As public opinion of the pharmaceutical and biopharmaceutical industry continues to worsen in Gallup polls (1) in the United States, drug prices remain a hot button political issue. This is particularly true for insulin. In a formal statement in December 2018 (2), former FDA commissioner Scott Gottlieb referred to insulin prices, which have increased nearly 600% between 2001 and 2015, with the average US list price rising by 15% per year between 2012 and 2016. He suggested that biosimilar versions of insulin would be likely to reach the market over the next few years.

	Almost a year after FDA formally issued its Biosimilars Action Plan (3), with the challenging goal of balancing patient safety with innovation, the spring and summer of 2019 brought several US congressional hearings on drug prices and greater focus on biosimilars, including an FDA meeting devoted exclusively to the future of biosimilars for insulin.

	Since 2015, when FDA approved its first biosimilar, Sandoz’s filgrastim-sndz (Zarxio), 19 biosimilars have been approved in the US. Thomas Linneman, head of regulation CMC customer solutions at Solvias, divides them into monoclonal antibodies, hormones, and Fc fusion proteins and cytokines (4). Although approved, these drugs are not “interchangeable” with name-brand biopharmaceuticals, as that term has been defined in the US. Unlike small-molecule generics, biosimilars cannot automatically be dispensed at the pharmacy level, in place of name-brand products, without a physician’s specific prescription. Given the relative novelty of biosimilars in the US, studies of physician patterns have found doctors to be conservative about filling patient prescriptions with biosimilars (5–7).

	Establishing interchangeability has required additional clinical studies. So far, Boehringer-Ingelheim has been the only company to have pursued a US strategy that combines basic biosimilar approval and approval for interchangeability for its adalimumab (Humira) biosimilar, Cytelzo (8). It began clinical interchangeability studies for the drug back in 2017.

	Biosimilar advocates have been pressuring FDA for more guidance on biosimilars interchangeability, and an update to draft guidance that the agency had issued in 2017. In August 2018, in a letter (9) to former Commissioner Gottlieb, representatives from hospitals, insurance companies, and patient groups complained that the lack of final guidance would discourage biosimilar development and leave the public without lower-cost options to name-brand biopharmaceuticals.

	A flurry of guidance documents in May and June

	With the clear intent of removing perceived roadblocks to biosimilar development, FDA issued a number of guidances in May and June 2019, including long-awaited guidance on interchangeability (10).  Acting FDA commissioner Ned Sharpless commented in an FDA press release, the guidance will enable biosimilar or interchangeable insulin products to enter the market (11).

	The agency also published draft guidance on the analytical methods to be used to establish biosimilar comparability and quality (12). According to reports on May 13 and May 21 in the 

Regulatory Affairs Professional Society Journal

, the draft updates 2015 guidance on quality that FDA had withdrawn after receiving critical comments from the industry and also replaces 2017 draft guidance (13,14).  It recommends best practices for chemistry manufacturing and control (CMC) programs for biosimilars, provides detailed recommendations for assessing lot-to-lot variability in product that might be traced to manufacturing variables, and also spells out clear requirements for nine crucial analytical steps, which include manufacturing process, stability, target binding, impurities, and references.

	In June, FDA also issued clarification and a proposed revision to regulations that have not allowed biosimilar developers to refer to drug master files (DMFs) in applications (15). The proposed rule, now in the 

 for industry and public comment, would affect 89 compounds, mainly enzymes and hormones, including insulin, that are currently licensed under the Food, Drug and Cosmetics (FD&C) Act, but that will transition to licensing under the Public Health Service Act in March 2020 (16).

	Intended to prevent potential shortages, the rule would allow developers of biosimilars in these categories to reference data on intermediates, drug products, or drug substances contained in DMFs, even after their drugs have been licensed. The proposal’s language suggests that, for the products involved, which have been established and regulated under FD&C for decades, any potential risk would be minimal. 

	Is FDA moving too quickly on interchangeability?

	But in a broader sense, given the complexity of biomolecules when compared with small molecules, there are questions of what interchangeability should mean for biosimilars, particularly more complex molecules, because the term cannot mean what it does for small molecules. “I understand regulators’ and Congress’ urge to make biopharmaceuticals less expensive,” says Sheila Magil, managing director of the BioProcess Technology Group, part of BDO USA, who stresses that these are her personal opinions and do not represent her employer’s. “Biopharmaceuticals may be costly, but they are also very expensive to develop, “she says.

	In the current environment, however, there is a danger of oversimplifying issues in the interest of speeding products to market without sufficient caution. In turn, lay people (e.g., most members of congress and many executives on the payer side) may be put in the position of having to make decisions on complex issues that they may not fully understand, Magil says. In general, she says, “I appreciate FDA’s goal for interchangeability, but at this point I am not entirely convinced that current guidance and regulations will get us there.”

	For instance, she explains, politicians, patient groups, and payers may view biopharmaceuticals the same way as they do small molecules, without realizing the potential risks entailed with large molecules, and issues such as immunogenicity and variability. For small molecules, where bioactivity is found in the first dimension, interchangeability is not a problem and it is easy for different manufacturers to produce virtually the same product and to achieve product purity levels of 99% or higher, she says. But she notes that is not always true for biopharmaceuticals, for which biological activity is in the third dimension. “With biopharmaceuticals, a great deal depends on how the individual molecule is folded and how it is affected by its environment,” Magil says.

	In its new guidance, FDA has acknowledged that these issues need to be recognized, says Magil. But that still doesn’t make it correct to use the term “purity” for biopharmaceuticals in the same manner as it would be used in the small-molecule world, at least not without specifying how that purity was determined, e.g., whether measured by ion exchange, spectroscopy, or some other analytical method. “It’s not the same for biopharmaceuticals as it is for small molecules, and there is great complexity in existing forms,” Magil says.  “We might not know how to test for those differences,” she says, since a change or structural difference that might seem very minor in a biomolecule can have a major biological impact, and that impact may not be understood or known before problems occur.

	Magil recalls a biopharmaceutical product that she had worked with when she worked as a scientist in drug development in the industry. As this particular product aged, its structure unfolded and it became more active. “If one were to develop a biosimilar for that molecule today, one would most likely choose to test and evaluate it at the point of product release, and thus would be very likely to miss this critical characteristic,” she says.

	These issues could be less of a concern for a company that had developed a branded biopharmaceutical and then opted to develop a biosimilar to that product later on, Magil says. “In that case, they would have all the original cell lines. But another company developing a biosimilar would be using a different cell line, posing potential risk because of the inherent variability in complexity for biosimilars, specifically in the drug substance,” says Magil.

	“I’m not sure that this issue is fully covered in FDA’s guidance,” she says. “I also have big concerns about immunogenicity. We don’t yet have a good way to test for that in-silico, to look at a molecule and gauge its immunogenicity, so we have to test the molecule in people, not even in animals, and there is not enough data out there at this point,” she says.

	Bioanalytical techniques and associated software have become much more powerful, and far beyond what was available in the early days of innovator molecules, Magil says, and techniques such as mass spectrometry make it possible to evaluate the higher order state of biomolecules. However, she suggests, “At this point, there are still too many things that we cannot measure or control, to be talking about interchangeability.”

	Magil also questions the idea of biosimilar developers relying on DMFs for product, ingredient, and other data, unless they have already developed all of this data internally and are able to compare it. “Doing this could be dangerous, since a company might wind up pointing to data that refute its own product data, unless they had made head-to head-comparisons between the data they had with data in the DMF.”

	Although the current aim is to encourage the development of biosimilars for treatments such as insulin, FDA and industry clearly face challenges in meeting the goals outlined in the Biosimilars Action Plan for novel and complex formulations. 

