Manufacturers exploring opportunities in global markets face dynamic demographic and disease trends, changing market demands, and evolving regulatory requirements—all of which differ from one country to another. While emerging markets continue to represent a new channel of demand for pharmaceutical products, manufacturers have quickly realized that significant attention must still be given to more established markets, such as Germany. It’s no longer sufficient to expect that regulatory data from prospective randomized clinical studies alone will be enough to convince payers to provide a price premium for new products. In this article, the authors take a look at some of the recent developments in the German pharmaceutical market, identify pricing and reimbursement challenges, and discuss strategies manufacturers should consider for sustainable success.


Germany is the third largest pharmaceutical market worldwide and accounts for approximately 23% of the European pharmaceutical market (1). Of equal importance to manufacturers, drug prices in Germany are often used as reference prices in 19 other countries. Taken together, gaining access to this market at an acceptable price has major implications for long-term success. In a cost-constrained economy, the German government has taken steps to reduce healthcare spending, and unfortunately for manufacturers, one way they have sought to do this is through The Act on the Reform of the Market for Medical Products (Arzneimittelmarkt-Neuordnungsgesetz—AMNOG). This piece of legislation was enacted in 2011 and mandates a more rigorous benefit evaluation procedure for new pharmaceuticals (and their subsequent label expansions), thereby adding significant challenges to the market access process.

In AMNOG’s new system, manufacturers continue to set the initial price for new prescription drugs after regulatory approval. This price is valid for one year. During this time, the manufacturer’s value dossier is reviewed by the Federal Joint Committee (G-BA), which represents the self-governance structure of the German healthcare system. With the help of the Institute for Quality and Efficiency in Health Care (IQWiG), an expert Health Technology Assessment (HTA) body, the G-BA determines the level of added benefit of the new drug relative to the appropriate comparative therapy (ACT) chosen by the G-BA as well as the certainty of evidence presented by the manufacturer. A brief description of these two inputs that go into the G-BA’s final decision is included in 

. Patient-relevant criteria for additional medical benefit include improving health, extending survival, shortening the burden of illness and reducing side effects, or improving quality of life, and are outlined in the IQWiG methodology paper (2). Originally, the law was written to include the possibility that patented products already on the market would have to undergo retrospective benefit assessments; however, manufacturers were recently spared from this potential headache (3).

Decision criteria for G-BA’s/IQWiG’s benefit assessment.

Factors shown to impact categorization   decision

Number   of appropriate clinical studies available; Use of valid measurement   instruments; Risk of bias (e.g., non-blinding); Endpoint type (e.g., hard,   surrogate)

Major,   considerable, minor, non-quantifiable, no added benefit, or reduced benefit

Effect   size at outcome level, confidence interval associated with effect

Once the G-BA reaches a final conclusion, pricing negotiations begin with the statutory health insurance (SHI) umbrella organization, GKV
  SV (Gesetzliche Krankenversicherung-Spitzenverband), which negotiates on behalf of all SHIs and also private sickness funds in Germany. It is important to highlight that the ACT is not only used by the G-BA in its assessment, it is also the price anchor for these negotiations. For drugs found to have no added benefit, their price is capped in reference to the price of the comparator. Reference pricing to the comparator can be either advantageous if the ACT is already a high-priced therapy or devastating if the ACT is a generic drug. Thus, manufacturers looking to enter breakthrough therapeutic areas where the current standard treatment is a generic will be particularly challenged. Therapeutic products that demonstrate added benefit are priced above the ACT but may still face a minimum price reduction of 7% (formerly 16%) by law, unless it has been replaced as part of the price negotiations. Unfortunately for manufacturers, while the number of products that have gone through the entire assessment and pricing process remains small since the law was enacted three years ago, early evidence suggests only limited correlation exists between benefit ratings and price reductions. Rather, G-BA’s benefit rating has primarily served as a door opener for price negotiations (4).

Snapshot of Benefit Assessments and Pricing Negotiations under AMNOG


As of May 2014, 79 products have been assessed by the G-BA within 169 patient subpopulations. More than 50% of these early benefit assessments have led to ‘no added benefit’ rulings by the G-BA (see

) (5). Not surprisingly, there has been considerable variance observed in the levels of additional benefit reported by pharmaceutical manufacturers and the G-BA. In contrast, the G-BA’s decisions have more closely resembled IQWiG’s recommendations with a few notable exceptions (e.g., belimumab, cabazitaxel, eribulin) (6). Reasons the G-BA has offered for ‘no proven added benefit’ include inadequate comparator, lack of a relevant study to back claims of benefit, lack of data on potential harms, and omission of data for relevant patient subpopulations. A more detailed breakdown of the G-BA’s negative rulings as of October 2013 is presented in   

 G-BA decision breakdown on “Degree of Added Benefit” for new pharmaceuticals within a specific patient subpopulation (N=169).

 illustrates, even though the outcome of benefit assessment is a key driver of price negotiations, manufacturers have not always prepared dossiers adequately. This, in turn, compromises the outcome of the assessment and subsequent price that is agreed upon. Such problems with incomplete or inadequate evidence may be overcome as drug makers gain better understanding of the requirements. To this end, the Federal Joint Committee offers consultation opportunities for manufacturers to give advice on trial design (e.g., patient segmentation, comparator selections, and endpoints). Not all manufacturers, however, have communicated with the G-BA to discuss the technical issues and challenges around preparation of the benefit dossier, or earlier to discuss the clinical trial (6).

Despite the market access challenges that manufacturers have experienced so far, only a few products have been pulled despite negative G-BA rulings and demands for substantial rebates. Novartis became the most recent manufacturer to do this, withdrawing its oral anti-diabetic Galvus from the German market after failing to secure a mutually beneficial price with payers (8). Given the size and revenue potential associated with this marketplace, the fact that few products have been pulled is not surprising. However, Hagen Pfundner, the chairman of Germany’s pharma lobby VFA and head of Swiss drugmaker Roche’s German unit, believes that a recent update to the AMNOG legislation could lead to “an increasing number of opt-out decisions” (9). In the past, a product’s original launch price in Germany served as the published list price for other markets. This approach is no longer the case. Since April 2014, the newly negotiated reimbursement amounts are published as visible ex-factory prices, effectively replacing the original list prices as source for referencing. Drug makers fear the information could challenge the entire pricing framework within Europe (and globally) either causing a downward spiral in reimbursement across the region or heavy parallel exports from Germany to other countries. If this new law is not overturned, it poses a significant risk to profits in the industry, especially considering that price discounts have been as high as 70% following G-BA’s benefit assessment and range on average between 1630% (10).  More broadly, the new rule reinforces the importance of manufacturers understanding the nuances of this market and having strategies in-place to achieve successful market access.


  While Germany is certainly not the only market that is enforcing more stringent pricing and reimbursement policies for new therapeutic products, it is perhaps scrutinizing the quality and appropriateness of clinical data more so than any other country. Taken together with the fact that other markets look to Germany to determine pricing, manufacturers should place a greater emphasis on G-BA’s recommendations when structuring clinical studies. To date, the majority of AMNOG submissions did not have much guiding input from the G-BA prior to clinical study design and were based on evidence generated on a pre-AMNOG world. Moving forward, proactively consulting with the G-BA to define the comparator(s), clinical endpoints and patient cohorts will be essential. Manufacturers need to do this early in product development, especially prior to starting Phase III trials. Internally, manufacturers should also enhance communication between regulatory and market access teams to strategically plan for both market authorization requirements and G-BA’s requirements.

