GE’s Sievers M9 Total Organic Carbon (TOC) Analyzer is designed to measure TOC in a range of samples from ultrapure water to process waters. GE offers the Sievers M9 TOC Analyzer in three versions: portable, online, or laboratory. The M9 Analyzer increases productivity by producing TOC results in two minutes. According to the company, the M9 enhancements promote simple operation and optimal data utilization. In addition, the M9 Analyzer can measure conductivity in grab sample or autosampler modes, an upgrade from the 900 TOC Analyzer series. 

Articles

Products

TOC Analyzer Increases Productivity

Several recent developments are leading to an increase in adoption of single-use technology in downstream processing, and more progress can be made, but it is important for providers to offer the right technologies at the right scale, according to Helene Pora, vice-president of single-use systems with Pall Life Sciences. “As a whole, suppliers are developing single-use technologies that are continuous in nature, and these advances have helped the adoption of single-use technologies in downstream applications,” Derek Masser, sales director with ASI-Life Sciences adds.

“In reality, however, areas such as tangential flow filtration (TFF) and chromatography have not seen a great deal of development in terms of single-use, ready-to-use, integrated models,” notes Pora. “There is still a lot to prove regarding reliability, particularly at large scale, although Pall has made the first steps with the introduction of ready-to-use gamma irradiated TFF modules and a single-use platform that offers cross-functional downstream solutions for running depth filtration, virus filtration, virus inactivation, and polishing applications with membrane chromatography.”

EMD Millipore also offers fully-automated single-use TFF and chromatography systems that have been designed for both operational flexibility and scalability, according to Willem Kools, head of field marketing and the biomanufacturing sciences network. “The modular system can be used for both TFF and chromatography with a minimum number of component changes, reducing both capital investment and product development times, and as a result, has attracted attention from contract manufacturers and biopharma companies installing pilot-plant operations,” Kools says. EMD Millipore has also introduced new high-performance single-use process consumables, including clarification filters and flocculants for handling cell removal and initial purification, as well as pre-packed columns.

Additional column sizes are also becoming available, and some vendors are now willing to work with any resin type, which is increasing the flexibility component for chromatography, according to Christine Gebski, product manager for bioproduction at Thermo Fisher Scientific. She adds that customers are also mapping the total time spent on the actual supportive (non-producing) workflow associated with column packing and testing and are realizing the potential efficiency gains associated with moving to pre-packed columns. Thermo Fisher has developed high-performance resins that increase efficiency, throughput, productivity, and costs for downstream processing and pre-packed, limited-use columns for more rapid validation and pilot scale-up studies and early-phase clinical manufacturing.

ASI has introduced a disposable heat exchanger for use in continuous downstream processes where jacketed systems are currently employed. The exchanger can be easily set up and integrated and, in addition to providing more efficient and continuous heat transfer for accurate temperature control, it also helps reduce bioburden concerns, according to Masser. 

Masser also notes that recent advances in single-use sensing technology have made it possible to provide individualized sensing capabilities that are beneficial for downstream manufacturing because the sensors are individualized to the process. In addition, new bioprocessing films with reduced extractable and leachable profiles are also helping biopharmaceutical companies meet regulatory requirements, according to Millie Ullah, senior product manager for single-use technologies with Thermo Fisher Scientific.

In 2013, Pall expanded its product offerings in purchasing Medistad Holding, SoloHill Engineering, and the LifeSciences business of ATMI, while Thermo Fisher acquired Life Technologies.

Downstream Processing

Industry suppliers describe new technologies to facilitate downstream processing.

A Sampling of New Tools for Downstream Processing

, which is celebrating its 50th anniversary in 2014, has a worldwide reputation for its monographs on APIs and excipients. Owing to its close collaboration with European regulators, the 

 is well aligned with regulatory developments and needs, and reflects state-of-the-art technologies and requirements. 

 includes amongst its texts a wide range of general dosage-form monographs and has taken measures to stay abreast with scientific and technical developments, for example, by fostering the implementation of quality-by-design principles and relevant modern analytical technologies, the Ph.Eur. Commission has been somewhat reluctant in the past to initiate extensive development of specific dosage-form monographs. Until recently, such monographs have only been established in certain specialized areas (e.g., for vaccines, blood products, insulin, and radiopharmaceuticals).