	1. J. McCarthy, “Big Pharma Sinks to the Bottom of US Industry Rankings,” 

, Sept. 3, 2019.
	2. FDA, “Statement from FDA Commissioner Scott Gottlieb on New Actions Advancing the Agency’s Biosimilars Policy,” Press Statement, 

, Dec. 11, 2018.
	3. FDA White Paper, The Biosimilars Action Plan, July 2018. www.fda.gov/media/114574/download
	4. T. Linnemann, “Regulatory Strategies for the Analytical Development of New Biological Entities and Biosimilars,” a Solvias Webcast, 

, April 17, 2019.
	5. E. Leonard et al., Journal of Managed Care and Specialty Medicine 25 (1) 102–112 (2019).
	6. J. Waller et al., 

 11, 519–530 (2017).  
	7. A. Gaffney, “How Comfortable are Physicians with Biosimilars? Not Very (Yet),” 

, Oct. 24, 2018.
	8. Boehringer-Ingelheim Press Release, boehringer-ingelheim.us, July 27, 2017, www.boehringer-ingelheim.us/press-release/boehringer-ingelheim-starts-clinical-study-interchangeability-between-its-adalimumab
	9. 

, Aug. 23, 2018.
	10. FDA, Guidance for Industry: Considerations for Demonstrating Interchangeability with a Reference Product, (Rockville, MD, May 2019).  
	11. FDA, “Statement from Acting FDA Commissioner Ned Sharpless on Policy Advancements to Help Bring Interchangeable Insulin,” Press Statement, 

, May 10, 2019.
	12. FDA, Draft Guidance for Industry: Development of Therapeutic Protein Biosimilars: Comparative Analytical Assessment and Other Quality Related Assessments (Rockville, MD, May, 2019).
	13. Z. Brennan, “FDA Replaces Withdrawn Biosimilars Guidance With New One On Quality Related Consideratons,” RAPS.org, May 21, 2019.
	14. Z. Brennan, “Updated: Interchangeable Biosimilars: FDA Finalizes Guidance,” 

, May 13, 2019.
	15. FDA, "FDA Takes New Step to Help Advance Transition of Certain Medical Products, 

, June 27, 2019.
	16. US Federal Register, “Proposed Rule, Biologics License Applications and Master Files,” 


	Vol. 32, Issue 10
	October 2019
	Pages: 38–42

	When referring to this article, please cite it as, A. Shanley, "Driving Improved Access to Biosimilars," 

Articles

Features

New guidance from FDA, including standards for interchangeability, is expected to speed development of biosimilars, but experts warn against oversimplifying risk to reduce costs.

Driving Improved Access to Biosimilars

As perfusion-enabled bioreactors become better established, upstream biopharmaceutical manufacturing is shifting from fed-batch to continuous mode, harnessing improvements that have been made to alternating tangential flow (ATF) cell retention devices and tangential flow filtration (TFF). “The ratio between fed batch and perfusion is changing, and there is more interest in continuous due to the reduction in operating scale and the efficiency improvements that perfusion approaches bring,” says Christine Gebski, vice president and general manager of cell culture at Repligen Corp.  The company has been supporting perfusion cell culture approaches for nearly a decade with its XCell ATF and KrosFlo TFF cell-retention systems. 

	The key benefit of operating upstream continuously in perfusion mode stems from the increase in productivity per liter of bioreactor volume that it allows, which can be 10 times that of a fed-batch bioreactor run, says Andrew Bulpin, head of process solutions at MilliporeSigma. “The cost for this additional productivity comes largely from the need for additional cell culture media. Most perfusion bioreactors are run at rates of one- to two-vessel volumes per day, which requires a significant increase in media production and downstream volume processing,” he says. However, Bulpin predicts that leveraging automation in the preparation of cell culture media and intensification of the downstream capture chromatography step will significantly debottleneck overall operations and lead to broader use of perfusion. 

	Applications beyond traditional biopharm

	Increased use of the technology is already being seen, not only for traditional monoclonal antibody and recombinant biopharmaceutical development but also for viral vectors and vaccines, says Gebski. “The technology has penetrated contract manufacturing and contract development and manufacturing organizations (CMOs and CDMOs) and Tier 1 to 3 producers and is finding less resistance as more people understand the process and manufacturing benefits,” she says. 

	Continuous platforms are also being adapted for use in manufacturing innovative therapies by companies such as Biosana, an Australian company with cell therapies in Phase I clinical trials. In March 2019, The New Jersey Institute of Technology’s New Jersey Innovation Institute and Pall Corp. set up a venture that will establish two new centers for continuous process development of cell and gene therapies (1), augmenting a previous $1.9-million collaboration launched in November 2018, in which Pall, Cobra Biologics, and the Cell and Gene Therapy Catapult in the United Kingdom will develop new continuous cell and gene therapy manufacturing processes (2). 

	A number of technology advances are driving the industry to improved continuous processing, including use of perfusion upstream.  One area of focus has been in optimizing approaches to allow for cell retention in single-use bioreactors, says Melisa Carpio, global technology consultant for cell culture technologies at Sartorius Stedim Biotech. 

	Within the past year, she says, a number of enhancements have been made to existing single-use bioreactor systems to enable continuous bioprocessing. At the small scale, she says, both the ambr 15 and ambr 250 have additional options that can be added to existing systems to enable perfusion.  The ambr 15 cell culture system has two new tools that help perform perfusion mimic: a rapid vessel drain option and custom centrifuge adaptors that allow direct centrifugation of ambr 15 vessels.  The ambr 250 high-throughput system now also features a perfusion option that allows for true perfusion (both ATF and TFF) to be run at the 100 – 250 mL scale.  At larger scales, Carpio says, both the rocking motion (BIOSTAT RM) and stirred tank reactors (BIOSTAT STR) now incorporate features that permit perfusion process scale-up.  A new BIOSTAT RM bag with integrated perfusion membrane was launched at the 200-L scale (with 100-L of working volume) so that perfusion can be run in rocking motion bioreactors ranging from 1-L to 200-L.  The BIOSTAT STR now also features a bag configuration that can directly connected to cell retention devices such as Repligen’s ATF system, Carpio says.  

	Atul Mohindra, R&D director for biomanufacturing at Lonza Pharma & Biotech notes that technology advances and greater process knowledge have been key breakthroughs driving use of perfusion. 

	Improvements in disposable technology offerings, particularly in the cell-retention devices that are fundamental to perfusion have been instrumental, he noted. “Until now, this unit operation had not been robust, but the new generation of ATF and TFF devices are far more robust and perform better than previous technologies,” Mohindra says.

	He also cites significant advances in sensors for at-line and in-line testing, which allow manufacturers to have better control over critical quality attributes (CQAs). At Lonza, Mohindra says, R&D has focused on a number of in line sensing technologies including the use of Raman spectroscopy to enable very precise measurement of viable cell concentration and individual metabolites such as glucose and lactate. 

	Changes in cell line selection criteria allowing production of more stable clones better suited to perfusion processes also has advanced adoption, noting that Lonza is also looking to develop small-scale models which allow us to assess and select clones which are best suited for continuous processing.”  

	In addition, Mohindra notes, better understanding of the interactions between cells, media components, and disposable technologies, permit the development of new engineering solutions that enable easier handling of media for perfusion processes.