Even before G-BA consultations, manufacturers need to give greater consideration to the product’s indication. While the indication does not determine how the benefit assessment is conducted, completed assessments indicate that it can influence the G-BA’s comparator choice and a pivotal trial’s relevance. For products with broad indications, IQWiG and the G-BA are demanding clinical data for multiple patient subpopulations and against multiple ACTs. For example, these commissioning bodies recently distinguished between four subpopulations and ACTs for Roche’s Kadcyla (see 

) (11). Manufacturers that have failed to anticipate and include such data are seeing a significant reduction in the overall level of added benefit granted to their products (e.g., Pixuvri and Inlyta) (12). Moving forward, manufacturers will need to ensure that all relevant subpopulations have robust and statistically significant clinical endpoints to support reimbursement, with increasing demand for quality of life data. If the resources required to study multiple populations using multiple comparators are too great, pursuing a narrower indication for a clearly defined patient population and against a single comparator serves as a viable alternative.

Manufacturers will also have to keep in mind that every new indication for a product will trigger another round of benefit assessment and a new price negotiation. Here, attempts to negotiate a higher price with subsequent indications may be met with resistance by the GKV-SV and prove to be a significant challenge for manufacturers. Thus, drug makers should give greater consideration to the trade-off between a fast product launch and the highest value launch.

Aside from data collection and defining a product’s indication, manufacturers must also develop a solid pricing strategy earlier on in the product development process and have capabilities to better predict a products negotiated price. There is currently no formula regarding the rebate amount the pharmaceutical company has to grant on its retail price.  Furthermore, while evidence demonstrating added benefit is required to avoid reference pricing, it is by no means the sole determinant of pricing decisions. Rather, the extent to which a product’s price will be marked down following a positive benefit assessment is largely dependent on its original launch price as well as the market size and price of the appropriate clinical comparator. As more products go through the product review and pricing process and as more data become available, manufacturers should consider developing algorithms to forecast the benefit assessment outcomes and the product’s final net price.

The first three years of the AMNOG reform have proven that demonstrating added benefit is no small feat for manufacturers, and while complaints continue to be heard from pharmaceutical stakeholders, the law shows no signs of slowing down. AMNOG has not only set new rules for reimbursement and pricing of pharmaceuticals in Germany, it also forces the industry to adapt at a global level, rethinking how it brings new products to market. Manufacturers that prepare thoroughly for benefit assessment and the negotiation processes within it will be best positioned for success.


  The authors would like to thank Stefan Seliger, PhD, for his review and insights.

German Pharmaceutical Industry Association/BPI, based on IMS World Review 2012.

IQWiG, General Methods Version 4.1.  (Nov. 28, 2013).

A tweak or an outrage? Germany passes new pricing law

LEO Pharma Germany, It´s difficult to meet HTA criteria according AMNOG — reason why?  (June 10, 2014).

 PRMA Insights Focus: Pricing and Reimbursement Success in Germany

F. Kermani, “Novartis Pulls Galvus From Germany After Failed Price Negotiations,”  

Germany’s stance on pricing threatens drug firm profits

,” Reuters, Feb. 18, 2014, accessed Aug. 14, 2014.

Comparing the Effectiveness of Prescription Drugs: The German Experience

” (May 21, 2014),  accessed Aug. 14, 2014.

Institute for Quality and Efficiency in Health Care, Trastuzumab emtansine 

 Benefit assessment according to §35a Social Code Book V

C. Henry, “Oncology drugs under AMNOG,” 


Jill E. Sackman, DVM, PhD,  senior consultant, and Michael Kuchenreuther, PhD,  research analyst,  at Numerof & Associates, Inc., St. Louis, MO, 

Articles

Features

The authors take a look at some of the recent developments in the German pharmaceutical market.

Germany Post AMNOG: Insights for BioPharma

Cellular therapies are positioned to be the next revolution in the healthcare industry. Disciplines such as bio-printing and tissue engineering also require high quality living cells and can be considered a part of the overall cellular therapy industry. Multiple diseases that were traditionally incurable are being addressed by cellular therapies with promising early clinical results. Both large and small companies are developing cellular therapies, and currently, more than a thousand clinical trials are being conducted (1).

Stem cells are unique in their ability to divide and regenerate themselves and in their ability to remain unspecialized or differentiate into specialized cells that can perform critical tasks in the human body. Because of these unique traits, these cells hold a great deal of promise in helping researchers learn about disease and develop treatments. The regenerative properties of stem cells are proving useful in developing treatments for common conditions such as diabetes, heart disease, Parkinson’s disease, spinal cord injuries, and strokes.

There are two primary categories of cellular therapies: autologous and allogeneic. Autologous therapies are patient- or donor-specific; allogeneic therapies generally do not have a specific donor or source requirement. Each therapy presents opportunities for treatments, but also faces unique challenges to overcome before it can get to the clinic. Autologous therapies typically are not scalable; allogeneic therapies are scalable from a manufacturing perspective. The number of product doses that can be manufactured or required for a single patient for an autologous therapy are limited compared to a scenario where multiple patients can be treated using the same off-the-shelf allogeneic therapy.


Although recent scientific and technological advances in the cellular therapy arena portend great therapeutic promise, the final objective is to accelerate the development path of these therapies to the clinic and make them universally available to all patients. Several steps along the pathway are fraught with challenges that institutions and biopharma will face in the attempt to commercialize these products.

One approach is to segment the various steps of the development process into discrete unit operations. This approach has been successfully implemented in the manufacture of chemicals, automobiles, and food, as well as other pharmaceutical industry sectors. The lessons learned can be applied to the development of cellular therapies. It is here that cell biology meets engineering with the application of sound manufacturing principles. These unit operations range from science and discovery to the successful execution of clinical trials and commercialization of product. This article focuses on the manufacturing operations that include supply chain management of raw materials, manufacturing processes with facility capabilities, quality control, quality assurance release, regulatory requirements, and shipping and distribution.

Each unit operation needs to be scalable to effectively meet clinical demand. The volume and complexity to make product for an early-phase clinical trial is different when compared to what is required at commercial scale-up. There is a need for process development within each unit operation and while the process starts out being flexible, it needs to be locked in by Phase III and before commercial launch. An important component of this overall strategy is to identify each of these requirements early in the process.

Cost drivers increase as the product moves through the different phases of clinical development with the majority of the costs closer to commercialization. While safety and efficacy are primary drivers, the cost of the final therapeutic product is also important for therapies to be universally accessible; there should be no limitations in getting these therapies reimbursed and delivered to patients (2).


Regulatory requirements for different cell therapies are at different stages of development, and national and international regulatory agencies are discussing guidance documents for the industry to follow. The regulators are willing to provide significant support to drug developers and will work with organizations to determine the correct path forward, resulting in a clear regulatory path to the clinic. Japan’s regulatory agency, for example, has recently retooled its drug authorization framework through legal reform to produce the world’s fastest approval process specifically designed for regenerative medicine therapies (3).


Supply chain management of key materials is the first unit operation within manufacturing. Cellular therapies depend on the acquisition of the right source material that, in most cases, is derived from tissue or blood. A good donor program needs to be established to ensure that reproducible procedures are in place using standard methods to procure material.

While there are several options that work well, there is limited standardization in approaches. Using a standardized accreditation process for blood and tissue sourcing, entities will help establish that the process being followed is uniform and consistent. Donor-to-donor variability will exist, so eliminating the collection process as a variable will improve reliability.

Another issue is a short supply of serum, a critical raw material in the supply chain. Projections indicate that there is insufficient serum to meet the commercial demands for cell therapy (4). Using synthetic biology approaches with computational modeling can identify substitutes to serum that are more abundantly available. A synthetic biology approach also offers better characterization and chemical definition, which will help deliver a consistent product and streamline the regulatory pathway. Many media developers have launched serum-free media, which are also under evaluation to address this bottleneck. Using raw materials and consumables that are well characterized and from qualified suppliers is important. Suppliers should have quality systems in place and open to an audit.


