gene Therapies,” Press Release, Jan. 15, 2019.
	2. A. Philippidis, 

Genetic Engineering and Biotechnology News 

39 (7) (2019).
	3. FDA, “2018 New Drug Therapy Approvals,” 

,” Presentation for the Alliance for Regenerative Medicine, Feb. 27, 2019. 
	5. ARM, “

,” June 2019.
	6. B. Taylor and D. Clarke, “Why is Healthy Donor Tissue So Important for Cell and Gene Therapies?” 

38 (6) 503–511 (2018).
	8. L. Jamshidi and A. Seif, 

19 (2) e4955 (2017).
	9. R. Chinnadurai, et al., 

34 (9) 2429–2442 (2016).
	10. B. Antebi, et al. 

17, 297 (2019).
	11. D. Clarke and B. Taylor, 

, pp. 24–28 (2019).
	12. HemaCare, “HemaCare Details Expanded GMP Capabilities at CAR-TCR Summit in Boston, MA.,” Press release, Sept. 4, 2018.
	13. C. Bennett, “Cell Therapy Manufacturing: The Supply Chain Challenge,” 

, PhD, btaylor@hemacare.com, is global marketing director, and Dominic Clarke, PhD, is global head of cell therapy, both at Hemacare Corporation, 8500 Balboa Boulevard, Suite 130, Northridge, CA 91325. 

To whom correspondence should be addressed.


	Vol. 32, No. 12
	December 2019
	Pages: 30–35

	When referring to this article, please cite it as B. Taylor and D. Clarke, “The Evolving Role of Starting Materials in Cell and Gene Therapy,” 

Features

Accelerated approval pathways and growing demand for cell and gene therapies are putting pressure on providers of cellular starting materials, and they must ensure a steady supply.

The Evolving Role of Starting Materials in Cell and Gene Therapy

	Health organizations, medical professionals, caregivers, and patients mark World AIDs Day-Dec. 1, 2019-to increase awareness of this decades-long public health concern. While current statistics demonstrate that progress has been made in the diagnosis, prevention, and treatment of human immunodeficiency virus (HIV)-related illnesses, the toll the virus has taken on the world population-an estimated 32 million deaths-is still profound.

	The World Health Organization (WHO) estimates that, at the end of 2018, 37.8 million people globally were living with HIV. Of those infected, 79% had been diagnosed, 62% received treatment, and 53% had achieved suppression of the HIV virus (1). The research and development efforts of the bio/pharmaceutical scientists were instrumental in this progress to combat the virus.

	The first AIDS cases were diagnosed in the United States in 1981. In 1987, FDA approved the first therapy against the disease, azidothymidine, a nucleoside reverse transcriptase inhibitor originally developed to treat cancer. Drug-resistant variations of the virus necessitated the development of other antiretroviral drug classes including fixed-dose combination drugs. In 2018, the first biologic treatment, ibalizumab, was approved (2).

	Research continues today on antiretrovirals to treat HIV, prophylaxis drugs for HIV-negative people at risk for HIV, and vaccines to prevent HIV or provide a cure for those living with HIV. FDA lists 34 investigational drugs for HIV/AIDS and related infections (3).

	While diagnostic tools and therapies have helped make HIV a chronic condition-not a death sentence-for many infected patients, there are gaps in treatments. In a statement to mark World AIDs day, the National Institutes of Health (NIH) reported that approximately 38,000 people in the US were diagnosed with HIV in 2018; and while the rate of HIV incidence has declined significantly from a peak in the 1990s, progress on further reducing that rate has stalled (4).

	Ending the HIV Epidemic: A Plan for America is a US Department of Health and Human Services initiative to reduce the incidence of HIV domestically by 75% in five years, and by 90% by 2030. Under this initiative, NIH plans to fund HIV prevention, diagnosis, treatment, and care efforts and will expand partnerships with agencies, community organizations, health departments, and other organizations to determine the best implementation of research advances.

	NIH also noted the efforts will ensure that underserved communities benefit from these strategies and other challenges faced by those vulnerable to HIV- including housing and food insecurity; lack of healthcare access; and discrimination-are addressed (4).

	On a global scale, WHO reports that in 2018, 62% of adults and 54% of children living with HIV in low- and middle-income countries were receiving lifelong antiretroviral therapy; however, there were 1.7 million new infections in 2018 and 770,000 people died from HIV-related causes (1).

	NIH and the Bill & Melinda Gates Foundation recently announced investments of $100 million over four years to develop affordable, gene-based cures for sickle cell disease and HIV (5), an example of the ongoing efforts to help those afflicted-and eradicate additional infections-that deserves year-round attention.

Antiretroviral Drug Discovery and Development

,” Press Release, Nov 25, 2019.
	5. NIH, “

NIH Launches New Collaboration to Develop Gene-Based Cures for Sickle Cell Disease and HIV on Global Scale

	When referring to this article, please cite it as R. Peters, “Fighting the Good Fight,"

From the Editor

Nearly four decades after the first diagnosis, the fight to treat HIV/AIDs continues.