	Apart from reducing bioreactor size-and with it, both capital expenditures and footprint- continuous perfusion systems boost flexibility, Mohindra says, by allowing companies to manufacture diverse product types, a crucial ability for any CDMO. “Traditionally, perfusion processes have been used for labile products but there are also advantages for non-labile proteins. Customers come to us increasingly with more complex formats and perfusion can increase the robustness of manufacturing bispecific antibodies and other new molecular formats. Better control of CQAs means that we can achieve a more stable process and better regulate the final product,” he adds. As a result, duration of the process can be modulated to increase or decrease output in line with changes in demand, (e.g., if more is needed for clinical trials or if regulators grant expedited review of a drug candidate). 

	However, Mohindra notes that perfusion doesn’t yet have the track record of fed-batch manufacturing. “Even certain terminology in regulatory and quality submissions is still geared towards fed-batch processes and needs to be adapted to include continuous processes,” he says. However, he notes that FDA has clarified that lot sizes may be defined based on either mass or time, and have stated that continuous manufacturing is fully consistent with the pharmaceutical quality-by-design concept.

	Another challenge Mohindra notes is that continuous processes blur the lines between the skillsets required for upstream and downstream processing, which have traditionally been handled by separate groups. This means reskilling some teams and changing operational setups. At this point, however, only a few companies are trying to integrate continuous processing both upstream and downstream. 

	“A lot of new technology has been brought to the market recently, so there is a much larger toolbox available to developers and manufacturers, and it’s not all about going straight to the fully continuous route,” says Peter Levison, executive director of business development at Pall. “Companies are considering a hybrid approach, in which they look at individual unit operations and replace the most troublesome ones, rather than going straight from batch to fully continuous,” he says. Generally, he notes, there is growing interest in perfusion and a lot of development work going on, but it will likely take years before it moves fully into large-scale upstream processes.

	CDMOs invest in continuous, and N-1 seed train platforms

	Nevertheless, continuous processing is moving into larger-scale projects and new therapeutic areas and CDMOs including Lonza,  MilliporeSigma, Fujifilm Diosynth Biotech, and Samsung Biologics have embraced perfusion-based continuous processing. In June 2019, Fujifilm Diosynth Biotech invested $10 million in a continuous processing facility in the UK, which is expected to come onstream in the autumn of 2019; the facility will use continuous upstream processing for pre-cGMP process development (3). 

	Whether in stainless-steel or single-use environments, continuous systems are being scaled up and out, and integrated with automation and process control and improved media, to boost cell density and product yields. Seed train, or N-1 approaches, in which cell densities are first built up in one or more smaller bioreactors with ATF or TFF, and then moved to a full-scale bioreactor, have become more important as a way to speed processing.

	In August 2019, Samsung Biologics scaled up the world’s largest ATF-and-perfusion installation to date, using an N-1 approach, at its facility in Songdo, South Korea (4). The company claims that the new installation, which uses a 15,000-L stainless steel bioreactor and Repligen ATF technology, will reduce production time by 30% and allow for a 10-fold improvement in cell culture while retaining viabilities of over 98% at the seed stage. Samsung Biologics has been able to maintain a perfusion rate of up to three-vessel volumes per day by using three 11-m2 X-Cell ATF-10 systems, developed by Repligen. The perfusion process was automated by integrating the ATF system with bioreactor control, the company reported, and validation and other tests were completed within six months.

	For die-hard fed-batch users, Repligen introduced a High Productivity Harvest clarification application in 2019, Gebski says, which allows users to run their process in fed-batch mode, then utilize XCell™ ATF for a short duration harvest during which cell viability is maintained and cell density increases. The application allows the output of a fed-batch reactor to be doubled or the duration of the run 

	Lonza has also realized benefits with upstream continuous processing. “We have been working on process intensification at the inoculum stage, which should allow us to significantly reduce the number of cell-expansion steps and improve the overall space and time yield in our facilities,” Mohindra says. “Simplifying the N-1 stage means that we can inoculate the production bioreactor at a higher cell density. Consequently, we can cut down the time required in the production bioreactor by about 50%, which brings significant time savings to our customers,” he adds. 

	Another benefit he says has been improved ability to control CQAs, as a result of work that is underway at Lonza’s R&D labs in Bend, OR, Mohindra says. “We see an increasing number of new molecular formats in clinical programs-for example, bispecifics-and we are looking to re-tune the process to improve the consistency and quality of products. This also means linking new in-line spectroscopic technology (e.g., Raman sensors) with data handling and analytics.”

	All of the company’s recent facility expansions, including that of its Ibex Solutions facility in Switzerland and its midscale facility in Portsmouth, NH have been designed to accommodate N-1 perfusion processes, Mohindra says.

	While efforts continue to improve data access and analytics, vendors are fine-tuning process-control systems and integrating them with ATF and TFF technologies, and perfusion systems. “A data-rich, continuous operation like perfusion cell culture is ripe for control optimization through process and data analytics, and several layers of integration offer value,” says Bulpin. “First, the use of real-time sensors (e.g., capacitance, Raman, and NIR types) provide a much better picture of the health of the cell, the concentration of nutrients and byproducts, and product and cell titers. Integrating the bioreactor and cell-retention system control strategy will allow us to use this data to maintain high productivity processes within a tight band of control,” he says, noting the potential for machine learning algorithms to be applied to optimize production and even utilize predictive control. 

	Models from a number of other industries offer biopharma developers a glimpse of what is possible, says Lisa Graham, vice president of analytics engineering with Seeq, which offers a browser-based application. Connecting with data historians, the app makes data available for users to perform near real-time data analytics and monitoring. In biologics, she says, there is a great need for real-time data and feedback when shifting from batch to perfusion mode, in order to maintain a steady-state process, she says. 

	A number of projects are also tackling better process control and automation. Sartorius Stedim and Repligen, for example, established a collaboration in 2018 to codevelop and integrated perfusion bioreactor. Zeroing in on single-use perfusion bioreactors in the 50Â­–2000-L range and using next-generation ATF technology, the goal is to integrate XCell ATF hardware and control logic directly into the bioreactor. The goal, says Carpio, is to integrate the bioreactor and cell-retention controller into a single interface that would allow users to control cell growth and perfusion rates in continuous and intensified bioprocessing. 

	At Sartorius Stedim, work is ongoing to enhance continuous processing capabilities via integrated analytics and advanced recipe control, says Carpio. In one recent example, the company’s Corporate Research team automated inoculation from an n-1 intensified culture in the BIOSTAT RM to the production BIOSTAT STR. Using an integrated biocapacitance reading (via Sartorius’ BIOPAT Viamass), the team programmed the controller to transfer culture from the seed to the production reactor automatically, once the Viamass reading reached a specified threshold. Data showed that the growth and productivity of the automatically inoculated bioreactor was comparable to that of a bioreactor inoculated using the traditional manual inoculation method.  “Applying this approach to the manufacturing process will allow for shorter timelines as well as reduced error due to the automation,” Carpio says.

	Mohindra sees advances in process control and data analytics as one of the most exciting drivers of continuous upstream bioprocessing. Driving these advances, he says, is the need for improvement in speed, cost, and quality. “Where we used to be able to measure basic parameters such as dissolved oxygen or temperature, we can now measure individual amino acids in line. The goal is to be able to measure conditions directly where the magic happens in the cell and this is really challenging the industry,” he says. 

	He expects the industry to be able to test by exception, to reduce the need for lab testing significantly. “Here, we’re lifting some of the tools and data-handling experience from small-molecule manufacturing, where real-time release (RTR) is happening. Even though I don’t think we’ll realistically get to RTR in mammalian cell-based biopharmaceutical manufacturing, there is the potential to improve timelines significantly,” he says. Ultimately, he expects to be using machine learning and predictive analytics, along with strong data sets based on past projects, to ensure process improvement.