The manufacturing facility needs to be built and equipped with the appropriate infrastructure and environmental controls. Options include building a facility, contract manufacturing, and using modular pods that are prefabricated and can be implemented quickly to meet requirements. Autologous cell- therapy production may be biased towards having a modular facility for each product dose to minimize cross contamination. Allogeneic facilities can be designed in a ballroom arrangement, as each production lot represents multiple doses made at larger scales.

Because terminal sterilization for cellular therapies is limited, it becomes important to minimize open system processing where a contaminant can be introduced into the system and lead to failed product batches. Open-system processing requires a higher degree of personnel training, aseptic process validation, stringent environmental controls, and increased costs. Using methods to ensure maximum closed-system manufacturing with automation will reduce some of the requirements on the facility. 
  
  

Most manufacturing processes start off being manual as they are developed in a laboratory-based environment with only small quantities required for preclinical testing. These processes are seldom scale-up friendly and require significant process development efforts to get them ready for large-scale production. Organizations face this dilemma when they review the process development costs versus moving the process as-is into clinical manufacturing. Technology transfer of the laboratory based process may work in the short term, but creates issues when the only option available is scale-out to achieve required product volumes.

Research and development scientists require insight into what scale-up options exist with protocols and application notes. These protocols will provide early opportunities to identify processes that can be easily translated into large-scale formats with some process development. Process development and technology transfer will always be required but using standard methods will streamline the process. When developers are working with T-flasks, they seldom think about transitioning into multi layers vessels and bioreactors. Producing 10 million cells can be achieved in a laboratory environment but when cells have to be produced at the billion to trillion scale, technology requirements are different. Some of this development can be done via computational modeling or small-scale experiments using well-characterized methods that have been qualified for their scalability potential. Large-scale experiments are expensive to run and while large, well-funded organizations have the resources, smaller organizations will find it cost prohibitive. Cost-of-goods modeling is required as the drug products approach commercial launch. By understanding the drivers of cost early in the development phase, the process can be optimized while there still is some flexibility. Once the process is locked down, much work and expense is required to establish equivalence with the previous process.

Automation options should also be evaluated to determine which steps of the process can be automated. Several automation options can be plugged in without extensive customization and process development. For example, automated and semi-automated filling systems for the final dose of the product are not only a faster and scalable than manual processes, they also minimize the opportunity for contamination. Other options from the pharmaceutical industry and other ancillary industries can be leveraged. Several academic and industrial consortia are being created to generate standardized platforms and methods to be used across a wide spectrum of requirements.


Intermediates used in the manufacturing process and the final product must go through quality-control testing prior to release. Multiple assays and methods are available to address sterility, safety, and efficacy. Many assays may seem relevant but have no consequence on the drug product. The development of the acceptance criteria should be relevant. Doing more assays does not necessarily impact outcome but doing the correct assays with the relevant acceptance criteria is important.


The drug product must reach clinical sites without compromising its sterility, safety, and potency; therefore, bio-preservation and cold chain management need to be addressed. Standard off-the-shelf bio-preservation options have been successfully used with supporting regulatory documentation (5). Evaluating existing options from the industry is recommended before designing custom options and will help in the regulatory submission.

Most cellular therapies are shipped and distributed under liquid nitrogen at -80°C or -20°C conditions. Many vendors and service providers meet national and international requirements. In addition, new technologies stabilize these cells at higher temperatures without compromising viability, shelf life, and efficacy. Final shipping and distribution costs can constitute more than 25% of the product cost, a factor that should not be underestimated during the budgeting process.


Challenges will always exist when launching new therapies and technologies. Established expertise from other fields can be leveraged and applied to cellular therapies. Options and tools are available to address each of these challenges. Execution of a commercialization strategy can be done in multiple stages over time and, most importantly, when required. In some cases, the investment needs to be made early enough to be ready for the next stage.

Accelerating a cellular therapy to clinic can be executed by identifying the relevant unit operations; identifying the major components within the unit operation and those that need the most attention; leveraging existing expertise or standards; implementing the right solution; and validating the implementation.

It is only a matter of time before treatments become available for diseases that were considered difficult to cure. The industry is focused on the promise of delivering effective cellular therapies worldwide.


  1.		Alliance for Rengerative Medicine, 

 (January 2014), accessed Oct. 15, 2014.
  2.		R. Shaw, B. Hampson, C. Betz,

, 13 (2), 26-31 (2014).
  3.		D. Cyranoski, 

. 11 (9), 510-513 (2013). 
  4.		S. Jung, K.M. Panchalingam, R.D. Wuerth, L. Rosenberg, L.A. Behie,

. 59 (2), 106-120 (2012).
5.		AJ Mathew, 


Uplaksh Kumar, PhD, MBA,  cGMP expert, AABB Consulting Services team
Naynesh R. Kamani, MD, division director for the Center for Cellular Therapies and Research, AABB Center for Cellular Therapies

Cover Story

Cellular therapy developers learn process development strategies from pharma industry experiences.

Standardizing Practices for Cellular Therapy Manufacturing

The past decade has seen considerable growth in the Indian pharmaceutical industry. India has emerged as a key supplier of high quality and affordable medicines not only to the developing world, but also to developed economies as well (

 ) (1). This sector has recorded a compound annual growth rate (CAGR) of 13.5% over the past five years and is projected to reach $45 billion in 2020 (2). With the ongoing shift from small molecules to biologics, the Indian biopharmaceutical industry is also set to replicate the success received by the Indian pharmaceutical companies (

) (1). The biopharmaceuticals market has witnessed the fastest growth in the 2013 financial year as compared to other biotech markets (e.g., Bio-Agri, Bio-Services, and Bio-Informatics).

Growth in this sector has also burdened the public institutions that are responsible for ensuring that the marketed products are safe and efficacious. In India, the Drug Controller General of India (DCGI)-led Central Drugs Standards Control Organization (CDSCO) together with the State Authorities are primarily responsible for ensuring the quality of biotech therapeutics post-approval. The Indian Pharmacopoeia Commission (IPC), which creates and publishes the   

 (IP), however, plays a key role. The IP, as per the Drugs and Cosmetics Act of 1940, prescribes the standards for drugs produced and/or marketed in India and, therefore, plays a significant role in the control and assurance of the quality of the pharmaceutical products (3). Unlike a lot of developing and developed countries, the standards stated in the IP are authoritative and legally enforceable by the Indian regulatory authorities (CDSCO and State Drug Regulators). In this 31st article in the “Elements of Biopharmaceutical Production” series, the authors report on the evolution and future of IPC. The focus of the article is on biotech therapeutics.


  The IPC was constituted in 1948 with publication of   IP  defined as its primary function. The first   IP  was published in 1955 followed by a supplement in 1960. This pharmacopoeia contained both western and traditional Indian pharmaceutical products commonly used in India.

Thereafter, the IP has been published at frequent intervals. The frequency has increased with time due to the phenomenal growth in the number of pharmaceutical products over the past five decades as well as the diversity in their origin and content. Over time, the IP committee has deleted monographs for products that have become obsolete and added monographs based on the therapeutic merit and medical need (3).