Fighting the Good Fight

Viral vectors must be free of viral contaminants, both adventitious agents introduced via raw materials and those generated during processing. Because conventional viral clearance treatments would degrade or destroy many viral vectors, assessment of the potential risk for contamination and the implementation of prevention and control strategies are essential. Detection can be an issue, too. Multiple analytical methods must be used, many of which have performance limitations, adding to the challenge.

	Regulatory authorities require testing to be performed at every stage of the manufacturing process. Cell substrate banks, viral seed banks, raw materials of animal origin, bulk harvests, and the batches of the final manufactured clinical product all have to be tested, according to Francesca Vitelli, head of process development, for viral vectors at Lonza. Both adventitious viral agents and replication-competent viruses are a concern, adds Tony Hitchcock, technical director with Cobra Biologics.

	While there are no regulations solely dedicated to the viral safety of viral vector products, several guidance documents from various agencies provide relevant information on ensuring the viral safety of biological products. There are also guidance documents specifically for cell and gene therapies, according to Karen Tiano, spokesperson for MilliporeSigma, and some do mention that testing for adventitious viruses is expected. Select guidelines for viral contamination testing are listed in 

Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines
					of Human or Animal Origin

Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) 

Characterization and Qualification of Cell Substrates and Other Biological Materials Used
					in the Production of Viral Vaccines for Infectious Disease Indications, Guidance for Industry

 5.2.3 Cell Substrates for the Production of Vaccines for Human Use

 2.6.16 Test for Extraneous Agents in Viral Vaccines for Human Use

, The Rules Governing Medicinal Products in the European Union, Volume 4,
					Good Manufacturing Practice, Guidelines on Good Manufacturing Practice Specific
					to Advanced Therapy Medicinal Products (Effective May 2018)

					FDA Draft Guidance
					for Industry

Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy
					Investigational New Drug Applications (INDs)

Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up 

	Many viral contaminants may be present in viral vector products. The risk for specific contaminants is dependent on the raw materials and the process, according to Lorraine Borland, product manager for regenerative medicine–cell and gene therapy in the cell line, media, and testing solutions business of Sartorius Stedim Biotech.

	“The highest risk pathways for introduction of adventitious viruses are typically raw materials used in the upstream portion of the manufacturing process,” asserts Morven McAlister, senior director of regulatory and validation consultancy at Pall Biotech. “Raw materials such as cell-culture media and trypsin typically cannot be autoclaved and so represent the highest risk of entry of these viruses. This risk is heightened with the use of serum. The cell line is also a potential source of adventitious viruses. To reduce the risk associated with the cell line, it must be very well characterized and both the master and working cell banks thoroughly tested,” she adds.

	“The manufacturing process, if not carried out in a closed system, may also have multiple aseptic process steps where breaks in the process or transfer of the intermediate material may increase contamination risk,” Borland notes. For instance, Hitchcock notes that producer cells and viral seed stocks can also become contaminated when used. The generation of replication-competent viral vectors during the manufacturing process is also a key concern.

	For raw materials, McAlister adds that contaminants can be placed into two categories: adventitious viruses that enter the viral preparation inadvertently through raw materials or poorly characterized cell lines and helper viruses such as baculovirus, adenovirus, or herpes simplex virus, which are deliberately used to stimulate expression of the virus of interest (generally adeno-associated virus [AAV] produced via transient routes).

	Efforts to control for the presence of viral contaminants have focused on testing for retroviral contamination and species-specific viruses when animal-derived raw materials are used, according to Vitelli. Examples include bovine or porcine adventitious agents and species-specific viruses such as rhabdovirus, polyomavirus SV40, and arboviruses for other animal and insect cells.

	“Where bovine serum or porcine trypsin has been used in the process, there is a risk of introducing bovine and porcine viruses such as bovine polyomavirus and bovine and porcine circovirus,” notes Borland. “Minute virus of mice and vesivirus are viruses that have emerged in recent years as contaminants of bioreactors, and screening for both is now included as standard for rodent cell lines.”

	Detection is more complicated with viral vectors compared to antibodies, according to McAlister, because human cell lines are generally used for their manufacture. A typical panel for a human cell line could include herpes simplex virus, HIV-1, hepatitis A virus, porcine parvovirus, and possibly encephalomyocarditis virus. “When working with human cell lines-for example HEK293 cells-besides standard viral safety issues, special precautions should be taken because this cell line is susceptible to infection by human viruses,” she observes. Other human viruses that may need to be tested for include cytomegalovirus, human T-lymphotropic virus, Epstein-Barr virus, hepatitis B virus, human papillomavirus, and hepatitis C virus.

	It is easier to identify residual helper viruses such as baculovirus, adenovirus, and herpes simplex virus because they have been deliberately introduced and are also usually present at much higher concentration, according to McAlister. Due to that fact, however, the challenge becomes removal rather than detection, she comments.