	The building blocks needed for end-to-end continuous operation are already there, says Levison, but process control and analytical systems are lagging behind. “There is a need for unit operations to be controlled in a way that they talk to each other, and facilitate feedback control so that, for example, if a single component needs changing this can be ascertained online,” he says. New analytical methods are available that will require the ability to store and analyze vast amounts of data, and more automation and control systems will be needed to do this. However, he notes, great strides have been made over the past 18 months, and he expects to see more in the near future.

	Technology providers are also responding to the need for better buffer and media handling systems for continuous perfusion processes. This year, MilliporeSigma introduced the BioContinuum buffer delivery platform, which includes a configurable offering of buffer concentrates, a precision dilution system, and single-use fluid management assemblies, says Bulpin. Its aim, he says, is to reduce the floorspace, labor, and process time required for process buffer preparation. The company also introduced EX-CELL Advanced HD perfusion media, specifically formulated for perfusion bioreactor processing, to improve results typically seen with recovered fed-batch media. 

	1. Pall, “NJII and Pall Corporation Form Agreement to Advance Cell and Gene Therapy Manufacturing,” Press Release, March 19, 2019.
	2. Cobra Biologics, “Cobra Biologics, Pall Corporation and the Cell and Gene Therapy Catapult Win £1.5M Innovate UK Grant to Investigate Continuous Manufacturing for Gene Therapies,” Press Release, Nov. 26, 2018.
	3. Fujifilm, “FUJIFILM Corporation Announces the Introduction of a Novel, Fully Integrated Continuous Production System for the Manufacture of Biopharmaceuticals,” Press Release, June 5, 2019.
	4. Samsung Biologics, “Samsung BioLogics Implements Large Scale N-1 Perfusion for Commercial Application” Press Release, Aug. 12, 2019.


	Vol. 32, No. 10
	October 2019
	Pages: 12–16

	When referring to this article, please cite it as A. Shanley, "Technology Redefines Continuous Processing Efficiency," 

Cover Story

Used with perfusion, alternating tangential flow and tangential flow filtration are redefining upstream efficiency.


Technology Redefines Continuous Processing Efficiency

	A recent Gallup poll concluded “Americans are more than twice as likely to rate the pharmaceutical industry negatively as positively.” In an August 2019 Gallup assessment of perceptions of US business industry sectors, 1525 adults ranked pharma last among 25 industries; 58% of the respondents gave a negative rating and only 27% gave pharma a positive rating. This grading-pharma’s lowest net rating ever in nearly 20 years of the survey-ranks it behind often-criticized business segments such as airlines, banks, oil and gas, the legal field, healthcare, and advertising and public relations. Even the federal government was viewed more positively than drug manufacturers (1).

	Other studies by business and reputation experts ranked pharma in a similarly negative light. In a 2018–2019 global study conducted by PatientView, 41% of patients surveyed rated the pharma industry’s corporate reputation as “excellent” or “good,” down slightly from the previous study. And, pricing appears to be a major factor in the perceptions that patients form about drug companies. Only 9% of the respondents rated pharma’s fair pricing policies as excellent or good (2).

	The reputation of individual pharma companies does not fare much better. Only four pharma companies are listed in the top 100 of the Reputation Institute’s Global RepTrak 100, compiled from a survey of 165,000 individuals. Johnson & Johnson was the highest ranked pharma company, placing at number 82 (3).

	While some executives may take the path of satisfying shareholders over patients or policymakers, positive reputations can be crucial for securing investments and partnership and sustaining innovation. Press releases, lobbying, or social responsibility mission statements can whitewash over some public perception problems. Resolving core issues-such as quality problems and drug shortages-requires investigations of the root cause of the problem and permanent fixes.

	As confidence in drug companies falters, the industry becomes less attractive to skilled researchers, who may turn to more respected fields in science and innovation. Patients who lose faith in pharma’s ability to provide quality medicines at affordable prices may look elsewhere, turning to online or overseas pharmacies that sell products of questionable quality. Problems with mainstream, regulated drugs, however, can also undermine patient trust in the pharma industry.

	Drug development and manufacturing professionals do not have the authority to influence drug pricing or other business decisions. They can, however, make a difference in the quality of drugs produced. A quality culture should be adopted throughout an organization, said Patrizia Cavazzoni, deputy director of operations at FDA’s Center for Drug Evaluation and Research, at the September 2019 PDA/FDA Joint Regulatory Conference. Executive management is ultimately responsible for assuring reliable manufacturing operations and high-quality standards, Cavazzoni said, noting that warning letters are directed to the top company executive to emphasis the importance of quality throughout the organization (4). 

	Organizations should plan for and design quality into all systems, invest in manufacturing technologies, guard against decay in operational effectiveness, and ensure suppliers are aligned with your quality practices, she recommended.

, Sept. 3, 2019.
	2. PatientView, “Corporate Reputation of Pharma in 2018-the Patient Perspective,” Press Release, April 18, 2019.
	3. Reputation Institute, 

2019 Global RepTrak: The Most Reputable Companies in the World

 (2019).
	4. P. Cavazzoni, “Advancing Drug Innovation Through Investment in Quality,” Presentation at the 2019 PDA/FDA Joint Regulatory Conference (Washington, DC, Sept. 16, 2019).

	When referring to this article, please cite it as R. Peters, “Image Repair Must Start with the Root Cause,"

From the Editor

Climbing out of a reputation hole can start with adopting a quality culture.

Image Repair Must Start with the Root Cause

Cell lines determine the performance of bioprocesses and the quality of the biologic drug substances they produce. As such, selection of the optimum cell line for each specific biologic is essential. Given the number of potential factors that can influence cell-line quality, the increasing complexity of biologic drug substances and growing regulatory requirements, identifying the optimum cell line can be challenging.

	A high-quality cell line will demonstrate high stability, scalability, and high titer, so that it can provide reproducible results-with all product quality attributes consistent with the desired profile-with high manufacturing efficiency, according to Ian Collins, senior director of process development at Catalent. “A high-quality cell line should be well-rounded among the critical chemistry, manufacture, and control (CMC) production cell line attributes,” agrees Jill Cai, vice-president of cell and protein sciences with WuXi Biologics.

	Before scaling up to bioreactors, the productivity level for monoclonal antibodies (mAbs) should be 2–7 g/L (fed-batch culture in shake flask) or greater, observes Selexis CEO Igor Fisch. Once scaled up, the mAbs should be 5 to >10 g/L in the bioreactor. “This high level of productivity must also be maintained for more than 60 generations at the high-density and in the defined media conditions of large-scale manufacturing,” he says. In addition, the parent cell line must be characterized and maintain consistency between each cell-line development campaign. Similarly, production cell lines must be well characterized so that scale-up from shake flasks to large bioreactors does not require extensive optimization.

	“Equally important,” stresses Fisch, “is the quality of the recombinant DNA expression and transfection systems. Incorporating recombinant DNA stably into a cell line’s genome in a manner that promotes high-level, stable gene expression while minimizing gene silencing is non-trivial. Therefore, both the cell line and the vectors systems must be optimal in order to achieve optimal results.”

	These aspects are in general desirable for both new biomolecules and biosimilars, although cells expressing biosimilars should be able to produce the target proteins comparable to the cells expressing the original therapeutic proteins. Special attention in this case should be paid to critical quality attributes such as glycosylation, which can add a variable to clonal selection and process development and can lead to a scenario where titer may have to be sacrificed for comparability, Collins notes.

	It is also desirable to leverage a cell-line development technology that has already been utilized in clinical trials or in approved products so that there is a precedent for regulatory filings, he adds.