The IPC was established in 2005 and the first addendum, which was published by IPC in 2005, included a large number of antiretroviral drugs and raw plants commonly used in making medicinal products not covered by any other pharmacopoeias, which attracted much global attention. Similarly, IP 2007 contained 271 new monographs with focus on those drugs and formulations that cover the National Healthcare Programs and the National List of Essential Medicines. The addendum in 2008 contained 72 new monographs (4). The commission had become fully operational as an autonomous body under the Ministry of Health and Family Welfare, Government of India in 2009 and released 

 2010 (5). 
  IP  2010 contained monographs on antiretroviral, anticancer, antituberculosis, and herbal drugs. It also included monographs of biologically derived products such as vaccines, immunosera for human use, blood products, and other biotech and veterinary (biological and non-biological) preparations. Addendum 2012 to the IP 2010 incorporated 52 new monographs (

: Evolution of India Pharmacopoeia (IP) over the past five decades. Source: 

 The primary objectives of IPC include the following:

Develop comprehensive monographs for drugs and upgrade them as needed

Accord priority to monographs of drugs included in the National List of Essential Medicines 

Collaborate with other pharmacopoeia (e.g., 

United States Pharmacopeia[USP], British Pharmacopoeia [BP], Chinese Pharmacopeia [ChP], European Pharmacopoeia [Ph. Eur.], 

 [JP]) to attempt harmonization with global standards

Prepare, certify, and distribute IP Reference Substances

Organize educational programs and research activities to generate awareness about the need for quality standards for drugs and related articles/materials (3).


  The scope of IPC has been extended to include products of biotechnological origin, indigenous herbs and herbal products, veterinary vaccines, and additional antiretroviral drugs and formulations, inclusive of commonly used fixed-dose combinations. IP 2014 had 577 new monographs and included monographs of API [134], formulations [161], excipients [18], new drug substances [43], antiviral [11] and anticancer [19], antibiotics [10], herbal [31], vaccine and immunosera for human use [5], insulin products [6], and biotechnology products [7]. IP 2014 also included 19 new radiopharmaceutical monographs and a general chapter on radiopharmaceutical preparations (4). 

 presents a comparison of monographs related to biotech therapeutics across the different international pharmacopoeial bodies. It can be seen that IP compares well with other leading global agencies. 

 illustrates the evolution and key milestones of IPC since formation.

The authors take a look at the past and future impact of the Indian Pharmacopoeia Commission.

Setting Standards for Biotech Therapeutics in India

Removal and inactivation of adventitious and endogenous viruses have traditionally been achieved during the downstream purification of biologics. Viral clearance is assessed in small-scale tests, and regulatory guidance documents provide a general framework for how these tests should be performed (1). Even with guidance, however, demonstrating an appropriate level of viral clearance can be challenging for biologics manufacturers.


Using media with a low level of viral contamination can lead to major problems in biologics manufacturing. Cell-culture media and other raw materials, particularly those derived from animals, should be subjected to rigorous testing and viral inactivation treatments, according to S. Steve Zhou, director of virology and molecular biology with the MicroBioTest Division of Microbac Laboratories. In addition to treatment with heat and ultraviolet radiation, virus retentive filtration is recognized as one of the most efficient and robust virus removal steps, and implementation in the media feeding process could decrease the risk of contamination significantly, according to Horst Ruppach, senior director, viral clearance with Charles River Biologics Testing Solutions. “Current filters can be used in this application, but they were not designed to run under continuous and high throughput conditions like those required for media supply,” he observes. Filter vendors are currently being asked to modify the filter design for this specific approach. Viral clearance studies will also need to be designed differently in order to address the specific conditions associated with media supply, Ruppach adds.


Conditions throughout the commercial manufacturing process must be constantly monitored to avoid viral contamination events. One major concern for biologics production is the entry of a small, non-enveloped, chemically resistant virus into the manufacturing system, according to Zhou. “Good facility design and strict compliance with good manufacturing practice is the best approach to avoiding this problem,” he says. Equipment and process failures, such as inadequate raw material screening or failure of an upstream inactivation method, particularly those that go undetected, are also a concern with respect to potential viral contamination. In these cases, close monitoring of the process, preferably using automated systems, and a good quality assurance/quality control program are essential for preventing such problems, according to Zhou.


As purification templates are established, the cost of goods is of particular importance because there is often pressure to limit operations to those that are necessary for biotherapeutic purification rather than include operations that confer a marginal improvement of product quality, according to Trish Greenhalgh, virology manager for EMD Millipore. Processes with fewer purification steps present a challenge to virus clearance, however, because higher removal claims are expected with individual unit operations. “Access to high titer virus preparations in testing labs is one way to achieve high clearance levels, and if complete clearance is observed across the unit operations, high log reduction values (LRVs) will be obtained,” says Greenhalgh. “Meeting clearance targets by evaluating a minimal number of steps that each deliver high removal can be a risky approach, and a more thorough evaluation of additional purification steps expected to contribute to virus removal is preferred by regulatory agencies,” she continues.

More recently, there has been a focus on understanding the impact of small changes in the operating window of individual unit operations on product quality.  “There is an expectation that the impact of these changes on the virus clearance capabilities for unit operations, particularly chromatographic purification steps, will be understood,” Greenhalgh explains.  Historical virus clearance data with the same template can be invaluable in setting realistic expectations for unit operation clearance. Ideally, however, she notes that clearance studies should also evaluate conditions outside the process center to more fully understand the robustness for virus removal.  Comprehensive risk analysis is often effective for highlighting and prioritizing such evaluations to streamline costly virus testing, according to Greenhalgh.


Another challenge for viral clearance validation is accurate process representation. “In order to perform high-quality viral clearance studies and avoid unreliable, inaccurate, or outdated viral clearance data, it is necessary to regularly review the manufacturing process and ensure that the current manufacturing process matches what was tested in the viral clearance study,” according to Zhou. A thorough definition of full-scale process conditions is particularly important so that they can be assessed in small-scale viral clearance tests.

These tests should also consider process excursions, including interruptions, depressurization events, and minor changes in feed quality from chromatographic unit operations, according to Greenhalgh. Zhou agrees: “In addition to having a thorough understanding of the process steps, particularly the process parameters that are critical for robust viral clearance, it is necessary to consider worst-case scenarios in order to accurately define the design space for viral clearance testing.” He also believes that it is important to update the design space as new knowledge develops. These assessments of process excursions require open communication between the biologics manufacturer and the testing laboratory to develop comprehensive evaluation plans, according to Greenhalgh.

For scale-down tests to provide reliable viral clearance data, the actual challenge feed must be representative of that in the full-scale process. Achieving this goal can be difficult, because the test material is typically frozen for shipping to the testing lab, potentially leading to changes in the process intermediates due to aggregation or product degradation, which can result in different performance for individual unit operations compared to that observed during process development. “Understanding the impact of shipping and extended storage on performance and the development of mitigation strategies are important for ensuring accurate virus clearance assessments,” Greenhalgh says.


Interest in continuous processing of biologics using single-use systems is gaining traction because this approach can significantly reduce production costs, particularly for smaller-volume APIs. Viral clearance becomes even more important under continuous production conditions, according to Ruppach. “Contaminants typically detected during the fermentation process may enter the purification process under fully continuous production conditions. Other questions must also be addressed: How can a potential contamination best be mimicked in a process where virus contaminants enter the downstream process under a constant flow? Will continuous processing impact the viral inactivation and removal capacities of downstream process steps for which effective inactivation or removal could be demonstrated under standard conditions like low pH inactivation, anion exchange (AEX) chromatography, or virus-retentive filtration? Further studies need to be performed to answer these questions,” he states.