	In addition to raw material and process risks, the facility, equipment, utilities, and people involved in the manufacturing the process are all potential sources of viral contamination, according to Borland.

	“The facility itself may be a point of entry for rodents, providing a pathway for viral and bacterial contamination to transfer to the process stream. If using stainless-steel bioreactors, the cleaning and sterilization regimes must be validated to ensure no cross-contamination of product. Such a risk is driving the movement to sterile, single-use systems. Water systems and non-sterile gas systems must also be taken into consideration,” Borland says. In multi-product facilities, there can also be opportunities for cross contamination by adventitious agents, according to Hitchcock.

	Operators, Borland asserts, may present the biggest risk to the process. “Viral manufacturing processes require aseptic manipulation within cleanrooms to controlled standards. People provide perfect reservoirs of bacteria, mycoplasma, and viruses, and therefore, the controls around people working on the processes must be to the highest standards,” she explains.

	One of the biggest challenges to assuring that viral vectors contain no viral contaminants is the fact that traditional viral clearance steps such as filtration and treatment with heat or strong acids cannot be used with many viral vectors such as enveloped viruses (e.g., lentiviruses), because they are too fragile and cannot withstand these processes. AAV is one exception; it is a relatively robust virus and so typically tolerates standard virus hold strategies to eliminate unknown adventitious agents in the preparation, according to McAlister.

	Neutralization of viral vector products without impacting the viral contaminants is also difficult, Hitchcock says. As a result, detection of unknown viral contaminants present at low levels is highly challenging.

	The range of potential viral contaminants and numerous sources, meanwhile, creates the need for a holistic approach to viral contaminant detection using a combination of methodologies including both general and specific assays, observes Vitelli. Examples are adventitious agent tests, usually non-specific tests capable of detecting a broad range of viruses; species-specific assays designed to detect the presence of identified potential contaminants (e.g., specific bovine or porcine viruses in serum or trypsin, respectively, or murine viruses in mouse cells); and tests for retroviruses.

	The limitations of the testing methods provide a huge challenge to the industry, Borland agrees. “Traditional 

 methods are broadly specific and will detect viruses that infect a range of cell types but may not detect viruses that do not cause cytopathic effects (CPE). Molecular methods such as real-time polymerase chain reaction (PCR) are more specific and have the ability to target those viruses that do not cause CPE in cell culture; however, they are limited by the fact they are specific. Methods such as [transmission electron microscopy] TEM, which is used to detect retroviruses, are limited by their sensitivity,” she explains.

	There are few tools available for accurate, efficient, and reliably precise assays to determine the activity of residual viruses, agrees Vitelli. “Generally, the limit of detection for these assays tends to be low, and more sensitive methods such as PCR-based assays can sometime result in false positives. In addition, risks are posed by potentially unknown viral contaminants that may be undetectable using current analytical tools,” she states.

	Borland notes that next-generation sequencing has been applied for general viral detection in biologics; technology advances and validation of this technique may bring it into routine testing use.

	Another concern for McAlister is the time required to test for the absence of viruses. “Current methods are laborious and may require more time to perform than the actual product is stable for. This issue is one of the biggest drivers for the development and acceptance of rapid alternative microbiology methods to allow testing within the shortest timeframe possible,” she comments.

	Once adequate testing is established, Vitelli notes that another substantial challenge is obtaining enough purified viral material for use in formal viral clearance studies, which is costly and time-consuming to generate. “These tests also typically require purified material that must be redirected from other potential uses that are critical to the product development cycle and timeline,” she observes.

	Tiano adds that there is only a finite amount of sample available for testing, which creates challenges, particularly when material is required for cytotoxicity pre-studies and viral interference testing in addition to virus spiking studies. “Reduction of the sample size for pre-studies is being made possible, however, with the development of sensitive molecular assays useful for testing cells, virus stocks, and raw materials used in the production of viral vectors,” she notes.

	Prevention, detection, and maybe removal

	The traditional approach to assuring that no viral contaminants are present in biologic products involves prevention, detection, and removal. Prevention involves ensuring that only high-quality, pure, contaminant-free raw materials are purchased from reliable suppliers, detection involves verification of the absence of contaminants in raw materials and process intermediates, and removal involves steps to remove or inactivate any potential viral contaminants that may be present.

	Because the latter step cannot be performed for many viral vector products, a risk-based approach must be taken, according to Borland. “Performing a detailed and relevant risk assessment is key to ensuring that an appropriate and comprehensive virus control strategy is employed,” adds McAlister. She also notes that because few viral vector processes have been performed at large scale, knowing how to do this risk assessment can be a challenge. “The good news is,” she observes, “that many years of experience have been developed in closely related processes such as monoclonal antibodies and vaccines, and this experience can be leveraged for these processes.”