	Many factors influence the selection of a quality cell line for a given biologic drug substance. First is the product itself, which can affect the choice parental cell line (e.g., Chinese hamster ovary [CHO] or other type). “The advent of more complex molecules rather than monospecific antibodies requires technology that can effectively express multiple genes, as well as titrate the genes as needed; this allows the correct product formation or the expression of other processing proteins to further ensure product quality,” Collins says.

	The capacity of the cell line to cope with the secretory stresses associated with the high level of protein production, particularly with many of the newer, non-natural proteins such as bi-specifics or multi-specifics, is also important, adds Fisch. “It is within the secretory pathway the proteins are properly folded, secondary modifications like glycosylation are added, and protein molecules are paired, when necessary, such as in monoclonal antibodies. If the secretory system cannot handle the secretory throughput, the biotherapeutic proteins can become misfolded or have improper secondary modifications,” he explains.

	This issue is particularly important for biosimilars, because the glycosylation patterns of biosimilars must meet biosimilarity specifications when compared to the innovator drugs, which can only be achieved when they are generated in cell lines with well-functioning and stable secretory machinery, according to Fisch.

	The plasmid ratio and medium are also important, Cai observes. “A balanced plasmid ratio usually has dramatic impact on some formats of bispecific antibody cell line development to minimize impurities,” she explains. Factors such as cell growth and metabolism, as well as product quality attributes like glycosylation distribution, are closely related to the culture medium, and thus many cell-culture media components can impact cell line and product characteristics, she also notes.

	Lastly, comments Fisch, optimized, well-defined, and maintained manufacturing conditions are absolutely critical to achieving the highest level of productivity of a high-quality product. “Feed streams, gas-exchange, metabolic bi-products, and temperature all play critical roles in the amount and quality of the biotherapeutic products. Only the highest quality manufacturing will result in the levels of productivity needed for clinical and commercial manufacturing,” he asserts.

	Challenges with variability, complexity, and regulations

	Selecting the optimum cell line must occur in the face of several Â­challenges. For Fisch, the biggest hurdle to overcome is the variability in the expression and secretion requirements of different biotherapeutic proteins. “Each cell-line development campaign brings its own set of challenges that can affect cell-line productivity and product quality,” he says. “Recombinant biotherapeutic proteins can misfold and stress the secretory pathway, leading to protein degradation or cell death, multi-chain biotherapeutic proteins may not pair properly, or perhaps glycosylation is incomplete. The challenge is to have a cell-line development platform that is robust and flexible enough to allow for troubleshooting across a broad range of production issues,” Fisch concludes.

	Added to this challenge is the strong focus by regulatory authorities on monoclonality, according to Collins. “Whichever method is used for clonal selection should be well validated and ideally utilized in programs that have gained regulatory approval,” he states.

	The sharp increase in the complexity of biomolecules is also increasing the difficulties associated with optimum cell-line selection, adds Cai. “Quickly developing and delivering high-quality cell lines is getting more and more challenging for bispecific antibodies and fusion proteins. It is difficult to rapidly screen more cell types clones to enable the selection of scalable cell lines capable of generating better cell productivity and product quality that are stable over extended cell-line generations and the long production cycle from vial thaw to harvest at large manufacturing scales,” she explains.

	Two specific challenges, Cai continues, include clippings for bispecific antibody and fusion proteins, which may require many constructs screening and extensive mass analysis or peptide mapping to detect the problem early on. Another challenge for bispecific antibody cell line development is the percentage of the desired product vs. byproduct impurities, which requires early downstream involvement to determine the types of impurities that are easy to remove, and consequently influences clone selection.

	The importance of an experienced team in cell-line development should not be underestimated, according to Fisch. “While there can be straightforward cell-line development campaigns with mAbs, there are also numerous examples of difficult-to-express mAbs. In addition, the newer bi-specifics and multi-specifics, which are non-natural proteins, can really challenge a production cell line’s capacity to produce and secrete,” he states. A team that has seen and solved many of the issues associated with difficult-to-express proteins has the know-how to identify issues early and a history of solutions to address them, Fisch asserts. Having an experienced team with access to the right technologies can save months on any cell-line development campaign, he adds.

	Bioinformatics tools can also be invaluable. “The ability to sequence the host cell-line genome, and more recently the transcriptome, along with the development of the bioinformatics to analyze that data has allowed for significant advances in cell-line development,” Fisch says. Bioinformatics analysis may help in getting genetic/epigenetic information that could offer insights into issues with protein production or cell stability in advance, agrees Cai.

	“Transcriptomics and proteomics can provide a more comprehensive and quantitative understanding of metabolic, signaling, secretory, and other pathways to determine whether a cell line is good for production,” Cai comments. “Additionally, Cai notes that bioinformatics methods can help rationally design a host cell through determination of ideal genome sites for target Â­integration and pathway engineering based on RNA sequencing results,” she notes.

	For instance, Fisch observes that secretory requirements can vary from protein to protein, and the genomic and transcriptomic analyses of the parent manufacturing cell lines helps to determine where there might be deficiencies or inappropriate expression of cellular proteins affecting a given biotherapeutic’s production.

	Detailed genomic data can also be used to assess cell lines for transgene integrity at junctions within the genome, transgene copy number and correct DNA sequence, number and locations of genomic integration loci, and possible adverse effects from transgene integration, according to Fisch. “[These] data, along with the right bioinformatics, can allow for direct assessment of the clonality of the biotherapeutic production cell line and provide traceability, validation, and origin of cells used to generate the production cell line-a vast improvement over the historical, indirect ways of determining clonality that makes it considerably easier to monitor drift and possible contaminations in production cell lines,” he remarks.

	The best instruments for selecting high-quality cell lines ideally enable the simultaneous screening of productivity (i.e., protein amount) and product quality (e.g., aggregation and cleavage), according to Cai. “Unfortunately,” she says, “currently available instruments mainly focus on productivity-based screening. Flow cytometry does, however, provide a relatively easy and economical way of screening high-producing cell lines.”

	New instruments based on microfluidic and nanofluidic technologies such as Cyto-Mine (Sphere Fluidics) and Beacon (Berkeley Lights), which allow the screening of cell-line productivity during the single-cell cloning step, are emerging, according to Cai, but such instruments are generally more expensive than flow cytometers and their reliability, time savings, and acceptance by regulatory agencies have not been widely demonstrated.

	Other new instruments designed for single-cell deposition are also available such as single-cell printers and verified in-situ plate seeding. Cai notes that these instruments are easy to use and may one day replace flow cytometry for single-cell deposition.

	Catalent has focused on the Beacon platform, which the company believes to be an emerging leader in the area of clonal isolation and selection. “The Beacon platform is an automated system that is able to select the highest expressing clones faster than traditional methods and provides sufficient data to allow for a single round of cloning, which can reduce timelines for early cell-line development,” Collins states.

	The best selection tools depend upon the cell-line development platform, adds Fisch. “In some platforms with lower transfection efficiencies, identifying high-expressing cell clones depends upon screening tens of thousands of clones, which is achievable using flow cytometry and high-throughput screening systems. When transfection efficiencies are high, identifying high-expressing and stable cell clones requires far less screening,” he explains. Selexis uses systems such as the Beacon optofluidic platform and ClonePix to provide faster, more efficient, multiparameter screening approaches for clone selection.

	Catalent is leveraging automation to create high-quality cell lines and timelines. The company combines its proprietary GPEx cell-line development technology with the Beacon platform to generate highly stable, high-titer clonal cell lines for even difficult-to-express proteins, while the ambr15 system (Sartorius AG) allows for early evaluation of the individual clones in a bioreactor environment, according to Collins.