Presently, according to Ruppach, the same set of model viruses (murine leukemia virus [MuLV], pseudorabies virus [PRV], reovirus [Reo-3], and minute virus of mice/porcine parvovirus [MVM/PPV]) is applied for the viral clearance validation of cell line-derived recombinant products. The achieved single reduction factors are summed up to an overall reduction factor that describes the general virus inactivation/removal capacity of the downstream process. For instance, he notes that it is accepted that AEX chromatography, when run under certain conditions, is highly effective in removing this set of model viruses. It is also known that low pH inactivation is an effective inactivation step for enveloped viruses when high reduction factors are demonstrated for the two enveloped viruses MuLV and PRV. “It is also known, however, that other model viruses behave differently. The Poliovirus, for instance, is not effectively removed by AEX chromatography under the same conditions, and HIV-1 and bovine viral diarrhea virus (BVDV) are much more resistant to low pH inactivation compared with MuLV and PRV. These varying responses raise questions about using the same model viruses for all steps, and a discussion should be initiated regarding how robust viral clearance can better be demonstrated,” Ruppach asserts.

In addition, because nanofiltration is known to be the most effective virus removal step and can remove both large (MuLV, PRV) and small (MVM, PPV) viruses, there is some discussion about the use of these small model viruses as surrogates for the larger viruses, according to Ruppach. He points out that some companies have in fact claimed MVM removal data as indicative of MuLV removal via virus-retentive filtration or used a fixed-reduction factor based on internal experiments. “This practice is not in full compliance with current guidelines, but some agencies have accepted this type of data for IND applications of mAbs [monoclonal antibodies] derived from CHO [Chinese hamster ovary] cell lines. It remains uncertain, however, whether all agencies will accept this practice and if it can be applied for late-stage viral clearance studies and other products,” he comments.

Another issue with viral clearance testing is the variable and often unknown quality of the virus preparations or spikes that are used in these tests, according to Greenhalgh. The Parenteral Drug Association 

 47 (2) provides a framework for the preparation and characterization of virus preparations, and individual testing labs have developed their own descriptions for virus preparations of differing purities.  Generally, Greenhalgh notes that virus purity has the biggest impact on the virus filtration step: less purified preparations are more likely to negatively impact the virus filter performance by reducing the volumetric throughput and ultimately resulting in over-sizing of the filtration area and an increased cost of goods for this dedicated virus removal step.  “Pre-studies to optimize both the feed and the virus spike level are critical prerequisites for ensuring that expected performance targets are met,” she states.


Viral clearance studies, when performed by contract research organizations (CROs), are frequently performed following good laboratory practices (GLP) principles. Recently, however, the question of whether viral clearance studies should follow GMP rather than GLP principles has been raised. “Audits performed by customers at CRO sites more often follow GMP rather than GLP processes, and typical GMP requirements, such as corrective action and preventive action (CAPA) procedures, are requested,” says Ruppach.

This discussion is additionally stimulated by the fact that viral clearance studies are required at different stages of the product lifecycle, from before the clinical phase through more comprehensive studies for marketing authorization. In the first case, a fully validated production process is typically not in place. In the latter case, a fully GMP-compliant validated production process must be in place. Accordingly, a GMP-compliant viral clearance study is more important for the late stage rather than the early stage of the product development process, according to Ruppach. “It is important, therefore, that CROs offering viral clearance services following GMP principles must not apply all of the measurements that are important for a production site that manufactures and releases pharmaceuticals for human use,” he asserts.


Meeting regulatory requirements for virus safety depends on well-designed studies that demonstrate virus clearance across multiple orthogonal steps with accurate scale-down conditions, according to Greenhalgh. “Comprehensive test design resulting from thorough process understanding and communication between the biologics manufacturer and the virus clearance testing lab is essential to meeting these goals.”

Guidance on Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin

Technical Report No. 47: Preparation of Virus Spikes Used for Virus Clearance Studies


Cynthia A. Challener is a contributing editor to 

Troubleshooting

Challenges remain for virus removal and validation.

Viral Clearance Challenges in Bioprocessing

At the 2014 CPhI trade show, held Oct. 7–9 in Paris, executives from CDMOs and contract labs whose operations are solely in Europe expressed their desire to expand their operations into North America. These executives, however, were stymied about how to do it.

Their interest in North America is not surprising: opportunities there are much greater than in Europe. There is far more venture capital financing in North America than in Europe and the level of drug development activity is significantly greater. Many European economies are contracting, and government budgets to pay for drugs are under stress. Further, with its large number of former bio/pharma facilities converted to contract services businesses, Europe experiences price competition in the drug-development market that seems more aggressive than in North America. 


European companies are challenged on several levels to enter North America. For one thing, European contractors don’t have a good understanding of the North American market. While some may have had an FDA inspection, many haven’t and don’t understand FDA requirements and how they differ from European requirements.

Further, they have minimal experience in trying to sell their services in North America; some may have one sales representative based in the US, but many have none. Sales and marketing in particular can be different in North America versus Europe. For instance, tradeshow marketing is important in Europe and cold calling is uncommon, but in North America it is the other way around. Even if they have sales representation in North America, European CDMOs suffer from low brand recognition and can have a hard time getting clients to travel overseas for services that are readily available domestically.

Another challenge for European contractors is that there is a higher barrier to market entry in North America than they are used to. Most European CDMOs were founded as buyouts of facilities that global bio/pharmaceutical companies wanted to get rid of. Facility closures are difficult in Europe because of labor laws and because of fears that mass layoffs could hurt companies tendering for government drug supply contracts. As a result, pharma companies have been willing to offload unneeded facilities to management teams or private equity investors at a fraction of their replacement value and with contracts to continue to manufacture legacy products for some period. This has made it relatively easy to enter the contract services business in Europe, although the resulting overcapacity has hurt pricing for contract manufacturing and development.

Opportunities to acquire facilities on such favorable terms are fewer and farther between in North America. Because North America consists of just two large markets, versus the 20-plus small to mid-size markets in Europe, there weren’t so many facilities built in the first place, so there have been fewer facilities to divest. As result, European companies looking to enter North America may have to “pay retail” to acquire a facility or services business. Acquisition valuations of CDMOs in recent years have been especially high.

European CDMOs are also hindered by a lack of capital necessary to acquire their way into North America. They are often poorly capitalized, given that their initial buy-in to the industry didn’t require a lot of investment. Further, their low profit margins mean they haven’t been able to accumulate a significant amount of equity through retained earnings. Also, some European CDMOs are employee-owned and may have particular challenges raising additional capital without threatening their ownership structure.


The barriers to European entry into the US CDMO market are high but not insurmountable. Examples of European contractors that have successfully established themselves in North America in the past 10 years include CDMOs Vetter and Almac as well as contract labs like SGS and Eurofins. Those companies, however, had favorable ownership structures (family trusts or publicly-traded) that gave them access to adequate capital.

Further, it is occasionally possible to acquire facilities in North America under favorable terms. In 2013, European CMO Unither acquired a dose manufacturing facility with contracts in Rochester, New York, from UCB Pharma; and Fareva was able to acquire a manufacturing and packaging facility with contracts in Richmond, Virginia, from Pfizer in 2011.

It’s also worth noting that North American companies have challenges similar to the Europeans’ when considering entry into Europe. A common problem has been the failure to realize that Europe is not a single market--not even the European Union--but rather a collection of more than 20 countries with different languages and cultures, and a wholly different concept of distance. So, for instance, North American companies have established facilities in Ireland intended to serve all of Europe, only to learn that continental companies view the Irish facilities as far away and foreign.

As the bio/pharmaceutical and contract services industries consolidate, having a global network of facilities, at least in North America and Europe, has become increasingly important for competitive success. Mid-size European CDMOs and contract labs must figure out how to overcome the barriers to their entry into North America if they intend to succeed over the long term.


Jim Miller is president of PharmSource Information Services, Inc., and publisher of Bio/Pharmaceutical Outsourcing Report, tel. 703.383.4903, Twitter@JimPharmSource, 

Perspectives on Outsourcing

European CDMOs want into the US market, but entry options are limited.

