Understanding the factors that impact stability and then generating product stability knowledge are key considerations detailed in the International Conference of Harmonization (ICH) Q8 (1) development standard as well as the Q1E standard. Accelerated stability analysis is a strategy used to quickly evaluate alternative formulations, packaging and processes; however, it is not always clear how to estimate expiry from the accelerated data.  

Understanding the product and process is becoming more of an expectation based on FDA guidance and technical presentations. Building representative product and process models for stability is now considered an essential part of product and process development. Once a product/process model has been established and verified it can be used to support and/or justify potential formulation, process, site, and supplier changes.

Linear and non-linear regression models are recommended in guidance from the health authorities for stability determination. Specifically, ICH Q1E 2.6 General Statistical Approaches states (2):

“Regression analysis is considered an appropriate approach to evaluating the stability data for a quantitative attribute and establishing a retest period or shelf life. The nature of the relationship between an attribute and time will determine whether data should be transformed for linear regression analysis. The relationship can be represented by a linear or non-linear function on an arithmetic or logarithmic scale. In some cases, a non-linear regression can better reflect the true relationship.”

Building a product or process stability model typically follows the following steps: 
  
  • State the purpose
  • Perform a risk assessment to identify the key factors driving the responses of interest (3)
  •	Develop a stability study design and associated power analysis (4)
  •	Collect the data
  •	Fit the model (linear and or non-linear)
  •	Evaluate the model’s usefulness, accuracy, and associated errors
  •	Evaluate the model’s predictive potential and associated errors
  •	Determine rate of degradation and expiry at nominal conditions and at accelerated conditions
  •	Verify any early accelerated predictions with long-term stability studies at nominal storage conditions.

Traditionally there has often been an attempt to use the Arrhenius transformation for all accelerated conditions. This approach is good when the assumptions associated with the Arrhenius equation are valid; however, often the Arrhenius transformation becomes the erroneous equation as it does not fit the data and the product or process assumptions cannot be satisfied. The health authorities have issued warning letters to companies that use Arrhenius transforms when the data does not support this method of stability estimation (5). This paper will outline an approach to model and predict linear and non-linear stability data under accelerated and nominal storage conditions. In general, the approach presented in this paper is to model the measured data, understand the scientific rational associated with the degradation pathway (what makes it degrade?), and then generalize the model for multiple accelerated conditions and at the nominal condition.

There are many factors other than environmental that may impact product stability.  These factors should be considered as well when developing accelerated and long-term stability and self-life claims:
  
  • Temperature
  • Humidity
  • API concentration
  • Water content
  • Amount of an excipient
  • Processing conditions and/or set points
• Packaging materials/method.


Proper design of experiments for data collection and factor effect isolation is crucial for building linear and non-linear stability models.  All factors should be orthogonal relative to each other and have zero correlation (or near zero). For this simple example, there are multiple time points at multiple temperatures. There are an equal number of samples at each time and temperature condition. Replicates are suggested at each condition to reduce the analytical method variation and to improve the precision of the estimates. In this study, the number of replicates was three at each time point. A power analysis (

) should be performed to assure the stability study has good statistical power (>0.95). This study is a 3*3*9=81 runs, three temperatures with three replicates and nine time points. Simulation can also be used to evaluate study plans and estimate their impact on stability.


The following is the step-by-step procedure for accelerated stability modeling and expiry determination:

1.Measure the data at multiple time periods and at multiple temperatures. Generate a plot of the data (

) to visualize the relationship of the curves over time.  Save the slopes from the curve and place them in a table.  Examination of the slopes will indicate if the environmental factor accelerates the rate of change/degradation.

 2. Use a multiple factor analysis of covariance (ANCOVA) model to fit the data. Examine the effects test (

) to make sure all terms in the model are significant. The inverse prediction (

) will provide the expiry and the 95% lower confidence interval. Also examine the whole model to determine the RSquare and the quality of the ANCOVA model. Check the residuals to make sure no hidden factor has entered into the study.

3. Save the expiry and the 95% predictions (

) at each temperature into a table.  Acceleration rate is the ratio of each slope at temperature to the nominal storage condition. 

) of how temperature accelerates the rate of degradation and expiry, fit a model of temperature to the coefficients (slopes), expiry, and 95% confidence interval (CI) and acceleration rate. Models may be linear or non-linear in their fitting parameters. Make sure the models selected makes good scientific sense and can be generalized.  Modern software programs make this easy to do.

5. Save the equations for the fit for the 95% CI, expiry, acceleration rate, and the slope:
  
  

Predicted Slope= (-0.0000009387967452312) + 0.0000001815600541022 * :Temperature + 0.0000000218633389171 * :Temperature ^ 2, Quadratic Model
  
  Predicted 95% CI= (-27.8898211558579) + 23.9294245426114 * Exp(0.0376760939674008 * :Temperature), 3 Parameter Exponential Model
  
  Predicted Expiry= 33.5848316003024 + 7328.12608635937 * Exp(-0.196329985274204 * :Temperature), 3 Parameter Exponential Model
  
  Predicted Accelaration Rate= 25.9675472550865 + 1008.32460629621 * Exp(-0.116629176337002 * :Temperature), 3 Parameter Exponential Model
  


  6. Check the model to make sure it matches the actual data. Correct any modeling errors.  
  7. Create a profiler (

) from the equation. This can be done using a modern statistical package such as SAS/JMP.

8. Predict expiry at any temperature using the profiler. A temperature of 4 C was not part of the study design; however, it now can be modeled using the prediction profiler.
  9. 95% CIs are often not helpful in estimation of long-term stability due to the limited sample size.  The sample size directly impacts the 95% CI but not the slope as much. Expiry based on the slope is typically the primary focus.  In this example, it is estimated at 4 C the product will be stable for 3375 days or 9.2 years.
10. Finally, long-term stability evaluation at nominal storage conditions will be used to confirm the early model prediction and will provide an independent secondary determination of stability and changes in dissolution.  Understanding rates of degradation should factor into shelf life and release specification limits (6).


  Reliable accelerated stability modeling and estimation has long been a problem in a variety of process and product modeling and prediction situations. The novel procedure discussed in this paper for the characterization of stability performance provides a clear, statistically defendable method for determining accelerated stability. Four primary tools are used in the generation of the analysis: design of experiments for the design of the study, ANCOVA model fitting, linear and non-linear model fitting of the coefficients, and profilers to integrate the equation and improve visualization and prediction. Long-term verification of accelerated conditions should always follow early determinations of expiry, acceleration rates and rates of degradation


  1.	ICH Q8 (R2) Pharmaceutical Development (ICH, 2009).
  2.	ICH Q1E, Evaluation for Stability Data (ICH, 2003).
  3.	ICH Q9 Quality Risk Management (ICH, 2006).
  4.	ICH Q1A(R2) Stability Testing of New Drug Substances and Products (2003).
  5.	FDA, FDA Warning Letter to ACell (April 26, 2013), www.fda.gov/ICECI/EnforcementActions/WarningLetters/2013/ucm352061.htm
  6.	ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (ICH,1999).


  Thomas A. Little, PhD is president, Thomas A. Little Consulting,12401 North Wildflower Drive, Highland, UT  84003, 

Articles

Analytical Best Practices

Characterization of stability performance provides a clear, statistically defendable method for determining accelerated stability.

Accelerated Stability Modeling

Vaccine development is on a roll, boosted by biomedical research uncovering new molecular targets for preventives and treatments, as well as innovative techniques for enhancing vaccine potency and production. There is high demand for new vaccines to prevent deadly tropical diseases, illustrated by the recent Ebola virus outbreak, and for capacity to respond quickly to global pandemics and bioterrorist attacks at home and abroad. More manufacturers seek to devise new versions of vaccines for pneumococcal disease, meningitis, and more potent influenza preventives, encouraged by positive coverage and reimbursement decisions. 

At the same time, early optimism about developing vaccines to prevent and treat HIV/AIDS has diminished, along with hopes for prompt discovery of a vaccine against drug-resistant tuberculosis. There has been progress in research on therapeutic vaccines for cancer, but so far these are highly targeted and expensive. The ability of manufacturers to produce high-quality, safe and effective vaccines to meet public health needs remains crucial to building public confidence in vaccination schedules that have expanded with the approval of more effective products.