	WuXi Biologics carefully considers each and every step involved in cell-line development using its WuXia platform to ensure the generation of high-quality cell lines. The strategy, according to Cai, typically starts with the molecule of interest and a codon optimization step to optimize the sequence of the gene(s) of interest. Proprietary vectors are then specifically engineered to enhance the expression level of the target genes. The company uses a tandem pool development and clone-screening approach to maximize the chances of deriving high-producing and high-quality cell lines. “We work closely with our analytical and process development teams to ensure the developability, monoclonality, and scalability of our cell lines and to achieve the key product quality attributes that our clients and partners desire,” Cai observes.

	With the Selexis SUREtechnology Platform, Selexis scientists apply a modular approach to generate high-quality cell lines that meet the production needs of each recombinant therapeutic protein. At the foundation of each cell-line development program, according to Fisch, are Selexis SGEs (Selexis Genetic Elements), unique epigenetic DNA-based elements that control the dynamic organization of chromatin in all mammalian cells and allow for higher and more stable expression of recombinant proteins.

	A panel of tens to hundreds of high-expressing clones are assessed for production levels, product quality, and cell viability. If any of these parameters are suboptimal, Selexis has a suite of solutions for addressing them. With this approach, Selexis is able to express almost every protein, including difficult-to-express proteins that have failed in other systems, Fisch says.

	Selecting cell lines for CAR-T therapies

	As opposed to a synthetic drug, chimeric antigen receptor (CAR)-T cells are living therapies that are sensitive to manipulation and difficult to manufacture with consistent quality. As a result, the process of preparing them so they are safe and effective is multifaceted and complicated, according to Kris Simonyi, global marketing manager with Bio-Rad Laboratories. “One must confirm that all replication-competent viruses have been removed before injecting CAR-T cells into the patient, that the transgene has been inserted in the right location in the genome with the right dosage, and that the cell line is free from microbial contamination,” he explains.

	As a result, a high-quality CAR-T-cell line should not contain any replication-competent viruses or microbial contaminants, but should contain one to four copies of the CAR gene (more than four copies exposes patients to the toxic effects of T cells, which include organ dysfunction and death).

	One of the biggest challenges in selecting high-quality CAR-T cells, Simonyi notes, is that even if a transfection is technically successful, it does not mean it will provide a clinical benefit. “Transgenes integrate into random sites and may have detrimental effects on a patient,” he says. For example, a CAR gene could integrate into and activate an oncogene and lead to cancer. It could also integrate into a non-coding region of DNA and never be expressed.

	To detect low levels of CAR genes and microbial contaminants and precisely quantify CAR genes, Bio-Rad’s Droplet Digital PCR (ddPCR) technology is of increasing interest. The technology selectively amplifies targets of interest including genetic alterations and nucleic acid contaminants, making them easy to identify, according to Simonyi.

	Samples are partitioned into 20,000 nanoliter-sized droplets and a polymerase chain reaction (PCR) takes place in each droplet. A fluorescent probe binds to the target DNA, making it fluoresce when it gets amplified. The nucleic acid fragments that do not contain the target sequence do not get amplified and do not fluoresce. By counting the number of fluorescent droplets versus the total number of droplets, the concentration of the target DNA strand can be determined.

	Custom probes can be used to select for cells containing the CAR gene and bacterial contamination. Because the system performs absolute quantification without the need for a standard curve, it can also measure transfection efficiency and precisely quantify the transgene copy number in CAR-T cells. “Our ddPCR technology can be 100- to 1000-fold more sensitive than quantitative PCR, enabling it to detect down to one CAR gene copy per cell,” Simonyi comments.

	It can also detect and precisely quantifying various types of DNA and RNA alterations that can factor into the quality of genetically engineered T cells, including minimal levels of nucleic acids of interest, such as genetic material from replication-competent viruses and other contaminating microbes. In addition, following next-generation sequencing to map transgene integrations, Bio-Rad’s ddPCR technology can be used to track the cells through the manufacturing process, according to Simonyi.


	Vol. 31, No. 10
	October 2019
	Pages: 18–21

	When referring to this article, please cite it as C. Challener, “Best Practices for Selecting a Top-Quality Cell Line,"

Leveraging automation and a step-by-step approach are keys to success.

Best Practices for Selecting a Top-Quality Cell Line

Because federal investigators cannot inspect every drug test site or production facility, FDA relies on manufacturers to submit complete and accurate information in all submissions and market applications, especially for innovative cell and gene therapies. Evidence that Novartis submitted manipulated data from certain preclinical tests related to its production process for breakthrough therapy Zolgensma recently produced a strong, public warning from FDA, with implications that the agency may pursue civil or criminal charges. 

also blasted Novartis and demanded a full accounting of its actions (1).

from FDA to Chinese over-the-counter drug manufacturer, Ningbo Huize Commodity Co., similarly cited egregious data integrity lapses (2). In a statement issued on Aug. 20, 2019, FDA said it was banning the import of all the company’s products following a plant inspection where local staffers provided FDA investigators with documents that were clearly falsified, including cleaning validation reports and batch production and control records for multiple drugs (3).

	In highlighting this enforcement action, FDA Acting Commissioner Ned Sharpless stated that efforts to “prevent, uncover, and combat data integrity lapses” is a continuing commitment of the agency and involves increased global inspections, updated guidance, and additional staff training. FDA published 

 in December 2018 for ensuring data integrity in compliance with current good manufacturing practices (CGMPs) (4), which emphasizes the importance of company top management establishing a “quality culture” that acknowledges the importance and value of data integrity in all aspects of drug development and production.

	Data integrity issues involving manufacturing activities are cited regularly in warning letters related to manufacturing facility inspections at home and abroad. A 

dated July 9, 2019 warned Indoco Remedies Ltd. of Mumbai, India of GMP and data integrity violations at a facility in Goa (5). It cited problems with batch production and control records for products intended for the US market. And a June 13, 2019 

 to Akorn Inc. raised concerns about the firm’s failure to ensure the accuracy and integrity of data to support the safety, effectiveness, and quality of its products (6). FDA cites a previous warning letter regarding inadequate quality controls at another Akorn production site and demands prompt correction of all cited violations or risk legal action and delays in product approvals.

	In the Novartis case, Peter Marks, director of the Center for Biologics Evaluation and Research (CBER), sharply rebuked the company, emphasizing the importance of FDA having confidence in all tests and data provided by sponsors, particularly to support the rapid development and accelerated approval of innovative therapies. The submission of “truthful, complete, and accurate data” is critical for FDA to be able to protect the public health, and the law requires it, Marks asserted in a statement on Aug. 6, 2019 (7).

	FDA’s pronouncements aim to provide clear warnings to manufacturers that data manipulation is a serious offense, and that data quality is critical for product approval and continued marketing. Marks said that Zolgensma would remain on the market, as the faulty information involved early animal studies related to developing the production process for Zolgensma and did not compromise product safety. But he acknowledged that if the agency had been aware of the data manipulation by Novartis’ AveXis unit, it probably would have delayed approval of the lifesaving $2-million drug.

	Although Novartis claimed that it quickly investigated allegations of data manipulation when the issues first surfaced internally, the timing and disclosure of these problems raise questions. The company evidently knew of the data errors as early as March 2019 but did not launch a formal investigation until May and did not reveal its concerns until June. And that was after the May 24, 2019 approval of Zolgensma, based on limited, but convincing evidence that it dramatically improved the health of infants suffering from the most severe form of the neurodegenerative disease spinal muscular atrophy.