	In addition, Hitchcock believes the best strategies clearly rely on understanding the vector design; selection of the appropriate producer cell line and manufacturing process, including the use of closed manufacturing systems, especially with regards to the production of replication-competent vectors; implementation of a stringent quality system and management of suppliers to ensure that the required control measures are in place to minimize the risks of adventitious viral contamination and for multi-product areas/facilities, adoption of procedures for prevention of cross-contamination and that demonstration of the removal of previously manufactured product from the cleanroom facility and shared equipment.

	“The over-arching requirement,” states Vitelli, “centers on good facility design, process design, material control, and strict compliance with good manufacturing practices to ensure safety and success. Fundamental facility controls are typically established during the facility design phase, such as the segregation of personnel, material and waste flows, and selection of cleanable materials of construction, and are propagated by validated facility management practices to include robust environmental monitoring, product changeover, and visual inspection.”

	Following the general viral safety strategy that incorporates prevention through safe sourcing, detection, and removal has resulted in safe biological products for many years, says Tiano. “Cell and gene therapies is an exciting and rapidly developing area. Although the therapies may be new, using this strategy for guaranteeing viral safety will help ensure that these new therapies are as safe as more traditional biological therapies.”

	There are limited options for viral clearance other than extensive testing of materials going into the process. Many of the same strategies used to inactivate/remove contaminating viruses can impact the quality/potency of the final viral gene therapy product. Manufacturers can look to adopting viral removal steps into their raw material entry points, Borland suggests. She points to next-generation virus filters that have been developed to offer a cost-efficient strategy for media filtration but notes that this technology is not yet adopted throughout the entire industry.

	Techniques such as nanofiltration, ultraviolet-treatment, gamma irradiation, and high temperature, short-time treatment can also be used to ensure that no viruses are present in starting materials, according to McAlister. She stresses, though, that it is important to ensure during process development that subjecting raw materials to these techniques does not affect their performance.

	Viral clearance technologies that are used in the manufacturing process for viral vectors that can withstand them are engineered into the manufacturing process and are most commonly heat inactivation and the use of viral retentive filtration, according to Vitelli. In some cases, such as for AAV products, affinity chromatography and exposure to acidic pH conditions (< pH3) may be employed.

	For removal of helper virus, McAlister notes that nanofiltration works well because there is a large difference in diameter between the virus of interest and the contaminating virus. She notes, however, that direct-flow filtration is not well adapted for the removal of viruses at high concentration. “It is generally recommended, therefore, that an initial virus removal step that can remove significant amounts of the helper virus, such as membrane chromatography, be performed first.”

	Some chromatography steps that are used in the purification of non-enveloped viral vectors may contribute to viral clearance for the vector as well, according to Tiano. “These steps may include an affinity resin or an anion exchange resin, which are optimized to purify the viral vector product and coincidentally may remove potential viral contaminants. Since the chromatography steps purify the vector product, viral clearance is only achieved when there is a difference in partitioning between the vector and potential viral contaminants,” she explains.

	Lonza’s R&D group is evaluating chemical additives and exploring different lysis reagents that have viral inactivation capabilities without risking impact on the quality of the viral vector product.

	As next-generation processes move toward commercialization, there has been a general shift away from adherent cell-culture processes in open, manual-intensive, flatware systems that are difficult to scale to automated, suspension cell-culture processes in closed bioreactors that are more efficient and readily scalable. This shift is also enabling the use of sterile, disposable systems, which is in turn enabling improved risk-mitigation strategies, according to Borland.

	Hitchcock notes that as viral vector manufacturing processes are scaled, the likelihood of the generation of replication competent viruses increases, which poses challenges for purification.

	Improving overall process yield will be an issue, in fact. McAlister points to downstream processing yields, typically around 30%, for AAV as an example. “Nanofiltration, if not done properly, can further reduce this yield. Appropriate filterability trials must be performed to ensure that a scalable scheme for virus control is designed,” she observes. Vitelli adds that as the per-dose demand for viruses increases, notably AAV, vendors of viral-retentive filters should continue to work closely with product developers and contract development and manufacturing organizations to provide scaling recommendations and new product sizing.

	For viral vectors that can withstand heat inactivation, these steps may also pose unique challenges with respect to scale up due to the need to adequately control both the temperature and duration of exposure in the liquid state, according Vitelli.

Vol. 32, No. 12
	December 2019
	Pages: 16–19

	When referring to this article, please cite it as C. Challener, “Prevent, Detect, and Remove: Viral Control for Viral Vectors,"

Ensuring that viral vectors are free of viral contaminants requires a focus on prevention and control.

Prevent, Detect, and Remove: Viral Control for Viral Vectors

Q. I work for a contract manufacturer and am responsible for performing investigations and reporting the results to the clients regarding their drug products. We have recently contracted with a client to manufacture a pre-filled syringe. Can you tell me what changes I should make to my investigation procedure when investigating deviations and complaints for this type of combination product?