Meanwhile, frequent vaccine shortages point to the need for more extensive and reliable manufacturing operations. David Swerdlow, associate director for science of the National Center for Immunization and Respiratory Disease at the Centers for Disease Control and Surveillance (CDC), emphasized the need to increase United States vaccine manufacturing capability at Terrapin’s World Vaccine Congress in Washington, D.C. in March. Swerdlow noted growing concerns about the MERS outbreak in Saudi Arabia and the Near East, and the potential for this and other deadly diseases to travel to Europe and elsewhere. 

Jesse Goodman, former chief scientist at FDA and now forming the Center on Medical Product Access, Safety and Stewardship (COMPASS) at the Georgetown University Medical Center, commented on the importance of vaccine supply chain security in assuring global access to treatment. He acknowledged considerable progress in establishing a more reliable influenza vaccine supply, noting advances in dual-use capacity and in developing cell-based products. Goodman also cited numerous challenges. Current flu vaccines have limited effectiveness, and health authorities have difficulty each year predicting which seasonal flu strains pose the most risk. Vaccine production capacity is limited, he added, and supply shortfalls occur all too frequently. 

The Biomedical Advanced Research and Development Authority (BARDA) in the Department of Health and Human Services (HHS) is responding with funds to support flexible manufacturing capacity that uses disposable technology to enhance US capability for fast scale-up of vaccines and treatments during a pandemic or health emergency. Such facilities also can provide support for biotech firms developing new vaccines and medical countermeasures. BARDA has awarded more than $400 million to establish three Centers for Innovation in Advanced Development and Manufacturing (CIADMs): Emergent Manufacturing is forming a biologics development and manufacturing suite in Baltimore; Novartis Vaccines and Diagnostics is expanding its cell-based vaccine production facility in North Carolina with a pilot plant to produce clinical lots of medical countermeasures; and Texas A&M University is establishing several development and manufacturing facilities. Each organization also is partnering with other biopharma companies and research entities to develop new pandemic influenza vaccine candidates. 

BARDA also is forming a Fill Finish Manufacturing Network with $40 million awarded to Cook Pharmica (Bloomington, Ind.), DMS Pharmaceuticals (Greenville, NC), JHP Pharmaceuticals (Parsippany, NJ), and Nanotherapeutics (Alachua, FL). The network will be capable of packaging 117 million doses of flu vaccine in 12 weeks and will support the CIADMs in building capacity for four-month production of 150 million doses of pandemic flu vaccine. The CIADMs will produce test countermeasures for clinical trials and provide commercial capacity to help remedy shortages in vaccines and biologics. A recent report on the program by the Government Accountability Office (1) notes that the value of this investment will begin to emerge as BARDA issues task orders this year to test the quality of CIADM core services and their success in spurring development of new therapies and vaccines. 


Another prominent strategy for expanding the nation’s vaccine supply is to develop adjuvanted products that require less antigen for comparable or expanded immune response. FDA is working with manufacturers to facilitate licensure of such products, noted Marion Gruber, director of the Office of Vaccine Research and Review in FDA’s Center for Biologics Evaluation and Research (CBER), at the Vaccine Congress. Such formulations may boost immune response in elderly and other special populations; achieve immunity with fewer doses; and reduce booster shots needed to extend immune response. While a number of adjuvanted vaccines have been approved in Europe, Gruber reported that FDA “finally” has done the same with the licensing of two adjuvanted products in the US.

Gruber explained that FDA has no special pathway for approving adjuvanted vaccines, as adjuvants are not active ingredients, but considered “constituent materials” to a vaccine formulation. The adjuvanted vaccine must be pure and of high quality, and it must demonstrate that the added ingredient does not reduce vaccine safety and potency. Manufacturers have to establish consistent production processes and provide a rationale for using an adjuvant, usually based on studies that compare a vaccine with and without the adjuvant to see what advantage is created. While FDA does not require an adjuvanted vaccine to demonstrate added benefit, such data may be requested if safety concerns emerge or if the manufacturer plans to make comparative claims for the reformulated product.  

FDA is willing to infer effectiveness of an adjuvanted vaccine from the immune response induced in populations in clinical trials and then confirm clinical benefit in post-marketing studies, as is usual for seasonal influenza vaccines. The agency wants to see 12 months follow-up to be sure to detect further adverse effects, but emphasizes that there’s no one-size-fits-all approach to developing and testing these products. 


Manufacturing innovation also can address many of the challenges in distributing vaccines to patients in third- world regions. While efforts continue to develop thermostable vaccines and medical products, Raja Rao, senior program officer at the Bill & Melinda Gates Foundation, noted at the Vaccine Congress that interest is growing in additional strategies. He discussed the need to mitigate the dangers of vaccine freezing under current distribution systems, which can greatly reduce potency, as well as identification of products that don’t need refrigeration if delivered to clinics in 30 or 60 days. Gates is looking more at improving supply-chain equipment and at devising more efficient vaccine delivery networks as less costly approaches than a “moon shot” investment in thermostability, Rao observed. 

Many of these initiatives are outlined in the March 2014 HHS 

, which cites efforts by BARDA and other government agencies to ensure stable supply and public access in the US to recommended vaccines (2). One innovation is the SMART vaccines program that uses software and other tools to help set priorities for developing and testing new vaccine targets. The report also describes federal programs for developing new vaccine production technologies, including use of a silk-based stabilizer to enhance vaccine stability in hot climates, and new vaccine delivery methods that conserve antigen and may be important during emergencies. 

The State of the National Vaccine Plan-2013

, Department of Health and Human Services, accessed Apr. 8, 2014. 

 Read Jill’s blogs at PharmTech.com/wechsler.

Regulatory Beat

New formulations and expanded vaccine production are encouraged.

Demand for New Vaccines Spurs Innovation

Exciting progress is being made in the development of consensus in best practice recommendations and standardization guides for single-use disposable technologies, facilitating implementation, quality, and safety.  These industry efforts include the Parenteral Drug Association’s (PDA) forthcoming new technical report; the BioProcess Systems Alliance’s (BPSA) new single-use guides; and in collaboration with the BioPhorum Operators Group (BPOG) and the American Society for Mechanical Engineering-BioProcess Equipment Committee (ASME-BPE), BPSA’s constructive discussions towards standardizing supplier extractables test data for single-use components.
  
  

Technical Report on Single-Use System Manufacturing Strategy

 is currently pending for final approval by PDA’s Scientific Advisory Board (1). This extensive guide, to be published by PDA in 2014, provides extensive recommendations on how to consider and adopt single-use technologies. These recommendations are written by a broad panel of industry experts from end-user and single-use equipment supplier companies, as well as regulators and consultants.  The goal of this document is not to be prescriptive, but rather to provide an understanding of key principles and concepts for selection, qualification, validation, and use of single-use technologies. It should serve to better enable people at various levels in an organization to ask the right questions and make better decisions for their individual situations. Organizations will be able to draw an effective road map to single-use implementation that suits them best, regardless of whether they are small or large, dedicated or multi-product, or contract manufacturers.

 The draft PDA single-use technical report has dedicated chapters for key topic areas including manufacturing strategies for multi-use versus single-use systems, an overview of the variety of single-use technologies, and a tutorial on business drivers influencing selection of multi-use and single-use systems.  Also included are dedicated sections on qualification of assembled single-use products and implementation of single-use systems in a manufacturing process.  The authors recognized that each area has a situation-dependent role in the decision process and may be weighed differently depending on the circumstances of the reader’s organization.

The manufacturing strategies section covers variables including process compatibility, facility requirements, and operational considerations. The business drivers section covers capital design, fixed and variable operating costs, and other factors. The qualification section includes recommendations on supplier qualification; verification of materials, components and completed assemblies, including extractables and leachables issues; risk evaluation (i.e., balancing pro and cons for multi-use and single-use systems); microbial control and sterilization; system process validation; filter integrity and leak testing; installation qualification and acceptance tests; and supplier audits. The implementation section introduces themes of stakeholder management, technical feasibility and risk management, scoping and technology survey, making the business case, and a project execution plan including user requirements. Once these are established, recommendations are provided for approaches to testing and validation, process control strategies, integration considerations (e.g., facility, equipment, and operational deployment), operator training and safety, supplier agreements, technical diligence, materials management, logistics practices, and waste management.
  