	FDA compliance officers continue to investigate whether Novartis and AveXis staffers intentionally hid evidence of data manipulation until after approval. According to press reports, FDA inspectors raised questions about a mouse essay at an inspection of a Novartis manufacturing facility in Illinois earlier this year, but company staff said they had been corrected. However, FDA’s 

 in July 2019 of AveXis’ San Diego control test lab found evidence that management failed to thoroughly review unexplained discrepancies in potency assays, had incomplete records, and failed to follow quality control and test procedures (8).

	Novartis dismissed the AveXis scientists linked to the data manipulation, but FDA holds corporate management responsible for ensuring adherence to rules at all levels. Although only a small portion of product test data in the market application appears to be manipulated, FDA says that a thorough assessment of all information will determine if further warnings or limits are warranted for this new therapy that has limited patient experience.

Warning Letter to NingBo Huize Commodity Co., Ltd.

, Aug. 2, 2019.
	3. FDA, “FDA Warns Company for Putting Consumers at Risk with Drug Manufacturing Data Integrity Violations,” Press Release, Aug. 20, 2019.
	4. FDA, 

Data Integrity and Compliance with Drug CGMP Questions and Answers, Guidance for Industry

 (CDER, 2018), www.fda.gov/media/119570/download.
	5. FDA, 

Warning Letter to Indoco Remedies Limited

, June 13, 2019.
	7. Dr. Peter Marks, MD, Statement on Data Accuracy Issues with Recently Approved Gene Therapy, Press Release, August 6, 2019.
	8. FDA, 


	Vol. 32, No. 10
	October 2019
	Pages: 9-10

	When referring to this article, please cite it as J. Wechsler, "Data Integrity Violations Draw Strong FDA Rebukes," 

Regulatory Beat

Falsified documents and manipulated test results prompt warnings and investigations.

Data Integrity Violations Draw Strong FDA Rebukes

Q: I am familiar with the term ALCOA as it relates to data integrity, but lately, I have heard people refer to ALCOA+. Can you explain what impact this new acronym has on my company’s data integrity program?

	A: Before we get into the reason for the additions to ALCOA, referred to as ALCOA+, we should review what both of the acronyms mean. The acronym ALCOA requires data be attributable, legible, contemporaneous, original, and accurate. The acronym ALCOA+ adds the concepts that, in addition to ALCOA, data also needs to be complete, consistent, enduring, and available. It is important to understand what each element of ALCOA and ALCOA+ mean in order to apply the concepts appropriately with respect to a company’s records. The following are some general definitions, paraphrased from the Pharmaceutical Inspection Co-operation Scheme (PIC/S) (1), that can be used for understanding the elements of ALCOA and ALCOA+:

		Attributable: The data generated or collected must be traceable back to the individual who generated the information.

		Legible: The data recorded must be readable and permanent.

		Contemporaneous: The results, measurements, etc. must be recorded at the time the work is performed.

		Original: Original or source data are the record, report, notebook etc. where the data point was initially recorded.

		Accurate: The data recorded must be complete, consistent, truthful, and representative of facts.

		Complete: Information that is critical to recreating and understanding an event. This would include any repeat or reanalysis performed on a laboratory test sample.

		Consistent: The data are presented, recorded, dated, or time-stamped in the expected and defined sequence.

		Enduring: The data or information must be maintained, intact, and accessible throughout their defined retention period.

		Available: The data or information must be able to be accessed at any time during the defined retention period.

	Data integrity has always concerned regulatory authorities, but it is important to understand what is prompting the renewed discussion of ALCOA and the introduction of ALCOA+ when discussing data integrity issues. Many of the concepts for ALCOA have been captured in the regulations as far back as 1978. Since that time, the industry has changed dramatically. The generic-drug industry has grown and in the United States alone accounts for more than 80% of the prescriptions written today (2). Coupled with the emergence of biosimilars, virtual companies, contract manufacturing organizations, rapid advances in automation and information technology, and the globalization of the industry have resulted in reinterpretation of the attributes associated with maintaining the integrity of data throughout the product lifecycle, whether those data are generated from electronic, paper-based, or hybrid systems. In addition, there has been an increase in citations internationally by the FDA, European Medicines Agency (EMA), Medicines and Healthcare products Regulatory Agency (MHRA), World Health Organization (WHO), and other health authorities. These changes and increased violations have brought about a resurgence and need to reeducate the industry on the basic principles and concepts regarding the proper control of data used to support the quality and safety of medicines.

	The ALCOA acronym has been used since the 1990s; however, the requirements governing data elements have been in regulations for a much longer period of time. 

chapter 4 states, “Suitable controls should be implemented to ensure the accuracy, integrity, availability, and legibility of documents. Instruction documents should be free from errors and available in writing” (3). The US 

) refers to these elements in various sections of the regulations (4–8). An example for language speaking to the element of attributable is in 21 

 211.194(a)(7), which states, “The initials or signature of the person who performs each test and the date(s) the tests were performed.” Language in 21 

 58.130 (e) addresses the elements of contemporaneous and legible by stating, “All data generated during the conduct of a nonclinical laboratory study, except those that are generated by automated data collection systems, shall be recorded directly, promptly, and legibly in ink. All data entries shall be dated on the date of entry and signed or initialed by the person entering the data.”

	Recent documents issued by WHO (9) and the PIC/S (1) have added to the original ALCOA attributes as indicated above. The PIC/S document actually states, “Some key concepts of GDocPs are summarized by the acronym ALCOA: Attributable, Legible, Contemporaneous, Original, and Accurate. The following attributes can be added to the list: Complete, Consistent, Enduring, and Available. Together, these expectations ensure that events are properly documented and the data can be used to support informed decisions.” WHO refers to ALCOA+ in the title of Appendix 1 to their 2018 document. The last two documents also address the concept of quality culture (10). The impact to your organization is that the quality culture must ensure that data supporting the quality and safety of your product must now meet the ALCOA+ elements in order to avoid regulatory citations for data integrity issues.

Draft Guidance: Good Practices for Data Management and Integrity in Regulated GMP/GDP Environments

 EudraLex,The Rules Governing Medicinal Products in the European Union, Volume 4, Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use

 11: Electronic Records; Electronic Signatures
	5.  21 

 58: Good Laboratory Practice for Nonclinical Laboratory Studies
	6.  21 

 211: Current Good Manufacturing Practice for Finished Pharmaceuticals
	7.  21 

 212.50: Current Good Manufacturing for Positron Emission Tomography Drugs
	8.  21 

 820: Quality System Regulation
	9.  WHO, 

Annex 5, Guidance on Good Data and Record Management Practices


	Vol. 32, No. 10
	October 2019
	Page: 54, 53

	When referring to this article, please cite it as S. Schniepp, "ALCOA+ and Data Integrity," 

Ask the Expert

Data supporting the quality and safety of product must meet the ALCOA+ elements in order to avoid regulatory citations for data integrity issues, says Susan J. Schniepp, executive vice-president of post-approval pharma and distinguished fellow, Regulatory Compliance Associates.

ALCOA+ and Data Integrity

Aluminum compounds have been used as adjuvants in human vaccines for more than 75 years (1), and they are the most widely used adjuvants in both human and veterinary vaccines. Billions of doses of aluminum salts containing vaccines have been administered safely to a diverse population of patients globally (2). Despite their widespread usage, there is little regulatory guidance or published literature regarding release and stability testing of these products. Though there have been great gains in knowledge of therapeutic protein chemical and physical stability, there are few published examples of the applications of bioanalytical stability methods applied to protein once adsorbed on to the aluminum adjuvant. This article provides an introduction to aluminum adsorbed vaccines, reviews studies of antigen stability, and proposes a panel of test methods for the analysis of aluminum vaccine release and stability analysis.