It has to be acknowledged, however, that there is a clear demand for these monographs. They are needed by official medicines control laboratories for their market surveillance studies; they support and facilitate the development of generic drugs, which are crucial to ensure the sustainability of today’s healthcare systems, and they may also be useful to regulatory authorities by facilitating the assessment of respective quality dossiers. Based on the feedback received from stakeholders in the context of its tri-annual scientific conference held in Prague in October 2010, the Ph.Eur. Commission has reviewed its policy. Following intensive discussions with the European Medicines Agency’s Joint CHMP/CVMP Quality Working Party, the Ph.Eur. Commission decided in 2012 to initiate a pilot phase on the feasibility of finished product monographs. A dedicated working party composed solely of representatives of licensing authorities and official medicines control laboratories was set up and assigned two tasks: to elaborate draft monographs on two finished products, one for which the API is still under patent in Europe (a so-called “single-source” product) and a second one for which generic drugs are already on the market (“multi-source” product), and to draft a guidance document to explain the rationale for finished product monographs and how to use them, intended for both regulators and the industry. 


  Since then, the pilot phase has concluded and the outcome was presented to the Ph.Eur. Commission for approval at its March 2014 session. Based on the commission’s unanimous decision, the draft monograph on sitagliptin phosphate tablets--a text elaborated by the working party in close collaboration with the innovator—is now published in 

 free online-forum, for public comments (1). For this monograph, like all other monographs based on a single-source product, broad public consultation is even more important than for multi-source products to ensure that the resulting acceptance criteria and test methods are indeed robust and applicable to a wide range of products. During the elaboration of the finished product monograph, a monograph for the active substance sitagliptin phosphate monohydrate was also drafted, for which the consultation period ended in March 2014. 

It is the Ph.Eur. Commission’s policy that each finished product monograph, taken as a whole, should provide a reliable basis for making an independent judgement as to the quality of the preparation. Finished product monographs will cover different formulations and strengths (whenever possible) of the same dosage form containing the same API. The Ph.Eur. Commission, as it does for all its API and excipient monographs, will only elaborate monographs on products that have been authorized in at least one of its member states and that contain an API for which a monograph has already been published in the 

 or which is on the work program. As for all monographs, the elaboration and revision of finished product monographs will be subject to public consultation and take into account current scientific knowledge and relevant medicinal products authorized at the time.

In addition, finished product monographs, like all other API and excipient monographs, while legally binding and reflecting the state of the art, are not intended to be a “regulatory straitjacket” that stifles innovation. In line with the requirements for all compendial APIs, a full dossier will need to be submitted to the licensing authorities for a finished product covered by a 

 monograph, detailing its composition, pharmaceutical development, manufacture, control, container/closure system, and stability. As analytical procedures may be affected by the presence of excipients and/or the manufacturing process selected, demonstration that the testing procedures described in a finished product monograph are suitable for the specific product will need to be included in the marketing authorization dossier. However, this is not an unusual requirement as any applicant referring to a compendial API has to demonstrate that the pharmacopoeial monograph is indeed capable of adequately controlling the quality of the substance, manufactured according to the submitted route of synthesis. 


  The EU pharmaceutical legislation, in directive 2003/63/EC detailing the requirements of marketing authorization applications (2), already provides for a feedback mechanism between regulators and pharmacopoeia authorities in case a compendial product is not adequately controlled by the respective monograph. This provision allows assessors to make sure that the specifications for a given product are indeed adequate for the specific purpose and provides the Ph.Eur. Commission with valuable information on the need to review and potentially revise the monograph. In addition, the 

 itself in its general notices offers much flexibility to users, from the notion of alternative methods—provided they lead to the same “pass/fail” result and are approved by the competent authority—to the possibility of using modern control strategies, including parametric release and real-time release testing or even using manufacturing flexibility, for example, that is allowed by the design space. 

While the pilot phase clearly demonstrated the feasibility of developing monographs for both single-source and multi-source products, the Ph.Eur. Commission felt it would be prudent to gather more experience with single-source products and has decided to start with the elaboration of single-source monographs on products that are potential future generic drugs. This does not, however, exclude the possibility of also including multi-source products in the work program. Such a decision will be based on a critical assessment of the usefulness of having a 

 monograph and its impact on products already on the market. 