  


  BPSA has also been active in preparing new best-practice guides. A key topic of discussion at the 2013 BPSA International Single-Use Summit (ISUS) was the concern for particles in SUS flow paths, especially those without inline filtration, as employed for non-filter sterilizable vaccines manufactured under aseptic sterile conditions, or simply downstream of sterilizing filters in single-use systems designed for filling of bulk or final dosage protein biopharmaceuticals and vaccines.
  
  To address these concerns and provide users and system suppliers with best practices to minimize risks of particles from the systems potentially reaching final dosages, BPSA has developed 

Recommendations for Control, Testing and Evaluation of Particulates in Single-Use Process Equipment

 (2). This document, developed jointly by technical and engineering representatives of user and supplier member companies, contains major sections on risks of particles, characterization and sources of particles in single-use technologies, chain of responsibility, measurement methods, and visual inspection (surveillance) programs. Also included are major sections on recommended methods for suppliers to control introduction of particles, best practices for handling single-use components to minimize risk of particulate generation, evaluation of single-use containers and filling systems for particulates during end-user manufacturing, and recommendations for developing a particulate deviation response and mitigation plan. 

The BPSA document will be of interest to vaccine manufacturers and final filling operators employing single-use disposable filling technologies. It will be published in July 2014 in coordination with the BPSA International Single-Use Summit annual meeting. It will also be made available via the BPSA website, www.bpsalliance.org.

BPSA Single-Use Component Quality Test Reference Matrices


  BPSA’s first best practices guide for single-use equipment, the 

BioProcess Systems Alliance Component Quality Test Matrices

, published in 2007, provided a supplier consensus of standards, regulatory guidance documents, and industry guides applied in the manufacture of single-use components for qualification, validation, and quality control to ensure product quality and performance claims, consistent with requirements of the biopharmaceutical industry (3). The document was divided into four sections for biocontainers, tubing, connectors, and filters.  
  
  In the ensuing years, the industry has been well served with this easy-to-use reference guide to supplier component quality testing. In the ensuing years, however, many newcomers to the field have been unaware of it, some new references have come to be implemented, and new components have come into broader use, that were not considered in the original document, namely single-use (or campaign) chromatography modules and single-use sensors for bioreactors and other systems.

Single-Use Manufacturing Component Quality Test Matrices 

will be published in July 2014 in coordination with the BPSA International Single-Use Summit annual meeting (4). It will be made available via the BPSA website, www.bpsalliance.org.

BPSA Single-use Equipment Quality Agreement Template


  BPSA has recognized a need among end-users and suppliers for a common template for quality agreements. End-users have been challenged to herd a variety of single-use component and system suppliers to agree to common requirements, or to managing different agreements among multiple suppliers. Likewise, suppliers engage in quality agreement discussions with end-users, many of whom have disparate sets of requests and requirements. This challenges suppliers to manage differing requirements and agreements among multiple customers, and often, instead, acts as a barrier to the establishment of effective quality agreements.

Consensus Quality Agreement Template for Single-Use Bio/pharmaceutical Manufacturing Products

 is intended to provide a common structure for quality agreements between suppliers and end-users of single-use products (5). The template is not intended to substitute for close dialog and negotiation between the two parties, but rather, it will hopefully serve to facilitate that discussion by providing a common structure to follow. The template’s content will better enable end-users to communicate specific expectations and for suppliers to communicate the attributes of their quality system and operating procedures, more readily providing the content for the final quality agreement between the two parties. 

As a template, the document does not proscribe all activities and specify provisions.  These still have to be negotiated between the parties to establish an effective agreement. There are areas where the template is left intentionally blank for the supplier and end-user co-authors to propose, negotiate, and either discard, change, or complete to establish a final agreement.  The template should support the process, however, and facilitate the establishment of quality agreements to build the partnership and transparency needed for successful implementation of single-use technologies. As with the BPSA 

Particulates Recommendations and Component Quality Test Reference Matrices

, the BPSA Quality Agreement Template will also be published in July 2014 in coordination with the BPSA ISUS annual meeting and it also will be made available from the BPSA website, www.bpsalliance.org.

Progress towards Extractables Standardization


  Early in 2013, the extractables committee of the BioPhorum Operators Group (BPOG), a consortium of end-user companies, released a proposal for standardized extractables testing and analysis it would like to see provided by single-use component suppliers (6). While suppliers recognized the benefit to end-users of standardized component extractables data packages, especially in terms of extraction sample size, solvents, and conditions, extraction temperatures and times, analytical methods and data presentation, there was also objection by suppliers to some of the test requirements and rationales, and the overall extent of testing proposed, which would demand extensive resources and time commitments. Supplier responses were consolidated by BPSA, and after meeting with BPOG representatives at the July 2013 BPSA ISUS meeting, negotiation between the two groups began in Autumn 2013 to harmonize a consensus proposal that could be submitted to a standards body such as ASTM. Negotiations have proceeded over the winter, and much has been agreed to, especially in regard to many of the extraction conditions and analytic methods. At time of this writing, it is hoped that a document that recognizes both the interests of end users (and regulators) and the resources of suppliers can be brought to consensus by the July 2014 BPSA ISUS meeting.

Other organizations are also looking at developing extractables testing standards. The United States Pharmacopeia (USP) has proposed a new section to its standards Chapter <661> on Plastic Containers currently under revision. A sub-chapter <661.3>, Plastic Systems Used for Manufacturing Pharmaceutical Products (differentiated from Sub-chapter 661.2 on Plastic Packaging Systems for Pharmaceutical Use), is currently on hold pending draft revisions of subchapters 661.1 and 661.2, along with evaluation of an ultimate BPOG/BPSA consensus proposal (7). Independently, ASME-BPE has approved an expansion of their Bioprocess Equipment Standard Part PM, Polymeric and Other Nonmetallic Materials, section PM 3.2 on Extractables and Leachables, which will be published at the end of this year (8). The ASME-BPE Standard Extractables and Leachables section 3.2 covers key concepts and includes a non-mandatory appendix suggesting possible extraction solvents. These general recommendations are useful in principle, but do not achieve the degree of supplier extractables data standardization proposed by the BPOG Extractables Committee. There will be more to come in this area, but real progress toward the standardization of supplier extractables data packages desired by end-users is being made. These efforts will ultimately serve to reduce a perceived barrier to further implementation of single-use technology.

Technical Report on Single-use System Manufacturing Strategy (DRAFT)

, publication pending Spring/Summer 2014,  accessed Apr. 4, 2014.
  2.	BPSA, 

Recommendations for Control, Testing and Evaluation of Particulates in Single-Use Process Equipment (DRAFT)

, publication pending Summer 2014, accessed Apr. 4, 2014.
  3.	BPSA, 

Bioprocess System Alliance Component Quality Test Matrices

, BioProcess International, April-May 2007, accessed Apr. 4, 2014.
  4.	BPSA, 

Single-use Manufacturing Component Quality Test Matrices (DRAFT)

, publication pending Summer 2014,  accessed Apr. 4, 2014.
  5.	BPSA, 

Consensus Quality Agreement Template for Single-Use Bio/pharmaceutical Manufacturing Products (DRAFT)

, publication pending Summer 2014, accessed Apr. 4, 2014.
  6.	Wong, K., “Extractable Protocol Standardisation Efforts for Disposable Systems from BPOG Working Group,” Biopharma Asia, Jan. 27th, 2014.
  7.	USP, Chapter <661> “Plastic Packaging Systems And Their Materials of Construction” (DRAFT), 

Bioprocess Equipment Standard Part PM, Polymeric and Other Nonmetallic Materials

, section PM 3.2 on Extractables and Leachables (DRAFT), publication pending Fall 2014. 


Jerold Martin is Sr. VP Global Scientific Affairs at Pall Life Sciences, Port Washington, NY and Chairman of the Board,  Technology Committee Bio-Process Systems Alliance, 1.516.801.9086, 

The Disposables Advisor

Progress is being made in the development of harmonized best practices for single-use systems.

Standardizing Practices for Disposables

Following the 2009 outbreak of the H1N1 pandemic flu and the numerous delays in producing vaccines against the virus, the US Department of Health and Human Services (HHS) recognized the need to invest in new vaccine technologies that can ensure national preparedness for a pandemic influenza or other diseases. Many other countries are focused on developing similar capabilities. There is also a significant requirement for therapeutic vaccines to treat existing and prevent further chronic viral infections. As a result, there is an urgent need to develop alternative vaccine production methods that can either generate large quantities of vaccines in a much shorter time and/or produce more broadly effective vaccines than is possible using traditional egg-based technology. Examples include cell-culture, synthetic DNA, chimeric antigen, and recombinant protein nanoparticle vaccine technologies. 