Outside Looking In

Eppendorf’s Cell Culture Consumables have an ISO class/GMP class C clean room production standard and a sterility assurance level of 10

The flasks feature air filter technology, with improved ergonomic design to increase handling efficiency. Each flask is given a 100% in-line pressure test for leaks. The resealable-top dishes include a corrugated ring to improve handling and transportation, with a SplashProtect ring inside the dish to protect against cross-contamination. The plates have been designed to minimize the “edge effect” with a chimney-well design in the outer moat of the 96-well plate.

Products

Eppendorf's Cell Culture Consumables have an ISO class/GMP class C clean room production standard.

Cell Culture Consumables Prevent Contamination

Ebola virus disease (EVD), which first appeared in Africa 38 years ago, generally was perceived by people in developed nations as a scary, deadly, but distant illness far removed from their lives.

EVD is a severe, often fatal disease that is transmitted human-to-human through bodily fluids. According to World Health Organization (WHO) data, nearly 2000 people in Africa were infected with EVD from 1976 through 2013 with an average fatality rate of 50%. The latest outbreak began in March 2014 in Guinea and spread to Sierra Leone, Liberia, Senegal, and Nigeria. As of Oct. 14, WHO reported 9216 confirmed, probable, or suspected cases of EVD worldwide and 4555 deaths.

When the first patient with EVD walked into a US hospital, the facility was not prepared; two nurses treating the patient were infected. Despite initial missteps, federal and local officials contained the outbreak to that single hospital. But it was too late to contain overreaction by the public, press, and politicians, which spread much faster and wider than the disease itself and distracted public attention from real health threats.

While the afflicted nurses battled the disease, politicians lined up to criticize the reaction of the federal government. Demonstrators outside the White House called for a halt to flights from West Africa. Schools closed. In one extreme example of undue caution, a school in Maine placed a teacher on a 21-day leave after she attended a conference in Dallas in mid-October, even though she had no contact with any people, transportation, or facilities involved in three EVD cases diagnosed in that area.

The 2014 outbreak clearly illustrates there is a need for effective vaccines and therapies for EVD; a handful of vaccines and treatments are in early development and testing stages. Questions remain, however, about the bio/pharma industry’s ability or desire to develop and produce therapies or vaccines cost-effectively.


While EVD generated alarm, two other health issues presented serious medical challenges for US patients.

Between mid-August and mid-October, the Centers for Disease Control (CDC) and state public health laboratories confirmed that 825 people, mostly children, in 46 states and the District of Columbia had respiratory illnesses caused by enterovirus D68 (EV-D68); seven patients died. Enteroviruses and rhinoviruses cause millions of respiratory illnesses in children each year; however, the 2014 outbreak of EV-D68 was much greater than that reported in previous years. There are no vaccines to prevent EV-D68.

Influenza is an annual threat to the health of the general public and patients with compromised medical conditions in particular. The October to March flu season is just underway and CDC estimates that 5-20% of Americans get the flu each year. While most people recover, CDC estimates that between 3000 and 49,000 Americans die from the flu or its complications every year.

The flu virus is contagious and, unlike EVD, can be spread through airborne transmission (a cough or sneeze) and by someone infected with the virus who is not showing symptoms. Unlike EVD and EV-D68, however, vaccines to protect against the flu are readily available; CDC says the vaccine reduces the chance of getting the flu by approximately 60%. Still, less than half of the US population gets innoculated.

Count me among those lining up for an annual flu shot. The vaccine does not come with a guarantee that I will avoid the flu, but a shot of prevention is worth more than a pound of paranoia.


Rita Peters is the editorial director of 

From the Editor

Ebola Virus Disease (EVD) is a severe, often fatal disease that is transmitted human-to-human through bodily fluids.

Hype vs. Health

The 30th anniversary of the Hatch-Waxman Act in September generated widespread recognition of how the legislation created today’s vibrant generic-drug industry. Generic drugs now account for more than 85% of US prescription drug use, and the development of biosimilars promises to extend this trend to large molecules. Such transformation was unanticipated back in 1984 by the authors of the legislation, Sen. Orrin Hatch (R-Utah) and Rep. Henry Waxman (D-Calif), who is retiring from Congress but continues to urge vigilance to ensure that Americans retain access to low-cost quality medicines.

A September conference commemorating Hatch-Waxman sponsored by the Food & Drug Law Institute (FDLI) described the world with few generic drugs prior to 1984 and how the legislation was born of a compromise between brand-name firms seeking patent extensions and consumers demanding a clearer pathway for bringing low-cost drugs to market. Former FDA officials recalled the challenges in establishing new policies for testing and evaluating generic medicines and how the program’s success has generated thousands of abbreviated new drug applications (ANDAs) and supplements that are requiring major operational changes at FDA. Hatch-Waxman also has generated a host of legal, regulatory, and economic issues that complicate generic drug oversight and distribution.


The Hatch-Waxman anniversary also coincides with the beginning of year three of the Generic Drug User Fee Act (GDUFA), the fee scheme approved by Congress in 2012 to speed up ANDA approvals. The program expands FDA review staff, supports timely field inspections, and funds research on developing more complex generic dosage forms. One result is the elevation of the relatively small Office of Generic Drugs (OGD) in the Center for Drug Evaluation and Research (CDER) into a “super office” to accommodate a larger cadre of scientists, reviewers, and policy experts to support new regulatory approaches.

GDUFA requires OGD to meet certain time frames for assessing and approving ANDAs and supplements, while also whittling away at a massive application backlog. Specific goals for reviewing ANDAs kick in this year, and manufacturers have concerns about OGD meeting the schedule. An unanticipated problem is that more than 1400 applications were filed this past year, many more than projected, making it even more necessary for OGD to implement a new review process that sets priorities based on product risk, medical importance, review goal dates, and internal workload factors.

Chemistry reviewers will provide “appropriate scrutiny” to high-risk areas, while streamlining the review of lower-risk products, explained Susan Rosencrance, then acting deputy director for generic drug chemistry in CDER’s Office of Pharmaceutical Science (OPS), at the FDA/PQRI conference in September 2014. The risk-based review approach has already paid off with a 50% reduction in chemistry review times this past year, she noted.

A main strategy for speeding up ANDA review and approval is to reduce the number of application review cycles. OGD is implementing a “real-time communication” program that allows staff to discuss minor deficiencies that can be resolved quickly with manufacturers. The agency also is adopting a “block review” approach that assigns a team to three or more ANDAs with the same reference listed drug or same drug master file. This system moves away from OGD’s random assignment approach, established to avoid any appearance of favoring one application over another. Block reviews aim to reduce repeated efforts, promote information sharing, and produce more consistent decisions.

CDER also is working hard to cut the massive backlog of some 5000 generic-drug manufacturing supplements. An efficient supplement review process is key to encouraging product modernization and quality improvements for marketed drugs, and Rosencrance’s office has almost halved the backlog through a “strategic plan of attack” by a special supplement review team of external and internal chemistry reviewers.

Not all is rosy with FDA’s GDUFA implementation process, though, as seen in concerns raised by manufacturers about still-slow ANDA approvals at a GDUFA public hearing in September. Small manufacturers asked FDA for reduced annual facility and filing fees, and several industry executives urged more clarity in how new guidance would be implemented. They offered specific comments on recent guidance documents on ANDA content and format and on the refuse-to-receive policy. The loudest complaints focused on OGD’s new communications policy ending informal phone conversations between FDA staffers and industry representatives. Manufacturers say such communication is key to resolving problems quickly and helps them plan for production changes and product launches. FDA officials maintain that constant queries are a burden, lead to inconsistent practices, and raise the specter of favoritism. But they now plan to review and revise the policy in response to industry concerns.