	A. Overall, the actual investigation process for a pre-filled syringe product should be similar to the process already used by you to investigate the drug products. The purpose of performing an investigation into a deviation is to determine why the deviation happened and what impact it has on the product quality. To determine the impact of the deviation on the product quality, it is important to determine the ‘root cause’ of the deviation. The process used in industry to determine the root cause is the investigation procedure. This procedure, regardless of the product you are investigating, should require the investigator to review various quality and manufacturing systems to determine whether they were the cause of the deviation under investigation. Each investigation must address the following elements: root cause, impact to the material or product, the immediate correction taken, the corrective action to prevent re-occurrence for specific product/operation, and the preventive action taken to prevent re-occurrence for all products/operations. The complication with combination products is that the investigation procedure needs to accommodate both the drug and device components of the product.

	It is important to consider a few general rules when performing an investigation regardless of the product being investigated. Remember, one size does not fit all. Simple errors require simple corrections while serious deviations require broader investigations. The complexity of the investigation is related not only to the seriousness of the investigation but also to the complexity of the factors that could influence the outcome.

	The best trait to have as an investigator is inquisitiveness. Continuing to ask questions and avoiding assumptions leads to better outcomes. Using other tools, such as fishbone diagrams and determination of most probable number (MPN), are to be encouraged, but they do not take the place of asking questions. In performing an investigation, it is important for the investigator to widen their perspective and look for ways to relate similar issues. The best way to ensure events are not related is to try and relate them, not the other way around. Keep in mind that human error is rarely a true root cause. There is usually something in the process that causes that human error. In addition, you should always verify the facts of the investigation.

	Once you develop an investigational history with the pre-filled syringe, you will also want to include a historical review in your investigation. This review should determine if the deviation occurred with this or other products, with the specific manufacturing line or other manufacturing lines, and/or with one group of  operators or multiple sets of operators. The historical review can help you prioritize the resources and aid in your detailed system review.

	In the situation you described, you can provide a historical review for the drug component part of the pre-filled syringe, but you will need to outreach to the syringe supplier and your client to start developing the historical review for the device component until you have a body of knowledge for this component. The best way to obtain necessary information for the investigation of the device component is to establish a communication channel between your company and the supplier of the syringe, with your client’s knowledge and approval. It would be wise to codify what information you would like to have access to through quality agreements with both the syringe supplier and your client.

	The detailed investigation should include a review of various systems. The systems most often reviewed are equipment and machinery, the manufacturing process, the raw materials used in manufacturing, the specifications, the environment, and finally, the operators. This is not to imply that these systems are the only areas you should look at during the investigation but that these are the most probable areas where you will uncover the root cause of the deviation. The raw material or supplier system may need to be updated to accommodate the syringe supplier. Rather than rely on the client’s evaluation and audit of the syringe supplier, it would be beneficial for your company to perform their own audit and assessment and establish a quality agreement. Inform your client that you want to develop your relationship with the syringe supplier to assure their assistance and timely responses for an investigation; make sure you inform your client when you do reach out to the supplier during an investigation. This will help facilitate both your client’s and the syringe supplier’s involvement and open the lines of communication should you require either of their assistance for an investigation.

	Once the aforementioned elements have been investigated, results from the investigation must be documented. The written narrative should clearly explain what happened, when it happened, and who was involved or observed what happened. The narrative documents the solution and rationale for the root cause that was determined through the investigation process. This should also include any action being taken by the syringe supplier should this component be implicated in the investigation.

	The key to any successful investigation is not stopping too soon and assuming you have the solution prior to completing the investigation. Ask questions until you can think of no more questions to ask and be sure to document the answers to your questions. If you follow your investigation procedure, make adjustments to accommodate the new component, and thoroughly document your results, you should have an acceptable investigation regardless of whether you are manufacturing a traditional product or a combination product.


	Vol. 32, No. 12
	December 2019
	Page: 37-38

	When referring to this article, please cite it as S. J. Schniepp, "Investigating Combination Product Failures," 

Ask the Expert

Investigating deviations or failures of combination products needs to accommodate both the drug and device components, says Susan J. Schniepp, executive vice-president of post-approval pharma and distinguished 

Investigating Combination Product Failures

	Pharmaceutical companies developed, and FDA approved, dozens of new drugs and biotech therapies in 2019, as new discoveries in genomics and drug development continue to bear fruit. Even if 2019 approval numbers don’t set new records, the list includes notable breakthroughs, including Novartis’ controversial gene therapy Zolgensma; Vertex’s triple combo for cystic fibrosis; and new treatments for cancer, migraine, light-sensitivity, Parkinson’s disease, pneumonia, rheumatoid arthritis, tuberculosis, and urinary tract infections. The steady rise in applications for investigational therapies has made it difficult for the Center for Biologics Evaluation and Research (CBER) to keep pace, while the Center for Drug Evaluation and Research (CDER) continued to reorganize its Office of New Drugs (OND) to better assess and process a growing volume of submissions requiring special attention.