  Traditional vaccine manufacture begins with a “seed” virus identified and provided by the Centers for Disease Control and Prevention. This virus is introduced into fertilized chicken eggs. It then reproduces and builds up in the white (allantoic fluid) of the egg, which is collected and purified. It takes two eggs to generate enough vaccine for one dose, and thus large numbers of eggs must be produced in advance, which is always a challenge. The limited availability of eggs prevents the rapid production of vaccines, which is a major concern in a pandemic situation. In fact, it takes many months to organize egg supplies, incubate the virus, and obtain produced vaccine that can be delivered, according to Novartis.

Another difficulty with this approach is the fact the seed virus is not always accurately replicated, and as a result, the virus in the vaccine may not be the same as the infective strain, which thus provides reduced immunity. Extensive DNA and protein analysis is required to help avoid this problem, and further testing is conducted to ensure that no pathogens from the eggs are transferred to the vaccine. All of this testing extends the production time and increases the manufacturing cost. 


Cell-culture technology enables the use of raw materials that are readily available and not threatened by pandemic events, as well as closed-system bioreactors that reduce the required biosafety level for the manufacturing space. In addition, it is possible to provide rapid response to potential pandemic influenza threats while fulfilling the need for seasonal influenza vaccines, according to Novartis. The company received the 2013 Facility of the Year Award issued by the International Society for Pharmaceutical Engineering (ISPE) for its flu cell-culture technology, which it has implemented at a new facility in Holly Springs, North Carolina.

In cell-based flu culture, immortalized canine kidney cells are typically employed; these cells can be stored in advance until needed and then rapidly amplified, enabling the production of large quantities of vaccine in a much shorter period of time than is possible using fertilized eggs. Cell-culture technology also enables more robust virus production, and therefore, the virus in the vaccine is more consistent and more closely resembles the seed strain, which leads to increased efficacy. In addition, the size of the bioreactors required to produce large numbers of doses is much smaller than the space required to produce a similar number of doses using eggs. Furthermore, the use of bioreactors ensures a closed, sterile, controlled environment, and thus the risk of potential impurities is reduced. As a result, Novartis’ Flucelvax vaccine does not contain any preservatives, such as thimerosal, or any antibiotics. Finally, unlike vaccines produced in fertilized eggs, cell-culture-derived vaccines can be administered to patients who are allergic to eggs.

Development of Novartis’ technology and construction of the plant were funded in part by the HHS Office of the Assistant Secretary for Preparedness and Response and the Biomedical Advanced Research and Development Authority. The Holly Springs facility has the capability to produce seasonal flu cell-culture vaccine, pre-pandemic vaccine, and 150 million doses of pandemic vaccine within six months of an influenza pandemic declaration, according to Novartis.  A fill-finish set up for the production of both flu and non-flu products has also been installed in the facility. The company received FDA approval for Flucelvax, the first cell-culture vaccine in the US designed to protect against seasonal influenza in individuals 18 years of age and older, in November 2012 and made its first shipments in August 2013. 


One of the biggest drawbacks of traditional vaccines is that for diseases that change rapidly and evolve into many different strains, the genetic makeup of the antigen introduced to the body by the vaccine may be different than what it encounters in an actual pathogen in the future, according to J. Joseph Kim, president and CEO of Inovio Pharmaceuticals. “Since the immune system isn’t looking for the mutated version of the antigen, it may not be able to prevent or fight an infection,” he explains. 

To address this problem, Inovio has developed synthetic DNA vaccines based on the genetic codes of viruses. “We can consciously manipulate and engineer protein sequences that are flexible and can recognize multiple viruses in a particular subfamily, which provides more comprehensive protection and is a more universal approach to the development of influenza and other vaccines,” Kim states. 

The synthetic DNA is encoded with instructions that enable cells in the body to produce only the targeted antigen relating to the pathogen or cancer of interest and cannot replicate or cause the disease. “This approach results in the body creating an immunogen capable of inducing strong, multi-faceted immune responses similar to actual pathogens,” Kim observes. As a result, the vaccines generate very strong T-cell immune responses, and particularly T-cells that are able to kill the targeted infected or diseased cells.

In animal models and early clinical studies, Inovio has shown that its H1N1 vaccine provides protection against all strains of the H1N1 virus identified over the last 90 years (since the 1918 Spanish Flu). “We believe the synthetic DNA vaccines are a paradigm-changing technology,” says Kim.

There are other advantages to the technology. The synthetic DNA vaccines are produced in pure water and do not contain any live virus, parts of a virus, adjuvants, or preservatives and are thermostable (i.e., no cold-chain requirements). The DNA plasmids are manufactured using conventional fermentation technology (i.e., no eggs) that is scalable, is engineered for maximum expression in the host cells, and can be rapidly produced in large quantities in a pandemic situation. Delivery is achieved through injection followed by 

 electroporation, which involves application of a brief low-voltage electric field (three pulses of 0.05 s) that causes the cell membrane to open and allow entry of the DNA plasmid. 

In addition to its H1N1 vaccine, Inovio has a therapeutic vaccine for cervical cancer in Phase II trials. It currently has a partnership with vaccine manufacturing company VGXI for clinical trial quantities, and will be selecting a contract manufacturer for the production of Phase III and commercial quantities later in 2014. Inovio is also participating in a National Institutes of Health malaria vaccine initiative focused on the development of novel technologies and is developing new candidate DNA vaccines for various cancers and other diseases, particularly prostate cancer and hepatitis B, which are being pursued in conjunction with Roche.

Mimicking natural antigen presentation pathways


The Chimigen vaccine technology developed by Akshaya Bio is based on chimeric antigens that bind to specific receptors on antigen-presenting cells, particularly dendritic cells (Fcγ and Lectin receptors), and mimic natural human antigen presentation pathways to generate antigen-specific, balanced, cellular (for clearing virus-infected and cancer cells) and humoral (antibody-mediated) immune responses, according to president and CSO Rajan George. As a result, the vaccines generate broad immune responses, “re-educate the immune system,” and “break tolerance” to chronic, infections and cancer. “Because a Chimigen vaccine has the characteristics of both an antigen and antibody in a single entity (i.e., it is a fusion protein), the technology is versatile and highly adaptable to disease-specific multiple molecular antigens and can be used to develop both prophylactic vaccines and immunotherapeutic agents,” he notes. The level of each type of response depends on the type of antigens used in the vaccine.

George adds that the incorporation of a xenotypic antibody fragment makes the entire molecule “foreign” and thus more immunogenic. Production of the vaccines in insect cells is also beneficial, because they impart non-mammalian glycosylation, thus increasing the immunogenicity and enabling the vaccines to be effective at low doses, according to George. Furthermore, adjuvant-related adverse reactions are not an issue with this technology, and cellular responses are promoted because no adjuvants are used in the vaccine formulations. 

The major technical challenge that Akshaya Bio is currently tackling relates to the production of the vaccine. “In heterologous protein production, the quantity of protein produced depends on the type of antigen (intracellular/extracellular) and the size of the protein molecule,” George explains. To date the company has been able to produce ~5-7 mg/L for vaccines with a size of ~75 kilodaltons (KDa) to ~3-5 mg/L for vaccines with a size large than ~250 KDa. Due to the high immunogenicity of Chimigen vaccines, however, George notes that the lower production levels are still economical.

The company’s lead candidate is a therapeutic vaccine for the treatment of chronic hepatitis B virus (HBV) infections, for which there is currently no effective treatment. The Chimigen HBV vaccine is in late pre-clinical development, having achieved ex vivo proof of concept and is ready for out-licensing to a pharmaceutical/biotech partner for clinical development, according to George. The vaccine also has prophylactic application in non-responders to currently available vaccines. Akshaya also has a Chimigen vaccine for hepatitis C virus (HCV), for which there are some newer treatments that are effective, but a vaccine is desirable for preventing new infections. The Chimigen HCV vaccine is ready for clinical development and Akshaya is looking for partnership opportunities. 