Meanwhile, the continued growth in generic drug market share has generated more legal challenges to product development and marketing. Recent Supreme Court decisions have raised questions about the adequacy of safety information in generic-drug labels, prompting FDA to propose a new rule on labeling changes that generics manufacturers strongly oppose as likely to lead to different labels on brands and generics. The Generic Pharmaceutical Association (GPhA) says FDA’s policy raises liability issues that could curb development of new low-cost medicines and has sought Congressional support for its position. The legal battle is escalating as brand manufacturers seek to avoid added liability for patients harmed by generic drugs, and generic-drug companies insist they can’t have labels different from the brand. GPhA has proposed that FDA itself establish a fast, efficient process for updating labeling changes on both brand and generic drugs, and to transmit such updates electronically to prescribers, dispensers, and patients.

Generic-drug makers also are fighting innovators over allegations that brands use risk evaluation and mitigation strategies (REMS) to block the development and testing of new therapies. GPhA backs legislation to make it harder for innovator firms to claim that a restricted distribution program required by a REMS prevents it from making available drug samples needed for bioequivalence testing.

The emergence of biosimilars continues to fuel debate over the assessment of similarity between reference products and follow-ons. Although there is general agreement that in-depth analysis of product structure and function plus pharmacology testing can combine to document similarity, there’s much disagreement over extrapolation of data by a biosimilar sponsor to support approval of additional uses and over product “naming” to distinguish innovators from biosimilars, as brands look to discourage interchangeability and substitution of future biosimilars.

Successful biosimilar development must consider manufacturing costs and quality commitments, pointed out Parexel Vice-President Cecil Nick at the September biosimilars conference sponsored by the Drug Information Association. In addition to assessing non-clinical and clinical data requirements, sponsors must weigh the costs and efficiencies of in-house production versus contract manufacturing in terms of production yield, media costs, disposable versus fixed production train, needed purification steps, and fill/finish arrangements.

The generic-drug industry is “at a crossroads,” commented Mylan Senior Vice-President Carmen Shepard at the FDLI conference. GDUFA promises to provide more predictable regulatory actions and to encourage high quality production. But manufacturers fear that an over-emphasis on meeting review goals could actually delay final approvals and end up with first filers losing exclusivity, undermining incentives for developing new generic products.

’s Washington editor, tel. 301.656.4634, 

Regulatory Beat

Manufacturers face regulatory overhaul, while brand-generic debates escalate over biosimilars and labeling changes.

New Era for Generic Drugs

Using a design-of-experiment (DoE) approach allows a formulation development scientist to examine the formulation design space in a statistically significant manner from the beginning of experimentation. In addition, a DoE-driven approach meets the International Conference on Harmonization Q8 guidelines for multivariate design space and the subsequent justification of formulation components when developing a drug product, according to Steven R. LaBrenz, scientific director, pharmaceutical development and manufacturing sciences with Janssen Research & Development. At the same time, DoE results depend on the selected critical parameters and the sample material used in the studies. In addition, a large amount of data is generated. Consequently, reaping the benefits requires a significant commitment to DoE, including the training of researchers in study design and data-processing techniques.


One of the major advantages of the DoE approach is the ability to study the effects of multiple excipients and their interactions in a formulation at the same time, unlike in the traditional one-factor-at-time approach, which is inherently flawed and biased, according to Mark Yang, director of fill/finish development for Genzyme. Phuong Nguyen, a senior scientist with Millennium Pharmaceuticals (The Takeda Oncology Company) agrees with this approach. “The quantitative models for these multiple quality attributes can then be used for global optimization of the formulation, leading to more robust formulation development,” she says. Nguyen also notes that depending on the data that are generated from an experimental design, it is sometimes possible to gain some mechanistic understanding of protein stabilization or behavior.

In addition, because multiple variables can be explored at once, fewer formulation studies are required and, therefore, formulation development is more efficient. An added benefit of this efficiency, Yang notes, is the reduced quantity of material needed for formulation development, which can be important early in the development of a biopharmaceutical product given the limited availability of the biologic API. Further, a DoE approach can be particularly useful for combination products that have device attributes that must be considered during formulation development, says Nguyen.


While there clearly are numerous advantages to using a DoE approach in the formulation development of biopharmaceuticals, it is important to bear in mind that there are also limitations to consider with this method.

Two main challenges, asserts LaBrenz, include the fact that sampling of an adequate design space is often difficult and there is an overwhelming amount of data that is generated with factorial design. Yang agrees that the typical subset of critical quality attributes of the formulated product that are assayed in a DoE study may not be comprehensive enough to cover some of the quality changes that can occur. Nguyen also warns that the models used for DoE formulation studies may include statistically significant parameters that may not be of any importance, such as when the resulting change from an input parameter range is within the error of the method or is not large enough to have a real effect.

In addition, Yang notes that the API material used in DoE studies comes from one or possibly a few selected primary production lots that are not necessarily completely representative of later production lots. “As a result, the conclusions drawn based on DoE formulation studies can be biased to some degree,” he says.

LaBrenz believes that the challenges of a limited design space and the management of large quantities of data can be ameliorated with the adoption of high-throughput screening (HTS) techniques that provide both full design space coverage and the ability to collect data in spreadsheets and, eventually, databases.

Even with the use of HTS, however, implementation of DoE for formulation development can be time consuming and resource intensive and can translate into more extensive experimentation than planned, according to Nguyen. “It is a challenge to balance the number of experiments required for a statistically robust DoE study with the available supplies, personnel, resources, and time,” she says. In addition, user training is a must, according to Yang. “DoE study design and data processing requires an understanding of statistics theory and models, which can be challenging for many new users,” he comments.


In most DoE formulations studies, data related to the critical quality attributes and the stability of the API, such as the protein melting temperature, secondary or tertiary structure, or other biochemical/biophysical characteristics, are generated. The ultimate goal for formulation development is to maintain the API in a stable state with respect to both structure and function during processing and storage, according to Yang. Nguyen points out that all of the previously mentioned parameters may serve as indicators for a stable state.

It is also important to reduce the data to single-value results as often as possible, such as the percent monomer or aggregate as determined using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), or the percent purity. For spectral data from characterization assays, Janssen calculates the Center of Spectral Mass Wavelength (or Mean Center Mass), which allows the creation of a relative stability profile for a given spectrum-based assay. “This single-point, continuous, data-point strategy is readily testable using most statistical methods,” LaBrenz explains. In addition, Nguyen notes that the generated stability data can be reduced down to a reaction rate constant or an output parameter used to develop statistical models. “Overall,” she summarizes, “data on quality attributes are generated that will provide an understanding of how the excipient or formulation within the design space affect these attributes.”

Because DoE studies lead to the production of large quantities of data, it is important to ensure that all of the data that are collected are actually relevant. “A thorough understanding of the API and desired formulation characteristics is necessary before initiating a DoE study,” says Yang. “With such knowledge, a scientist can make sound scientific judgments and consider the potential impact of new factors or excipients when designing a DoE study.”

LaBrenz adds that developing the proper experimental design space before setting up DoE experiments ensures that the parameter critical for the stability of an API is identified. More specifically, conducting a screening DoE to determine which product quality attributes (and the corresponding output parameters used to represent them) are relevant in the design space is helpful, according to Nguyen. Accelerated stability studies can also be performed to determine relevant output parameters.

Another key to generating appropriate and relevant data is to make sure that normal-distributed data, obtained either directly or after transformation, are used. If there is a major process change, it is necessary to evaluate and understand any impacts on the protein and the formulation, according to Yang, which may mean conducting comparability or bridging studies.