	At the same time, high price tags on transformative therapies heightened challenges for prescription drug reimbursement and payment strategies. Health plans, payers, and policy makers continue to debate multiple proposals for reforming drug coverage and pricing, ranging from wider importing of less costly products from abroad, federal government price negotiations, and specific limits on annual price increases and patient out-of-pocket costs. There’s much talk of “value-based pricing,” but legal and technical difficulties limit this approach. The resulting uncertainties make it difficult for manufacturers to plan research programs, devise pricing and marketing strategies, and commit to upgrades in production operations.

	A main cause of delay in bringing cutting-edge therapies to market in recent months has been difficulties in ensuring that production systems can ensure product safety and quality. The transition from pilot-scale to commercial manufacturing of gene and cellular therapies can be challenging and costly, observed CBER Director Peter Marks at the PDA/FDA Joint Regulatory Conference in September 2019. He emphasized the need for high-quality manufacturing of these new products, noting that treatments made from living cells or tissues often are not easily characterized, are susceptible to microbial contamination, and involve complex manufacturing materials and processes.

	CBER published several guidances on testing and producing new cellular and gene therapies and has promoted additional assistance for manufacturers of advanced technologies, Marks added. But limited gene therapy manufacturing capacity will continue to be a major problem as multiple new products gain market approval, particularly those for treating chronic conditions in larger patient populations.

	Such hurdles have prompted leading manufacturers such as Pfizer and Sanofi to make major investments in new biotech manufacturing sites. In addition, firms of all sizes look to work with contract manufacturing organizations with capacity for quality biotech production. To address these needs, as well as the difficulties of smaller developers of treatments for rare conditions, Marks backs the creation of a public-private consortium that can produce quality materials for preclinical and clinical studies more reliably and at lower cost.

	While manufacturing issues delayed access to new breakthrough therapies, such problems also continued to disrupt production of many common, low-cost medicines that are important for treating many serious health conditions. In response to concerns from Congress and the public, FDA issued a report in October 2019 examining the financial, manufacturing, and policy issues underlying short supplies of important prescription medicines in the United States. The analysts found that of 163 drugs that first went into shortage between 2013 and 2017, 62% of supply disruptions were associated with manufacturing or product quality issues; only a few had problems due to increased demand or natural disasters (1).

	A lead proposal from FDA officials is that manufacturers able to ensure a reliable supply of high-quality therapies should be able to command higher prices, an approach designed to encourage companies to invest in modern production systems able to avoid shortages, particularly for the low-priced, low-profit sterile injectables that companies often decide to abandon, rather than commit to the long-term upgrades needed to meet standards. To support this approach, FDA Acting Commissioner Ned Sharpless and CDER Director Janet Woodcock called for a system able to measure and rate a facility’s “quality management maturity” that can inform the public about its commitment to reliable quality production (2).

	In addition, the report included more common actions to prevent and address ongoing shortages, such as permitting longer expiration dates where justified or increasing penalties on firms that fail to provide timely and adequate notification of a likely interruption in production. FDA also could require manufacturers of certain drugs to conduct periodic risk assessments to identify vulnerabilities in supply chains and plans to mitigate such risks. And new international standards for harmonizing post-approval changes in drug manufacturing and formulation, as proposed in a new guideline (Q12) developed by members of the International Council for Harmonisation (ICH), would make it easier for manufacturers to modernize or alter a production facility that serves multiple markets.

	Manufacturers should “sell quality-not just medicine,” Woodcock stated in an FDA “voices” posting (3). Even though industry strongly opposed an earlier CDER effort to establish a system for rating companies on their ability to achieve quality drug manufacturing, Woodcock  maintained that a firm’s ability to reliably make a product in sufficient quantities and with sufficient speed to “ensure that supply consistently meets demand over sustained periods of time” should be a strong selling point. This discussion will continue in 2020.

Report–Drug Shortages: Root Causes and Potential Solutions,

, Oct. 29, 2019.
	2. N. Sharpless and J. Woodcock, “Statement on FDA’s New Report Regarding Root Causes and Potential Solutions to Drug Shortages,” Press Release, Oct. 29, 2019.
	3. J. Woodcock, “

To Help Reduce Drug Shortages, We Need Manufacturers to Sell Quality-Not Just Medicine


	Vol. 32, No. 12
	December 2019
	Page: 8

	When referring to this article, please cite it as J. Wechsler, "Quality Issues Offset Biomedical Advances," 

Problems in assuring reliable drug quality and supply dampens progress in bringing lifesaving therapies to market.  


Quality Issues Offset Biomedical Advances

A biotech company’s journey to develop a protein-based therapeutic drug from discovery, process development, manufacturing, and clinical trials to commercialization is long and complex. Multiple activities are involved, and many challenges are encountered when complying with chemistry, manufacturing, and control (CMC) requirements. Downstream process development and manufacturing play a crucial role to ensure the protein drug’s safety, quality, identity, purity, and efficacy (SQIPE) meet regulatory requirements for clinical trials and commercialization.