Additional earlier-stage pipeline products include products for various cancers, influenza, malaria, and HIV. The HIV vaccine, which is being tested in animals to evaluate immune responses, is being developed with support from a Government of Canada/Bill & Melinda Gates Foundation partnership and the National Resource Council Canada Industrial Research Assistance Program. The Chimigen malaria vaccine was initially developed using a Grand Challenge Exploration award from the Gates Foundation.

“The short-term goal is to establish proof of concept in humans for at least one of the Chimigen vaccines. In the long-term, we look forward to Akshaya’s products helping to alleviate suffering due to infectious diseases and cancer,” George remarks.


Novavax’s recombinant protein nanoparticle vaccine technology combines the flexibility and speed of genetic engineering with the efficiency of single-use disposable technology to produce highly immunogenic nanoparticle vaccines, according to the company, which is developing vaccines against viral, bacterial, and parasitic diseases.

With the Novavax technology, the genetic code of a virus of interest can be used to produce, within a few weeks, a vaccine candidate that is designed to generate protective immunity for that specific virus. Two different types of immunogenic particles are used: virus-like particles (VLPs) and recombinant protein micelles.  Novavax’ seasonal and pandemic influenza vaccines consist of VLPs or recombinant particles with matrix proteins that provide a structure onto which the surface proteins hemagglutinin and neuraminidase are incorporated. “VLP constructs resemble the virus they are designed to protect against; however, because they do not contain any RNA, they are not infectious, and thus are generally highly immunogenic,” states Novavax’s vice-president of Vaccine Development, Gale Smith.

The recombinant protein micelles are generally composed of a single target protein, engineered to assemble into stable nanoparticles that elicit an immunogenic response like the virus itself. For example, Novavax’s Respiratory Syncytial Virus (RSV) vaccine candidate is composed of recombinant micelles engineered as modified full-length fusion (F) proteins with the potential to induce protection against all strains of RSV, according to Smith.

The vaccine nanoparticles are produced in Sf9 fall army worm (Spodoptera frugiperda) ovary cells, which grow in perpetuity in special culture media. The cells produce recombinant proteins when infected with an insect virus (Baculovirus or BV) engineered to carry the foreign gene or genes of interest. “The Sf9/BV genetic engineering technology is now well established in the biopharmaceutical industry, and has for example been used for the production of a licensed human papilloma virus vaccine and a recently licensed influenza subunit vaccine,” Smith observes. In addition, Sf9/BV efficiently expresses large antigens and particles with proper folding, which promote superior immunogenicity and better functional immunity. Smith also notes that the adoption of single-use manufacturing technology accelerates process validation and analytical testing for Novavax’s vaccines and may allow for ultimate regulatory approval of the company’s vaccines derived from a common platform.
  Novavax has also produced new vaccine candidates for the prevention of SARS and the newly emerged MERS-CoV virus, malaria, rabies, and other diseases where new or improved vaccines are needed. 


Cynthia A. Challener is a contributing editor to

Cover Story

New approaches to vaccine production are targeting rapid supply for pandemic situations and broadly effective therapeutic treatments.

Novel Vaccine Technologies Meet the Need for Pandemic and Therapeutic Solutions

Vetter introduces two clinical syringe packages that can start syringe development earlier in the drug development process, potentially cutting up to 18 months off time-to-market. Vetter’s Clinical Syringe Standard Package includes all development activities through launch, while the Clinical Syringe Starter Package offers a range of testing without the financial commitment to a clinical fill. 

The standard package is an inclusive service, starting with the materials selection, which is a combination of syringe, needle, and stopper to meet a compound’s requirements and continuing through cGMP clinical syringe filling. The starter package begins with the same materials selection, proceeds through feasibility testing and regulatory consultation, and ends with a non-cGMP stability run. 

According to the company, these packages may make it easier to recruit medical clinics for trials that use prefilled syringes, since they require less handling and preparation than vials and have reduced risk of needle-sticks.

Products

Clinical Syringe Packages Increase Productivity

There are two topics in today’s pharmaceutical landscape commanding the attention of both the industry and the regulators: drug shortages and quality metrics. It is increasingly difficult to discuss one of these topics without discussing the other. Establishing, maintaining, and interpreting quality metrics to measure the suitability of pharmaceutical products and the capability of the manufacturer to provide these products consistently and without delay has become a high priority for the industry and FDA.


  The first piece of this puzzle emerged in 2012 when Congress passed the Food Drug Administration Safety and Innovation Act (FDASIA) enhancing FDA’s capability to proactively react to, prevent, and alleviate drug shortages. This direction is codified in the language contained in Title VII—Drug Supply Chain and Title X—Drug Shortages. Specifically, Title VII Section 705 of the Act states FDA “shall inspect establishments described in paragraph [1] that are engaged in the manufacture, preparation, propagation, compounding, or processing of a drug or drugs (referred to in this subsection as ‘drug establishments’) in accordance with a risk-based schedule established by the Secretary” (1). Additionally, this section also describes the risk factors to be considered in establishing the inspection schedule. The last risk factor listed in this section is “Any other criteria deemed necessary and appropriate by the Secretary for purposes of allocating inspection resources.” Section 706 of the same act allows FDA to request certain information from companies in advance of or in lieu of inspections by stating “Any records or other information that the Secretary may inspect under this section from a person that owns or operates an establishment that is engaged in the manufacture, preparation, propagation, compounding, or processing of a drug shall, upon the request of the Secretary, be provided to the Secretary by such person, in advance of or in lieu of an inspection…” 

The next piece of this puzzle is found in Title X section 506C-1 (Annual Reporting on Drug Shortages). Title X section 506C-1 requires FDA to annually provide Congress “a report on drug shortages…”

The third piece of the puzzle fell into place in a February 12, 2013 

 Notice. FDA asked the industry to “assist the Food and Drug Administration in drafting a strategic plan on drug shortages as required by the Food and Drug Administration Safety and Innovation Act…” This notice asked a series of thought-provoking questions including “What metrics do manufacturers currently use to monitor production quality?” and “How frequently would such metrics need to be updated to be meaningful?” (2).

The last piece of the puzzle revolves around the industry reaction to this information. Many trade organizations responded to the questions in the 

. Some prepared white papers while others held meetings to discuss the issue with their members. The general consensus, and the easy part from this activity, was that industry needed to actively engage with the agency in defining suitable quality metrics to provide information to the agency supporting their efforts to eliminate drug shortages and determine the appropriate quality metrics to be used in establishing a risk-based approach to inspections. 


  Companies currently use a variety of metrics to measure performance including, but not limited to, corrective action and preventive action (CAPA), out of specification (OOS), batch rejection rate, complaints, field alerts, recalls, batch yield, training effectiveness, and environmental monitoring excursions. The goal is to review and define which of these metrics can be used to measure performance based on product quality and suitability as opposed to performance based on compliance standards. The hard part is trying to define them so they are not subject to interpretation: they are simple but comprehensive and applicable to traditional, biotech, virtual, contract manufacturers, drug substance manufacturers, etc. The industry needs to ensure the metrics chosen provide meaningful data while avoiding unintended consequences. The metrics need to be simple so they do not divert or dilute a company’s resources from daily activities associated with producing and delivering high quality medicines.

So, what might these metrics be? The Parenteral Drug Association (PDA) held their Annual Meeting in San Antonio in April 2014 (3). During this meeting, there was an open member session where PDA proposed seven potential metrics for consideration. For each metric proposed, there was a definition to consider, a calculation, a recommendation on how to report the metric to FDA, and a discussion of potential unintended consequences that might result from the implementation of the metric. The metrics proposed by PDA were:

The PDA recommendations included some guidelines and principles clarifying the reporting of these metrics. Product metrics were defined as items with the same formula regardless of their final packaging configuration. Under this model, drug substance manufacturers would consider each drug substance batch as a product batch. Site metrics would be a compilation of the product metrics for the site and would combine drug-product and drug-substance metrics for sites that manufacturer both. The recommendation for reporting was to collect the data monthly and report annually, perhaps using annual product reviews or annual report dates to determine the cycle.

The metrics proposed by PDA seem reasonable but there is a missing piece to this puzzle. The most important metric used to determine a company’s well-being is a measure of their quality culture. The culture of a company dictates the veracity of their metrics. Achieving a quality culture requires management and employees to establish an environment where responsibility, accountability, and reliability are paramount, and to understand the role each person performs in delivering a high-quality product to the customer and sustaining that performance on a continual basis. Management must educate employees and provide the tools and environment where they can perform their functions in an atmosphere that encourages excellence and continuous improvement. 