	Early vaccines were prepared as alum precipitates, which could be heterogeneous. Preformed gels of aluminum hydroxide and, later, aluminum phosphate replaced alum precipitation and have resulted in standardized formulations of aluminum-adsorbed vaccines. Aluminum-based adjuvants continue to be used in new vaccine preparations as recombinantly expressed protein antigens are identified, which can serve as safer replacements for inactivated or attenuated pathogens.  Additionally, vaccines containing combination adjuvants linking aluminum with synthetic and natural ligands have been approved (2). The addition of adjuvants is often needed to induce a robust immune response and effective immunization to protein antigens, which may not possess strong immunogenicity. The physiological mode of action of aluminum adjuvants, however, is not completely understood (3). Potential mechanisms include that the aluminum forms a depot at the injection site from which the antigen is released slowly; that the particulate form of the adsorbed vaccine results in uptake by macrophages, neutrophils, and dendritic cells; and the immune system is stimulated directly. Additionally, it has been proposed that adsorption of the protein on the aluminum gel could result in structural destabilization increasing the protein’s immunogenicity (4).

	There are two common aluminum salt preparations used in human vaccines: aluminum hydroxide (Alhydrogel) and aluminum phosphate (Adju-Phos). The pH at which the net surface charge of the aluminum adjuvant is zero is called the point of zero charge (PZC), which is similar to the isoelectric point (pI) of a protein. The adjuvant surface is positively charged when the solution pH is below the PZC and negatively charged when the solution pH is greater than the PZC (5). Aluminum hydroxide has a PZC of approximately 11 and aluminum phosphate has a PZC of 4–5.5 (4), though consideration should be given to the buffer used in vaccine formulation as substitution of ions at the surface may lower the PZC and impact the adsorption of the antigen (5). For many proteins, antigen adsorption is best in pH intervals between pI of the protein antigen and PZC of the aluminum adjuvant because it is in this pH range that the protein antigen and the adjuvant will have opposite electric charges (1). For this reason, lysozyme, which has a pI of 11.35, has been used as a model antigen in studies of aluminum phosphate. Bovine serum albumin (pI of 4.7) and ovalbumin (pI of 4.5) bind more efficiently to aluminum hydroxide (4). Generally, Alhydrogel has a higher adsorption capacity than Adju-Phos, and the adsorption capacity of Alhydrogel decreases as its particle size increases and as the molecular weight of the proteins increase (6).

	The adsorption mechanism of proteins to aluminum adjuvants is not completely understood (6) and is likely a combination of physical phenomena including “electrostatic attraction, hydrogen bonding, apolar interactions, ligand exchange, and van der Waals’ forces” (4).  Studies of model proteins found that electrostatic interactions were the major mechanism for adsorption of lysozyme, human growth hormone, diphtheria toxoid, a monoclonal antibody, and PEGylated growth hormone, but that for one protein, ovalbumin, adsorption involved ligand exchange (6). The predominant mechanism for adsorption of HBsAg by aluminum hydroxide was determined to be ligand exchange between the phospholipids in HBsAg and the surface hydroxyls in aluminum hydroxide adjuvant (7). HBsAG was found to bind more tightly to an aluminum hydroxyphosphate sulfate adjuvant when the concentration of phosphate buffer was increased (8). The correlation of strength of adsorption to vaccine effectiveness is related to the adsorption mechanism. Phosphorylated proteins adsorbed via ligand exchange have an inverse relationship between high adsorption strength and poor antigen presentation and immune response (9). Studies of a trivalent Pneumococcal  protein vaccine demonstrated differences in immunogenicity resulting from selection of adjuvant impacted immunogenicity and found that lower microenvironment pH at the antigen surface and decreased strength of adsorption improved antigen stability (10).

	A significant challenge in vaccine formulation development is the understanding of the antigen stability following adsorption. While aluminum-precipitated and aluminum-adsorbed vaccines have been in use for 75 years, it has only been in the past 15 years that studies investigating the impact of adsorption on protein structure and stability have been reported, presumably as new techniques developed to characterize recombinant proteins were applied to vaccine research. In a review of the subject, Clapp and colleagues describe how the environment of the protein at the adjuvant surface differs from that in solution, potentially driving structural change (11). Specifically, studies of the rate of acid-catalyzed hydrolysis of glucose-1-phosphate (G1P) adsorbed to aluminum hydroxide concluded that the pH of the microenvironment at the surface of the adjuvant was approximately two pH units higher than that of the bulk solution (12). Differences in polarity and charge density on the adjuvant surface may also result in structural changes in the bound protein (11).

	Spectroscopic methods have been applied to the study of protein structural perturbations resulting from adsorption. Techniques have been well described in the literature and have included fluorescence spectroscopy (13), transmission Fourier-transform infrared (FTIR) spectroscopy (14), FTIR spectroscopy using an attenuated total reflectance (ATR) cell (13, 14, 15), and circular dichroism (CD) (15). Differential scanning calorimetry (DSC) has also been applied (13, 16). In studies of model vaccines, structural changes by FTIR–ATR, fluorescence spectroscopy techniques (13, 15), and DSC (13) were observed by following adsorption.  Studies using transmission FTIR (14) concluded that no structural changes resulted from adsorption in studies of six model proteins. Overall, a review of studies indicates that variation in the buffer concentration, buffer pH, and properties of the antigen itself may influence the structure of the adsorbed protein antigen. In studies of a hepatitis B vaccine, the interaction between the antigen and aluminum hydroxide adjuvant was modified by optimizing the phosphate ion concentration and could possibly ensure that the native concentration of the antigen was maintained (17). While no structural information was presented, the authors concluded that a formulation containing 40 mM phosphate had improved thermal stability by 

 antigen reactivity. Additionally, FTIR–ATR studies determined that the structural changes were dependent on the amount of protein adsorbed, with more native protein observed in samples with maximum adsorption (17). The authors theorize that at lower adsorption concentrations, a monomolecular layer is formed in which the confirmation of the adsorbed protein is affected by the properties of the adjuvant surface and are more denatured. At higher adsorption levels, the protein is more weakly linked and may retain the native structure. While the focus of aluminum-absorbed vaccine characterization studies has protein antigen structure, questions remain about the applicability of studies to individual vaccine formulations and the impact that antigen structural perturbations may have on vaccine effectiveness and stability throughout the shelf life of the product.

	Regulatory guidance for vaccine stability studies

	There is little general guidance on release and stability test methods for aluminum adsorbed vaccines. The FDA document, 

Guidance for Industry, Content and Format of Chemistry, Manufacturing and Controls Information and Establishment Description Information for a Vaccine or Related Product 

(18), states that identity, purity, and potency should be included in drug product release specifications but does not provide specific test methods or guidance. 

The authors provide an introduction to aluminum adsorbed vaccines, review studies of antigen stability, and propose test methods for the analysis of aluminum vaccine release and stability analysis.

BioPharm International-10-01-2019

Driving Improved Access to Biosimilars

Technology Redefines Continuous Processing Efficiency

Image Repair Must Start with the Root Cause

Best Practices for Selecting a Top-Quality Cell Line

Data Integrity Violations Draw Strong FDA Rebukes

ALCOA+ and Data Integrity

Stability Indicating Methods for Aluminum Adsorbed Vaccine Products

Using Software in Process Validation

Streamlining Operations in Fill/Finish

Outsourcing Early Development Operations

BioPharm International, October 2019 Issue (PDF)