	In this article, the authors analyze the challenges and seek effective solutions for CMC compliance (

), specifically focusing on protein complexity, raw material quality, upstream process (e.g., media source and titer), downstream process development (e.g., process capacity, efficiency, scalability, and control), manufacturing complexity, and product SQIPE.

Antibodies are complicated biologic molecules in their nature, including size (e.g., 150 kDa IgG–900 kDa IgM), heterogeneity (e.g., glycosylation, deamidation, oxidation), structure (e.g., monomer, dimer, trimer, hexamer), charge (e.g., pI of 5.0–9.0), hydrophobicity, and genetic source (e.g., rodent, human). Due to their intrinsic complexity, smart design for protein purification is necessary.

	Raw material screening and selection is the first thing to be considered for purification. It is best practice to select a high grade of raw materials (e.g., chemicals) when process development is initiated. For chemicals, multi-compendial grade is the first option. Single compendial grade-such as 

United States Pharmacopeia–National Formulary (USP–NF), European Pharmacopoeia (Ph.Eur.), or Japanese Pharmacopeia (JP)

-is acceptable if multi-compendial grade is not available. Finally, some chemicals may not be available in compendial grade. These need to be considered on a case-by-case basis regarding the purity, application, and option for substitution by a compendial-grade chemical.

	Using a high grade of raw materials can minimize negative impact to product SQIPE and avoids potential timeline delays and reduces cost from process development, technology transfer, and manufacturing, to regulatory filing. The raw materials used are for upstream process (e.g., cell lines, media components, additives), downstream process development (e.g., chromatography resins, chemicals, buffers, membranes, filters, column housings, tubing, bags, tanks, water), formulation (e.g., excipients, buffers), and fill/finish (e.g., vial, prefilled syringe). It is important to monitor and control raw material critical attributes, grades, impurities (e.g., trace elements, leachables, extractables), lot-to-lot variations, safety (e.g., viruses, bacteria, fungi, mycoplasma, and endotoxin), and storage during manufacturing. Due diligence of vendors for critical raw material supply chain from primary and secondary vendors for late-stage and commercial production is highly recommended.

	Harvest via clarification connects upstream and downstream processing. It is often conducted by centrifugation, depth filtration, microfiltration, or alternative tangential flow.

	Depth filtration, a disposable technology, is routinely used for cell-culture harvest (bioreactor ≤ 2000 L). It removes cells, cell debris, and other impurities by size and/or charge. A number of depth filters are available on market (e.g., Millipore, 3M, Pall, and Sartorius). Although depth filters share similar mechanisms for particle and impurity removal, each filter has its unique properties in grade (e.g., low extractable and endotoxin), media feature (e.g., activated carbon, cellulose, glass fiber), charged or non-charged (e.g., positively charged ligand), capacity, and pore size (e.g., 2–20 µm). A properly designed depth filtration step could result in high viral clearance (>4 log10 reduction) (1) and impurity removal (e.g., host cell proteins [HCP]).

	Disk-stacked centrifugation is designed for a large bioreactor harvest (e.g., ≥10,000L). A reliable scale-up and/or scale-down model between scales from bench, pilot, to manufacturing is essential for large-scale harvest and troubleshooting by small scale. A mature model can be established based on cell-culture conditions (e.g., cell line, cell viable density, cell viability, titer, and media osmolality), centrifugation system design, feed flowrate, bowl speed, sigma factor, discharge rate, and centrate quality (e.g., turbidity and/or lactate dehydrogenase activity). An automated centrifuge with a disposable flow-path marketed for cell therapy (Sartorius) eliminates cross-contamination, minimizes cell lysis, and enhances process efficiency, quality, and control.

The purification process for a monoclonal antibody (mAb) typically contains clarification, purification of capture, intermediate and polishing chromatography, viral clearance, final formulation, and bulk drug substance (BDS) filtration steps. The best downstream process should possess high capacity, efficiency, controllability, scalability, flexibility, cost-effectiveness, and biosafety (

	A platform protein approach for mAb purification offers advantages (Figure 3) and can save time and resources, as well as expenses related to raw material screening, process parameter selection, process development, documentation preparation (e.g., using template strategy), technology transfer, scale-up, product characterization, formulation development, and regulatory filing. A platform approach also can anticipate process yield, product purity, and quality.

A mAb-purification capture step often uses Protein A resin, a 42-kDa surface protein originally identified from 

 (2). Protein A resins are made of either native or engineered protein cross-linked to different matrix (e.g., Eshmuno A from Millipore, PraestoAP from Purolite, and MabSelect SuRe from GE Healthcare). This protein has a high binding affinity to mAb or Fc-fusion protein by hydrophobic interaction with Fc domain. To select a Protein A resin, one needs to consider the resin-binding capacity, specificity, stability, cleanability, sensitivity to caustic (e.g., sodium hydroxide) solutions, ligand detecting kit availability, flow capability, good manufacturing practice (GMP) supply, and resin cost. For a thorough review of the progress seen in Protein A chromatographic media, see Bolton and Mehta (3). The current major drawback from Protein A resin is its high cost (approximately $14,000/L for MabSelect SuRe).