The trouble with using quality culture as a metric is determining how to measure it. An unhealthy quality culture is easy to identify. People in a poor culture do not understand their job and its importance to the business. They often appear stressed, and they hide their mistakes or blame others for their errors. There is no evidence of teamwork. People work in silos and rarely, if ever, seek input or advice from others. The metrics that could potentially be used to measure a poor culture include a large employee turnover, an overabundance of deviations attributed to human error, and lack of pride in the performance of their jobs. In contrast, a robust, healthy quality culture can be evidenced by alignment of goals between quality and operations, self-sustained work teams that focus on continual improvement, and employees who incorporate quality into their jobs on a daily basis. They are not afraid to speak up and offer suggestions for improvement to their colleagues. People understand the importance of their jobs and respect each other and their management. This culture welcomes inspections and views these inspections as another tool to use in their continual improvement initiatives.

The establishment of simple quality metrics that not only measure the quality of the product but also reflect the quality culture of an organization is required to assist FDA in establishing a risk-based audit program. 

, Vol. 78, No. 29 (Feb. 12, 2013) 9928-9929.
  3.	PDA Annual Meeting, Apr. 7-11, 2014, Session titled Quality Metrics Update and Proposed Definitions.


Susan J. Schniepp is vice-president of quality and regulatory affairs at Allergy Laboratories.

Inside Standards

Industry players offer suggestions for quality metrics as FDA continues to try and solve the problem of drug shortages.

Linking Drug Shortages and Quality Metrics

Biopharmaceuticals are generally manufactured in established cell lines that are dominated by mammalian cells, particularly those based on Chinese hamster ovary (CHO) cells, but that also include 

 (yeast) cells. While these traditional cell lines are productive, their development can be slow and costly, and animal-derived systems provide non-human glycosylation patterns. Despite these difficulties, these traditional expression systems are generally used because they are approved by FDA, they are familiar, and there are established downstream purification processes for removing contaminants. Alternative expression systems, on the other hand, are being designed to avoid the performance issues associated with traditional cell lines. They offer biopharmaceutical manufacturers the chance to establish a stronger intellectual property position and improve their processes. New animal (including human), yeast, and plant-based expression systems have been shown to be effective for the production of biologics.

Human cells ideal for expressing human proteins


  Human cells present the proper background to express human proteins. They can add the correct posttranslational modifications to these proteins, according to Nicole Faust, senior vice-president of development and services with CEVEC Pharmaceuticals. “Having the correct posttranslational modifications is of particular importance when expressing complex glycoproteins; human cells only add modifications normally found in humans, while with rodent cells, foreign structures can be added to the proteins that are potentially immunogenic in humans, such as N-glycolylneuraminic acid,” she explains.

The use of human cells is also advantageous because any potential host-cell proteins (HCPs) present in the final preparation of the therapeutic protein will be of human origin and therefore present a far lower immunogenic risk than host-cell proteins derived from nonhuman cells. Such an immune response to contaminating HCPs has been observed with rhFIX produced by CHO cells, according to Faust. “Some human host-cell expression systems can also be used to propagate a variety of different human-pathogenic viruses, which extends the versatility of the expression host from recombinant proteins per se to relevant vaccines and viruses,” she adds.

Plant-based expression offers speed and simplicity


  With plant-based expressions systems, such as the transient expression system in whole green plants (Nicotiana benthamiana) from iBio, there is no requirement to identify, amplify, and adapt high-expressing cell clones to large-scale culture, saving a year to 18 months in product development time, according to Terence E. Ryan, iBio’s senior vice-president and chief scientific officer. With iBio’s system, production of protein can take place within 21 days of knowing a gene sequence, and this speed has attracted a lot of attention in the area of vaccine antigen production for pandemic diseases, particularly for influenza, according to Ryan.  

The process is also simple, with plant biomass grown over five weeks prior to vector infiltration. The plants are grown in hydroponic medium in a soil-free substrate under controlled conditions. Once the plants reach an appropriate size, the vectors are introduced by vacuum, delivering the vectors to all of the leaf cells at the same time. Once gene expression reaches its peak, the plants are ground and homogenized, allowing the desired protein to be purified by standard chromatography. “No bioreactors or staff skilled in aseptic cell culture are required, and thus this technology is highly attractive in areas of the world where capital and a highly skilled biopharmaceutical work force are in short supply,” Ryan says.  In addition, he notes that because the process is virtually the same regardless of the product (only the purification chromatography varies significantly), a single factory design can support multiple products, allowing for “campaign-style” production in one site.  

The speed and flexibility offers tremendous advantages, particularly at the early stages of development, by allowing product optimization and product risk mitigation in much less time and at much lower cost, according to Gene Garrard Olinger, a principal science advisor with MRIGlobal. He adds that in comparison with CHO, plant systems, specifically the rapid antibody manufacturing platform (RAMP) developed by Icon Genetics and Kentucky BioProcessing, offer the ability to control specific glycosylation patterns on the protein (1). “For antibodies, this specificity in glycosylation yields products with superior antibody-dependent, cell-mediated cytotoxicity (ADCC) profiles,” he observes. Ultimately, Olinger believes that with scale, the method should offer a significant cost advantage compared to cell-culture-based production.
  


  Plant-based expression systems are also valuable because plants are capable of producing just about every type of biopharmaceutical product, including vaccine antigens, enzymes, replacement proteins, monoclonal antibodies, and others, according to Ryan.  In addition, because no animal products are used in iBio’s technology, there is no risk of contamination by animal viruses or other adventitious agents. 

Meanwhile, the human amniocyte CAP cells developed by CEVEC, which are not derived from cancer cells or aborted embryos, are designed to efficiently produce complex proteins and glycoproteins at commercial scale, and therefore high product titers can be obtained even for difficult-to-express proteins, according to Faust. “We have found that in some cases, CAP cells have yielded 5- to 10-fold more protein than CHO cells. In addition, less or no proteolytic degradation of sensitive proteins was observed with the CAP system,” Faust observes. She adds that the cells are robust and easy to handle, and unlike traditional expression systems, the product quality is unaffected by small variations in the upstream process, making production runs highly reproducible. CEVEC has also developed a highly efficient transient protein expression platform (CAP-T cells) that is based on CAP cells and produces proteins with nearly identical post-translational modifications. “As a result, the transition from transient protein production in CAP-T cells at early project stages to stable production in CAP cells at later stages is very easy,” Faust says.

Further advances and demonstration of utility


  Going forward, Faust believes that an increasing number of genetically engineered cell lines that have a bias towards certain post-translational modifications will be developed. She also expects further improvements in expression levels of CEVEC’s human expression systems through optimization of vectors, the use of cells with altered metabolisms, and further improvements in chemically defined media.
  
  With respect to plant-based expression systems, Larry Zeitlin, president of Mapp Biopharmaceutical, expects rapid increases in yield to be realized. Zeitlin is hopeful that existing facilities such as the one at Kentucky BioProcessing, which can produce 10 kg of GMP protein per year, will be expanded to produce 100s of kg/year. “We also hope that the reduced capital costs to build plant manufacturing facilities will enable local production in developing nations to address their unique public health needs,” he says. Olinger adds that there is advancing work in tobacco with vaccines that use production of protein subunits to fully-formed, virus-like particles (VLPs), and he anticipates that with FDA approval of elelyso, which is derived from a carrot-based expression system, more plant-derived pharmaceuticals will be commercialized over the short and long term. 

Efforts at iBio are directed at demonstrating the utility of its expression systems in new biotherapeutic classes, which often requires the coexpression of heterologous proteins within the same cell to provide helper functions or enzymatic cleavages not normally accomplished by plant cells. “Many interesting biotherapeutics are naturally expressed as ‘pre-pro’ proteins whose active form is produced as a result of post-translational cleavage or other activation,” he explains. iBio’s long-term focus is on the recapitulation of the complete human glycosylation machinery so that plant proteins are identical at the glycoform level to human products, according to Ryan.