While the decision of the Ph.Eur. Commission to be more active in the field of finished product monographs will help to further broaden the scope of the 

, it is not a change in paradigm. The same general principles for monograph elaboration will be applied as for API and excipient monographs to ensure they are of the same high scientific quality. In addition, public consultation will provide feedback from a wide range of stakeholders and contribute to the texts’ usefulness and adequacy. 

Based on the promising outcome of the pilot phase and its decision to transform this into a routine work program, the Ph.Eur. Commission is looking forward to its future collaboration with sponsors who are interested in contributing to the elaboration of finished product monographs in the 

,  accessed May 9, 2014.
  2.  European Commission, Commission Directive 2003/63/EC, 


Susanne Keitel, PhD, is head of the European Directorate for the Quality of Medicines of the Council of Europe. EDQM is responsible for the 

Inside Standards

The European Pharmacopoeia Commission re-evaluates its policy on the development of monographs for finished drug products.

Finished Product Monographs in the European Pharmacopoeia

Few FDA officials discuss pharmaceutical regulation these days without reference to how the international reach of the biopharmaceutical industry has altered drug research, production, and regulation. At the annual meeting of the Food and Drug Law Institute (FDLI) in April 2014, FDA commissioner Margaret Hamburg concluded her keynote address by describing a “dramatically changing global marketplace” and its “huge implications” for FDA’s ability to ensure the safety and quality of products manufactured elsewhere. To better assess product risks, Hamburg proposed “enhanced intelligence” and more collaboration with regulatory partners through bilateral and multilateral agreements and through international organizations. 

In addition to conducting more inspections and oversight of foreign manufacturers, FDA is working with international organizations to communicate its policies and standards more clearly and to enhance regulatory capacity in emerging countries. In a March 2014 lecture to the United Kingdom’s Medicines and Healthcare Products Regulatory Agency (MHRA), Hamburg described efforts underway to form an International Coalition of Medical Regulatory Authorities (ICMRA), which would work to coordinate and enforce regulatory standards to ensure product safety and quality around the world. 

At the same time, FDA and the European Medicines Agency (EMA) annouced plans to expand their many collaborative programs, including renewal of a pilot program that allows for parallel review and consultation on quality-by-design (QbD) components to new drug applications and supplements, an initiative designed to provide consistency and clarity to QbD policies. 

At a London meeting in April, senior leadership from FDA, EMA, and the European Commission reviewed efforts to leverage each other’s inspection resources; collaboration to support global development of biosimilars; pediatric cluster meetings to encourage more common policies for pediatric studies and applications; information exchange by the EMA-FDA pharmacovigilance cluster; and broader cooperation related to veterinary medicines. The participants also discussed the creation of a EU-US Identification of Medicinal Products task force to explore adoption of US substances registration software in the EU, with an eye to forming a global identification system for medicinal products. 

And last month, Howard Sklamberg, deputy commissioner for Global Regulatory Operations and Policy (GO), Janet Woodcock, director of the Center for Drug Evaluation and Research, and Karen Midthun, director of the Center for Biologics Evaluation and Research, unveiled a new initiative to collaborate more with EMA and European governing bodies to enhance pharmaceutical quality. FDA will be represented by a European-based team, with the aim of moving beyond information-sharing to “strengthen mutual reliance” on trusted regulators outside the US.


At the MHRA meeting, Hamburg commented on her visit to India in February and FDA efforts to expand inspections and develop stronger ties with local regulatory authorities. The commissioner acknowledged that FDA has imposed import alerts on multiple drugs produced by leading Indian pharmaceutical companies such as Wockhardt Ltd. and Ranbaxy Laboratories, but emphasized that FDA was not “targeting” Indian manufacturers, but applying consistent inspection and quality evaluation standards to all products imported into the US. 

FDA is expanding its presence in China even more to monitor the rising volume of APIs and medical products produced for US markets. At a hearing before the US-China Economic and Security Review Commission, held in Washington, D.C. in April 2014 to assess the safety of medical products imported from China and other health-related trade issues, this Congressionally appointed panel queried experts on the difficulties faced by FDA and manufacturers in ensuring the quality of Chinese drug products and components. 

Christopher Hickey, the director of FDA’s China office, reported on increased FDA inspections in China—up to 84 in 2013 from 46 in 2010. Hickey acknowledged that his office has only two full-time field inspectors, but has plans to double FDA’s China staff, pending resolution of delays in obtaining necessary visas. He also noted formation of an FDA-China working group on economically motivated adulteration of medical products. 