 For DoE studies to be useful, the generated data must not only be relevant, but manageable. There are different approaches to addressing this problem.

The solution for LaBrenz is to use an open, read-only database that can be shared between multiple groups, such as preformulation, formulation, and analytical development groups, so that each unit can focus on particular aspects of interest in a DoE. “While a formulation scientist needs to know where the most stable space for an API resides, an analytical chemist may be more interested in the variability and reproducibility of results, both of which can be obtained from a well-designed DoE,” he says.

Nguyen remarks that using software to automate data collection and extraction of the relevant output parameters for statistical analysis is important. She also stresses that having an understanding of which output parameters to collect and limiting the excipients examined to a subset of approved parenteral excipients can reduce the amount of data that must be analyzed.

Training on data processing is also crucial, regardless of what approach is used. LaBrenz uses many different tools depending on the level of complexity required for a given project, and often the choice of software is determined by collaborators. “It isn’t the software package that actually matters. It is all of the extra training to develop my applied statistics skills, including six-sigma training and seminars, that helps me on the job,” he states.


  Cynthia A. Challener is a contributing editor to 

Defining critical parameters and processing large quantities of data can be a challenge.

DoE Provides Benefits, but Preparation Is Necessary

Gene- and cell therapy-based medicines are experiencing resurgence due to the introduction of “next generation” transfer vectors, which have demonstrated improved safety and efficacy. Patient samples are often extracted, expanded, and transduced using gene-therapy vectors, and the modified cells are re-implanted in the patient for therapeutic applications.

In this circumstance, the modified cell is the drug product rather than the gene-transfer vehicle, and it carries with it distinct regulatory challenges. An increase in the number of candidate molecules is anticipated due to the interest in the cancer treatments based on recent successes seen with chimeric antigen receptor (CAR)-based cell therapies. Although the end product of the manufactured viral vector is considered a drug substance, in the case of cell therapies, the scrutiny for testing for adventitious agents is high. As in many cases, the transduced cell products possess short shelf lives, often before all release testing can be completed. In addition, the use of plasmids for generation of viral vectors adds another layer of compliance and testing.

Given the complexities associated with new technologies, especially with “first-in-class” applications, the technical and scientific input can be the deciding factor in the success of a testing and/or manufacturing program. Both the manufacturing process and the performance of routine (e.g., adventitious agent testing, mycoplasma, sterility) and custom-designed (e.g., identity, potency) assays need to meet clinical and regulatory compliance standards.

The authors offer insight into cell-therapy manufacturing, vector production, and the regulatory requirements and safety aspects of testing for characterization.

To illustrate the manufacturing and testing strategies for a complex cell-therapy production process, the multilevel production required to generate a CAR T cell therapy product can serve as a good example. This production process starts with generation of cell lines and transfection of these cells with plasmids to produce a viral vector, which is then used to transduce patient T cells, before these are reintroduced into the patient. This is a long and complicated path that involves, in many cases, three or more different manufacturers (for cell banks, plasmids, lentiviral vectors and patient cell manipulation). The overall process is shown in 

 applications, gamma retroviral vectors, derived from murine leukemia virus (MLV), were the first to be used; however, lentiviral vectors, based on human immunodeficiency virus (HIV), have gained attention due to their ability to transduce non-dividing cells. While MLV has a simple genome structure (containing gag, pol, and env gene sequences), lentiviruses have a more complex one containing additional accessory genes that are able to manipulate host cell functions. The general strategy in designing lentiviral vectors for gene therapy is based on the deletion and alteration of the native viral sequences to prevent the generation of replication-competent retroviruses. To achieve this, the lentivirus components are segregated into three or four different plasmid constructs with the goal of preventing the possibility of complete recombination to a fully replication competent lentivirus (RCL). The viral vector genome contains at a minimum the transgene expression cassette, the long terminal repeats (LTRs), and the packaging signal. In most cases, three additional plasmids provide the factors required for virus production and packaging (e.g., gag, pol, env). The envelope proteins are typically replaced by a heterologous viral glycoprotein, most commonly vesicular stomatitis virus G-protein (VSV-G), to modify the host range of the vector. An important safety feature is also the deletion of the promoter-enhancer region from the 3’ LTR preventing transcription from this region and subsequent viral replication (self-inactivating vector; SIN).

 gene therapy involve cell isolation and 

 culture of the desired cell type to allow the selection, expansion, and differentiation either before or after the cell has been transduced with a viral vector. In the case of hematopoeitic cells, most of these steps can be performed in a closed system using single-use blood collection and processing bags.

For CAR T cell therapy, patient blood cells are harvested, and the desired T cell populations are selected and grown to the required levels. They are then transduced with a viral vector carrying the CAR gene cassette, followed by CAR T cell expansion to the billion-cell level. Lentiviral vectors have been shown to transduce T cells more efficiently and are, therefore, the preferred vector for introducing CAR into patient target cells. Expanded cells are then reintroduced into the patient. To move quickly into patients, the testing window is relatively short, and administration of cells may be initiated before all testing is completed. This requires a balance of product risk and timely treatment of cancer patients.


Lentiviral vector cGMP production is frequently accomplished using adherent HEK293 (human embryonic kidney cells immortalized using sheared Adenovirus 5 DNA), or HEK293T cells (HEK293 cells containing the SV40 large T antigen gene) transiently transfected with plasmids providing the relevant vector components. The critical raw materials usually needed for vector production are the cells, medium and serum, and the plasmids. Each of these must be sourced from approved suppliers and should have gone through a rigorous testing program to reduce the risk of introducing adventitious agents into the production process. Master and working cell banks (MCBs and WCBs) and animal-derived media components are also extensively tested.

If fetal bovine serum (FBS) and porcine trypsin are used in production, they should be gamma irradiated to reduce the risk of viral contamination. FBS also carries the risk for bovine spongiform encephalopathy (BSE) contamination and, therefore, should be restricted to negligible BSE risk countries, such as the US, Australia, and New Zealand. Plasmids being produced in bacteria are tested for sterility and endotoxin levels. Similarly, the vector product requires testing for adventitious agents, purity, genetic identity, as well as potency before its release as a drug substance for transduction of cells 

For lentiviral vector current good manufacturing practices (cGMP) manufacturing at the SAFC Carlsbad location, several approaches for scale-up are being pursued, including use of cell factories, Corning cellSTACK or HYPERStack. Depending on the size of the facility and the available incubator space, 20 to 40 cellSTACKs or up to 20 HYPERStacks can be incubated in parallel and still allow a consistent and reliable vector production. To allow consistent productivity, the plasmid transfections are done with polyethylenimine (PEI) as the transfection reagent, which does not require as high scrutiny with respect to pH and buffer consistency as the calcium phosphate based precipitation method. In many cases, the cells used for production are still grown in the presence of serum, which requires a higher scrutiny in testing, and efforts to implement serum-free production should be undertaken in sight of commercialization. Overall, efforts should be undertaken to move toward animal-free components, which apply to media components as well as cell propagation.

In comparison with other virus productions, where cell lysis is involved, retroviral or lentiviral vectors are harvested from supernatant, which may, dependent on the production system, also allow multiple harvests and thereby increase the overall yield. Supernatant volumes can reach 100 L or more, and require first a clarification and concentration step followed by column purification. The main criterion for purification is to obtain an acceptable vector concentration and assurance that the vector product has high transduction efficiency and does not compromise target cell functionality due to the presence of contaminants.

The authors offer insight into cell therapy manufacturing, vector production, and the safety aspects of testing for characterization.

BioPharm International-11-01-2014