Collaborating to advance novel expression technologies


  One area that iBio is particularly proud of is the use of its technology to develop a next-generation yellow fever vaccine in partnership with the Fraunhofer Center for Molecular Biotechnology, the Oswaldo Cruz Institute (Fio-Cruz), and Bio-Maguinhos, with the latter two entities from Brazil, according to Ryan. “Bio-Maguinhos is the largest manufacturer of yellow fever vaccines in the world, and the perceived safety advantages of a recombinant subunit vaccine over the current (but 70-year old) live attenuated virus vaccine has spurred the development of the new vaccine,” he notes. In fact, the design for a manufacturing facility in northeastern Brazil capable of processing 1000 kg of plant material per week is in the second of three planning stages under sponsorship by the Brazilian government. “Through this project, we have received major endorsement of our technology and been able to demonstrate how quickly it can be recognized, adapted, and brought into manufacture,” Ryan concludes.
  iBio is also collaborating with Caliber Biotherapeutics (College Station, TX) to establish a turn-key plant-based biopharmaceutical development capability, from the earliest stage of product selection and optimization through large-scale production.

For CEVEC, the growing market for complex, glycosylated molecules is reflected in the increasing number of worldwide licensees using its CAP technology, according to Faust. In addition to several top pharmaceutical customers, the company recently signed a licensing deal with Yuhan Pharma, one of the larger Korean drug manufacturers. A clinical Phase I study with CAP cell-derived human alkaline phosphatase was successfully completed in Q4 of 2013 in the Netherlands, and several customer molecules are nearing clinical trials. CEVEC also achieved two major milestones by showing excellent viral yields for influenza and RSV in addition to licensing its CAP cell line for the exclusive production of a cytomegalovirus vaccine based on dense bodies. 


  1.	Zeitlin et al. PNAS 108 (51), 18030-18035 (2011).
  
  


  Cynthia A. Challener is a contributing editor to 

Expression Systems

New human and plant-based expressions systems can enable faster product development and improve quality at potentially lower costs.

Improving Protein Expression with Novel Systems

The Steam-in-Place (SIP) connector product offering from Colder Products Company includes genderless capabilities for use on hybrid systems. The combined genderless AseptiQuik G sterile connector and Steam-Thru II SIP connector gives biopharmaceutical manufacturers flexibility in process design and plant configuration and can simplify systems integration, the company reports. A new combination connector allows users to steam in place the AseptiQuik G connector directly to stainless processing equipment. The genderless portion of the connector supports modular closed-system design and allows the same bag system and tubing manifold SKUs to be used on both hybrid and complete single-use systems. The AseptiQuik G STC Connectors can be mounted directly to a stainless steel vessel via either a 3/4- or 1-1/2-inch sanitary termination on the Steam-Thru portion of the connector. AseptiQuik G STC Connectors are offered in both a standard version for gamma sterilization and a high temperature version for gamma orautoclave sterilization.

Integrated Single-Use Sterile and SIP Connector Provide Flexibility

In virus challenge studies, healthy volunteers are administered a pathogenic or virulent strain of virus. Such strains can be attenuated viruses that produce a much milder set of symptoms compared to the naturally occurring or fully active virus. If the volunteers are administered an investigational drug (e.g., antiviral, vaccine, immunomodulatory drug) besides inoculation with the virus, the studies are called viral-challenge studies. 
  
In a historical context, the concept of challenge studies is not new. The experiments conducted by Louis Pasteur in the 19th century, where chickens were challenged with a weakened bacteria causing chicken cholera and immunized from further chicken cholera infection, can be seen as a type of challenge study. In the early 20th century, scientists used a self-challenge approach when developing vaccines and drugs

Viral inoculation studies have been performed in the United Kingdom since 1946 when the Medical Research Council established the Common Cold Unit (CCU) (also known as the Common Cold Research Unit [CCRU]) at Salisbury, Wiltshire (1, 2). The aim was to undertake laboratory and epidemiological research on common colds in view of reducing human and economic costs. Common colds account for a third of all acute respiratory infections and the economic costs are substantial in terms of days off work. The volunteers were infected with preparations of corona- and rhinoviruses and were housed in small groups of two or three, with each group strictly isolated from the others during the course of the stay. In 1989, the CCU closed down after failing to find a cure. 

In current clinical research practice, the use of viral-challenge studies as proof-of-concept (POC) studies is gaining wider acceptance. Healthy volunteers are inoculated with a challenge strain of a virus, usually influenza, and administered a vaccine or antiviral before or after the inoculation. Although viral inoculation studies can be performed with a wide range of viruses, this article will focus on respiratory viruses and influenza in particular.


The vaccine or antiviral first goes through a complete non-clinical development program to assess its safety and efficacy. Afterwards, it goes through a full Phase I, first-in-man, pharmacokinetic (PK) and tolerability study.

The influenza virus is isolated by a combined nasal/throat swab from an ill patient. An aliquot of this clinical sample is then used to inoculate specific pathogen-free eggs (SPF), which are grown through sequential passages. Another option is to grow the virus through a cellbank. The virus is then manufactured under GMP standards and ensured that it is free of adventitial agents and other pathogens. A non-clinical program with the virus is then initiated (see 

To establish the correct viral dose for the viral-challenge POC study, in which the vaccine or antiviral drug will be administered, a dose-finding study in humans must first be conducted. The virus is administered to the study subjects using intranasal drops. The viral dose to be selected for the viral-challenge POC studies is the dose at which 80% of the inoculated volunteers show clinical symptoms. After the phase I study with the vaccine or antiviral and the viral-inoculation dose-finding study, sufficient information is available to start the POC studies.

Two possible study designs are used for the POC studies—treatment and prophylaxis. In treatment studies, the volunteers are screened and randomized. They are first inoculated with the challenge strain and after a given incubation period, the investigational vaccine/antiviral or placebo treatment will be initiated (either to both symptomatic/asymptomatic or to symptomatic volunteers alone). Patients are quarantined during the study if indicated, depending on the type of inoculum (3–5). 

In prophylactic studies, the volunteers are screened and randomized. They first receive the investigational vaccine/antiviral, or placebo treatment, followed by inoculation with the challenge strain. Patients are quarantined during the study if indicated, depending on the type of inoculum. Depending on the indication, study considerations, and objectives, one of the two designs, or a combination of both, may apply.

Pharmacodynamic (PD) endpoints in challenge studies usually include measurements, such as clinical respiratory symptoms, nasal discharge weight, and quantitative measurements of viral shedding, and/or cytokines in nasal washes.

Challenge studies can provide useful exposure-response and safety information and the opportunity to demonstrate pharmacological activity in humans under controlled conditions. Data from challenge studies contribute to dose selection for Phase IIb and Phase III studies, and provide the opportunity to explore the effects of different times of drug initiation relative to virus exposure. Challenge studies can be used to assess a first efficacy.

Required quality information on the virus


  The influenza virus to be used first needs to be isolated from a patient showing clinical signs of infection with the virus. The virus is usually isolated by nasal/throat swab. Given that virulence decreases with age, the virus is usually isolated from young patients. The reduced risk of co-infection and a better-defined medical history also make young patients the preferred host to isolate the virus from. In addition to evaluating acute respiratory illnesses of the patient and his family members, information on current and past medical history, travel history, and social history should be systematically recorded. The sample should be tested by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of the desired virus.

To avoid co-infection of the sample, the sample needs to be screened for the presence of other viruses. In the case of isolation of an influenza virus, the sample should also be tested by PCR for human rhinovirus, respiratory syncytial virus (RSV), parainfluenza virus types 1, 2, and 3, human metapneumovirus, and adenovirus, and the results should be negative. Plasma samples from the patient should also be negative for human T-cell leukemia virus 1 and 2, human immunodeficiency virus type 1 or 2 (HIV-1 or HIV-2), HIV ribonucleic acid (RNA), hepatitis B surface antigen (HBsAg), hepatitis B deoxyribonucleic acid (DNA), anti-hepatitis C virus (HCV) antibody, HCV RNA and Hepatitis A virus. The patient, from whom the influenza virus has been isolated, will be followed to assess that he remains in good health. 

An aliquot of this clinical sample will then be used to inoculate SPF eggs that are then grown through sequential passages or using mammalian cells. The number of passages will depend on the required infectious virus titre for preclinical (i.e., ferret model) and human testing. The virus should be manufactured according to GMP requirements.