	Low pH retrovirus inactivation is usually conducted at low pH (3.3–3.6) for ≥60 minutes hold time if the mAb is stable at the test pH. Low pH retrovirus inactivation is a robust viral clearance step, which can reach >4 Log10 reduction value (LRV). It was reported >5 LRV under pH 3.9-4.1 (4). A previous study showed >5 LRV at pH 3.8 held for 60 min (unpublished data). It is known that pH, hold time, buffer matrix, acid (e.g., acetate, citrate, glycine), and temperature (15–30 °C) have impact on retrovirus inactivation kinetics (4). The lower pH, higher temperature, and longer hold time maximize the retrovirus inactivation, but this must be tempered by the sensitivity of the antibody. Current industrial practice is toward a modular approach based on the understanding and historical data from mAb, buffer type, salt concentration, acid type, and operating range for pH, temperature, and hold time. If mAb is unstable for low pH retrovirus inactivation treatment, detergent inactivation (e.g., polysorbate 80) or UV-C inactivation system (UVivatec, Sartorius) can be an alternative.

	This step can result in a significant impurity removal (>150-fold HCP reduction) due to impurity precipitation and/or adsorption by a charged depth filter. This may allow a two-column process. The intermediate depth filtration process design parameters include pH range, hold time, mixing, temperature, HCP testing method, and depth filter type.

	Cation-exchange chromatography (CEX) step

	In most cases, CEX step is conducted after the intermediate pH hold and depth filtration step, which provide for the needed pH and conductivity adjustment prior CEX loading. CEX is performed in a bind-and-elute mode, which removes both process- and product-related impurities (e.g., HCP, rProtein A, aggregates, and acidic species). The drawbacks from this step are from its efficiency (e.g., resin diffusion) and viral clearance capability (<3 LRV for parvovirus reduction). However, it is still possible to have >4 LRV retrovirus reduction when proper resin and operating condition are designed and developed.

	Anion-exchange chromatography (AEX) step

	In either resin or membrane format, AEX is often carried out after the CEX step to further remove process-related impurities (e.g., HCP, DNA, endotoxin) and viruses. The AEX step is an effective step to remove both retro- and parvoviruses. The CEX and AEX column order can be switched to overcome issues related to equipment, process, or facility. For example, if the CEX eluate salt concentration were too high, it would need a significant dilution to decrease conductivity prior AEX load and this could encounter limitation of equipment (e.g., tank size) or facility fit (e.g., suite size). Under these circumstances, AEX could be considered as second column step (unpublished data).

	AEX-membrane chromatography (e.g., Sartobind Q) has unique advantages in process efficiency, buffer consumption, disposability, and is ready-to-use. Its shortcomings are mainly from its low-binding capacity and cost (e.g., at large scale-up). Therefore, it is ideal to put it after CEX column, which will minimize impurity breakthrough and enhance viral clearance capacity. Natrix Separations’ 3-D hydrogel membrane columns (e.g., NatriFlo HD-Q) overcome the limitations from conventional membrane chromatography (5).

	Small viral retentive filtration is a robust viral clearance step (>6 LRV for parvovirus). A number of commercial viral filters are available (e.g., Sartorius, Merck Millipore, Pall, Asahi-Kasei). The viral filter selection depends on filter pore size (e.g., 15–35 nm), filter design (e.g., dead-ended, hollow-fiber), capacity (e.g., L/m2, g/m2), and cost. Process conditions (e.g., product purity, pH, conductivity, buffer matrix) and recovery must also be considered. Different viral filters have different load capacity (e.g., >10,000 g/m2 from Planova 20N BioEX) without significant flux decay (unpublished data). High capacity can reduce raw material cost significantly. To improve both viral clearance capability and process-related impurity removal (e.g., HCP, DNA), the authors expect vendors to develop innovative viral filters with dual functions of size exclusion and charge in the future.

	Ultrafiltration and diafiltration is employed for mAb formulation by protein concentration and buffer exchange into final formulation buffer. Different types (e.g., A, C, D) of membranes (e.g., regenerated cellulose or polyethersulfone) are available for screening and selection based on recovery, product quality, and process efficiency. For bulk drug substance with high concentration (>100 g/L) and viscosity (>10 centipoise), C or D type of membrane with special system set-up and design (e.g., retentate solution level higher than inlet of membrane) is recommended.

	Bioburden and endotoxin are strictly controlled to the lowest level during DSP and manufacturing to minimize safety risks to the patient. Bioburden and endotoxin excursions occasionally have been reported from poor environmental monitoring and control, insufficient equipment cleaning and maintenance (e.g., suite, biosafety cabinet), and lack of standardized in-process 0.22-μm filtration per unit operation. Personal hygiene and gowning are other possible causes.

Downstream process development and manufacturing play a crucial role to ensure safety, quality, identity, purity, and efficacy.

BioPharm International-12-01-2019