With some 4000 establishments producing medical products and substances intended for the US, however, Commission members concluded that FDA’s operation seems “woefully, inadequately staffed” for its job. Numerous API samples are falsified or substandard, according to research by the American Enterprise Institute. Alan Coukell of The Pew Charitable Trusts described rising concern over reliance on Chinese sources for key ingredients in older pain-relief medicines and antibiotics. He noted that more frequent FDA inspections of foreign drug establishments are important in prompting manufacturers to meet quality measures and agreed with Hickey that manufacturers of finished drugs need to do more to monitor suppliers and contractors. 


While inspections and import alerts attract public attention, FDA also emphasizes its role in China and other regions in educating public officials and industry leaders on US policies and standards. Sklamberg’s GO organizes a range of capacity building programs and collaborative activities designed to strengthen regulatory systems in developing countries and to explain US and international manufacturing policies and standards to local authorities.

Similarly, leading trade and professional organizations are broadening international outreach and educational programs to support these efforts. The Parenteral Drug Association (PDA) has a European subsidiary and chapters around the world, including a new one in Singapore. These entities hold conferences and training courses on quality manufacturing and testing, as seen in a program this month on parenteral manufacturing in Istanbul. 

The Drug Information Association (DIA) recently established its global headquarters in Washington, D.C. to better serve 18,000 members in 80 countries through operations in Europe, Japan, India, China, and Latin America. DIA’s chief executive, Barbara Lopez Kunz, came from Battelle with strong international management experience to oversee DIA’s worldwide growth, which will be highlighted at its 50th annual meeting this month. 

The International Society of Pharmaceutical Engineers (ISPE) is unveiling a Drug Shortages Prevention Plan this month, along with plans to collaborate on a similar proposal for the EMA. ISPE and PDA are working with European associations and regulators to craft a plan that addresses shortage-related quality issues relevant to the European market. 

Global expansion has been a prominent theme at the United States Pharmacopeia (USP), which now has offices and laboratories in India, China, and Brazil to provide local manufacturers with access to reference standards, test methods, and training programs on correct procedures for testing and ensuring product quality. USP’s Promoting the Quality of Medicines initiative, which is funded by the US Agency for International Development, also assists manufacturers in developing nations produce medicines that meet quality and safety standards; a recent five-year extension of the program will provide up to $75 million for USP to provide technical assistance to government regulatory systems in establishing domestic quality control laboratories, quality monitoring systems, and effective domestic regulatory authorities. 

Efforts to harmonize pharmacopeial standards in different regions over several decades have been stymied by differences in legal authority and traditional practices in different countries. Pharmacopeial harmonization now is shifting to a more prospective approach, working with the World Health Organization to develop common testing practices and standards-setting processes. 

These activities will be on the agenda at the next USP convention in April 2015, which will set the stage for USP programs through 2020, when the organization marks its 200th anniversary. To prepare for these and other challenges, USP’s new chief executive officer, Ron Piervincenzi, is consulting with stakeholders, reviewing the organization’s operations, and examining options for future growth and change.

 Washington editor, tel. 301.656.4634, jwechsler@advanstar.com. Read Jill’s blogs at 

Regulatory Beat

Regulators and industry organizations explain policies and standards to manufacturers and authorities in all regions.

Global Expansion Shapes Drug Oversight

There is a clear link between a country’s rate of economic development and the strength of its intellectual property laws. This is particularly true in knowledge-intensive sectors such as biopharmaceuticals.

The good news is that some mature and emerging economies are making growing use of patent systems to facilitate biotechnology research and commercialization. The bad news is that a number of countries have established bureaucratic and burdensome hurdles to patentability. This reality stands to harm innovation everywhere.


The Biotechnology Industry Organization (BIO) is concerned over a series of patent revocations including a decision by India’s Supreme Court to deny patent protection for a novel therapy on the grounds it did not demonstrate enhanced efficacy. Furthermore, Brazil’s unprecedented review of patents by the health regulatory authority, China’s increased data requirements, and Canada’s increased utility requirements all ultimately undermine the drug development and patenting process and may impede the delivery of novel medicines to market.

Despite the 2010 declaration by the then-President of India that the next 10 years will be a “Decade of Innovation,” the use of a compulsory license, a series of patent revocations, and weak enforcement efforts raise serious concerns about India’s commitment to promote innovation.