Non-clinical testing
Non-clinical testing of a virus requires both 

 pharmacology studies. In-vitro pharmacology in cellular-based assays includes in-vitro infectivity, antigen characterization, and susceptibility to antiviral agents.

non-clinical testing is usually performed in ferrets. Ferret models (Mustela putorius furo) have been established for numerous viruses that cause respiratory infections, including human and avian influenza viruses, coronavirus, nipah virus, and morbillivirus among others. Ferrets are an appropriate mammalian model for these studies because they show numerous clinical features associated with human disease, such as fever, lethargy, and sneezing. In addition, sick ferrets have the ability to infect healthy ferrets. Ferrets and humans share similar lung physiology, and human and avian influenza viruses exhibit similar patterns of binding to sialic acids (i.e., the receptor for influenza viruses), which are distributed throughout the respiratory tract in both species. Furthermore, their small size eases the logistic burden (6–9). 

 pharmacology studies, infectivity and safety are tested. Temperature and body weight changes, clinical observations (e.g., sneezing), and infectious viral load are monitored. Safety pharmacology studies are not needed and no formal toxicology is needed if the profile of the virus corresponds with the characteristics described in the literature. No reproduction toxicology studies are needed if appropriate contraceptive measures (i.e., double barrier method: chemical and physical contraception) are used in the clinical studies.

The non-clinical testing of the vaccine or antiviral to be used in the viral-inoculation study should be performed according to standard non-clinical testing requirements described in the International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use (10–12).


The regulatory requirements for viral-challenge studies in humans can be divided in two parts, the requirements for the studies and the requirements for the infrastructure (Phase I units) where these studies are performed. In general, given that the concept is rather new, very little guidance exists around this type of viral-challenge/inoculation studies. There is no guidance on this subject provided by the European Medicines Agency (EMA) or any national European authority. An FDA guideline on the development of influenza drugs briefly mentions this type of study (13). 

In the viral dose-finding study, healthy volunteers are inoculated with a virus to establish the dose for the POC study. The dose-finding study is usually an open-label study in which several cohorts of volunteers receive an ascending dose of the virus until the optimal dose is found. The optimal dose is the dose that has the appropriate safety and illness/infectivity profile to be used as an influenza virus challenge strain in future challenge studies. For each strain, only one dose-finding study needs to be done. The strain (in the optimal dose) can then be used in multiple challenge studies. 

Whether or not this study can be considered a clinical study in the strict sense remains a question for discussion. Because the objectives of the stand-alone experiment are not in line with the definition of a clinical study as described in Directive 2001/20/EC article 2 (a) (14), the classification as a clinical study can be challenged, and the study is not considered a clinical study as per Directive 2001/20/EC. 

Viral-challenge studies, in this case, would be regarded as an “experiment,” which will only require approval from the ethics committee and not from health authorities. Such studies, however, should be approached with care because although the European directive does not consider viral-challenge studies to be a clinical study, national legislations may not agree and may consider them as clinical studies. 

Furthermore, there is the risk that the dose-finding study may have to be repeated if performed without health-authority approval, especially if the chemistry, manufacturing, and controls (CMC) data of the virus are considered insufficient by the health authority at the time of submission of the POC study. It is, therefore, highly recommended that dose-finding studies are considered as clinical studies. 

The POC study (in which the virus and a vaccine or antiviral is administered) will in any case be considered as a clinical study according to Directive 2001/20/EC. The virus, however, does not need to be considered an investigational medicinal product (IMP) because it does not match the definition of an IMP given in Directive 2001/20/EC, Article 2 (d) (14) and the Guidance on Investigational Medicinal Products (IMPs) and Non Investigational Medicinal Products (NIMPs)(15). 

If an inoculating virus is used to evaluate the efficacy of an investigational product, the inoculating virus is classified as a “condition” and not as an “intervention” (i.e., the disease or the health issue worth studying in a clinical study according to FDA classification at ClinicalTrials.gov) (16). According to Eudralex Volume 10, guidance on IMPs and NIMPs, revision 1, March 2011 (15), the inoculating virus could be classified as NIMP in the European Union. In this case, the inoculating virus is a “challenge agent.” A challenge agent by definition is usually given to study subjects to produce a physiological response that is necessary before the pharmacological action of the IMP can be assessed. A challenge agent may be a substance without marketing authorization, but could have a long tradition of clinical use.

Information regarding the quality of the inoculating virus should be provided in the non-investigational medicinal product dossier (NIMPD). There is no standard format available for presenting the information regarding an inoculating virus; however, guidelines on the requirements for quality documentation concerning biological investigational medicinal products in clinical studies, EMA/CHMP/BWP/534898/2008 (17), could be used as a reference. 

The quality information regarding an inoculating virus can be presented in the sections 2.1.S, 2.1.P, and 2.1.A of the NIMPD (18), similar to the quality documentation requirement for IMPs (14). It is not necessary to provide extensive information, similar to a biological drug used for marketing authorization. The NIMPD should mainly focus on the quality attributes related to safety aspects, considering the state of development or clinical phase. Appropriate GMP requirements should be applied (19-21).

The quality part of the NIMPD should include information related to the quality, manufacture, and control of the NIMP. It is preferable to present data in tabular form accompanied by a brief narrative highlighting the main points. The information that should be provided include virus isolation, manufacturing process, control of materials, including master cell bank and working cell bank systems, control of inoculating virus (i.e., quantity, identity, and purity), analysis, and stability, among others. In section 2.1.A.2 of the NIMPD (adventitious agents safety evaluation) (22), information assessing the risk with respect to potential adventitious agents and other human pathogens contaminations should be provided (guideline on virus safety, EMEA/CHMP/BWP/398498/05) (23).

Another important issue is the regulatory and operational aspect of running the viral-challenge study itself. It is extremely important to avoid cross-contamination between the patients infected with the virus on one side and the study staff on the other side. Furthermore, a back-up plan to treat a patient with an antiviral agent or other drugs should he or she become too ill after the challenge should be available. This issue is equally true for the virus dose-finding study as for the challenge study together with the vaccine. The aim is to avoid the virus from being spread to the “outside world” and to avoid infected study staff to infect the patients and jeopardize the study results by infecting placebo patients.

Patients will need to be isolated in a specifically designed quarantine unit and will be treated according to the principle of “reversed-barrier nursing.” This method is comparable to barrier nursing used in an intensive care unit (ICU) setting, where the aim is to keep pathogens away from the ICU patients by creating a barrier between the outside world and the inside of a patient room by using gloves, masks, gowns, and disinfectants. With reversed-barrier nursing the aim is to keep the challenging agents confined in the facility using the same principles.


Recently, viral-challenge studies have become widely accepted as POC studies to demonstrate the efficacy of antiviral and vaccine therapeutics for RSV, influenza, and other common cold viruses. The regulatory framework for this type of studies has not been fully developed. The exact regulatory requirements for viral-challenge studies need to be discussed with the health authorities of the country where the study will be performed.

Due to their nature, viral-challenge studies can only be performed in specialized clinical pharmacology units. Conducting these studies in a controlled quarantine environment allows for a superior study design, which is more cost-effective. This approach critically accelerates the selection of a safe and effective dose and dosing regimen for a new antiviral drug or vaccine because it allows for early detection of efficacy. It, therefore, lowers the risk of a negative outcome when performing a large field-based Phase III study. 

Viral-challenge studies are performed under tightly controlled circumstances, and such studies do not seem appropriate to replace large field-based trials in “real-life” circumstances. In this respect, the use of viral-challenge studies in the framework of vaccine or antiviral drug development needs to be discussed further with regulatory authorities. 

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3.	J.A. Maher and J. DeStefano, 

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Review regulatory requirements and the use of viral-challenge studies in drug development.

BioPharm International-05-01-2014

Accelerated Stability Modeling

Demand for New Vaccines Spurs Innovation

Standardizing Practices for Disposables

Novel Vaccine Technologies Meet the Need for Pandemic and Therapeutic Solutions

Clinical Syringe Packages Increase Productivity

Linking Drug Shortages and Quality Metrics

Improving Protein Expression with Novel Systems

Integrated Single-Use Sterile and SIP Connector Provide Flexibility

Regulatory Requirements for Viral-Challenge Studies: Influenza Case Study

India's Developing Market Offers Opportunities

Stuck in Neutral

Biopharma Takes a Nervous Glance Over Its Shoulder

Aseptic Valve Range Increases Sterility

BioPharm International, May 2014 Issue (PDF)