The tragedy underlying this anti-intellectual property position is not simply that it diverts attention away from the real problems of access to health-care that millions of Indians face, but that it undermines the nation’s goal of becoming a healthcare and science innovator. There are companies around the world interested in collaborating in research, science, and the development of medicines in India. India’s anti-intellectual property position makes it difficult, and often impossible, for such deals to happen. 

It is imperative for governments that want to foster a robust biotech innovation environment to create a set of strong intellectual property standards--particularly those governing data protection, patents, and trade secret protection--that are relevant to biological products.


The biotechnology industry is a dynamic, job-creating industry and presents opportunities for every country. Indeed, the vast majority of biotechnology companies are small- and medium-sized enterprises. What these companies share is a philosophy that is crucial to the task of developing biotechnology products--a willingness to take huge risks and invest private capital in development of new technologies that will lead to new products and services that will improve people’s lives. 

With no source of product revenue and long development times before market launch, biopharmaceutical companies must leverage the strength of their global patent portfolio to raise the large amounts of capital required to get their innovations to the market.

Upholding the system of intellectual property rights is essential to guaranteeing future innovation and future jobs not just for US biopharmaceutical companies, but also for other countries and other industries around the world. 
  BIO believes that strong intellectual property laws create an environment that promotes collaboration and innovation across borders. We will continueto work around the world to champion intellectual property rights as a vital 
  means of creating jobs, saving lives, advancing global economic growth, and generating breakthrough solutions to global challenges.

The 2014 BIO International Convention, taking place June 23-26 in San Diego, will host an Emerging Opportunities in Global Markets Forum specifically designed to highlight biotech initiatives in global markets, looking at both emerging economies and global initiatives and trends that affect the industry’s landscape. The Forum will overview developments in key markets, cross-cutting policy trends, unique collaborations, and cross border initiatives to further innovation. To learn more, visit 


Jim Greenwood is president and CEO of the Biotechnology Industry Organization (BIO).

Guest Editorial

Protecting intellectual property rights is vital to biopharmaceutical innovation.

Bureaucratic Roadblocks Threaten Biopharma Growth

Biopharmaceutical companies have traditionally considered outsourcing as a way to control costs and manage their internal resources to make the core activities left in-house more efficient. In recent years, surveys of biopharmaceutical manufacturers by BioPlan Associates have shown that cost cutting is, by far, not the primary reason companies outsource anymore.  In fact, this factor ranks far below other measures. New data from the 

11th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production

 (1) indicate that cost control is becoming less of a driver of outsourcing activity. This reduction is due in part to outsourcing becoming more highly interwoven with higher-end, technical activities for which the right partner is more important than an extra dollar saved. This trend also dovetails with evidence of a significant expansion in planned funding for outsourcing activities.

As part of the study, BioPlan evaluated how companies are addressing cost issues in biopharmaceutical manufacturing through their actions. As with the past three years, the most common action being taken by respondents was to implement programs to reduce operating costs. This action was done by 70.3% of the more than 200 global respondents to this year’s study. The numbers are consistent with last year’s study, though down slightly from prior years, when budgets may have been a little tighter. 

Other common actions taken by companies to cut costs include reducing process development times and costs, implementing operational excellence programs such as Six Sigma, and implementing “lean manufacturing” programs, each by more than 4 in 10 respondents. Against that backdrop, the responses found specific to outsourcing were much less significant. In fact, of the 19 actions identified, the five specific to outsourcing were among the six least-taken. What’s more, after a couple of years of increases, the percentage of respondents this year having used outsourcing as a cost-cutting mechanism was for the most part either flat or down from last year’s study, for example:  

After doubling from 7% of respondents in 2011 to 14% last year (2), the share in this year’s respondents outsourcing manufacturing to domestic service providers specifically to cut costs fell to below 10%. 

After more than doubling from 5.7% of respondents in 2011 to 12.6% last year, the percentage of respondents in this year’s study who off-shored manufacturing to cut costs was essentially flat, at 13.1%. 
  

Those trends weren’t unique to outsourcing of manufacturing. Fewer respondents reported having outsourced jobs in process development and R&D to cut costs, while the percentage that outsourced jobs in manufacturing remained flat after two years of increases. These results indicate that while outsourcing can, and does, play a role in cost containment, it is no longer a top priority when facilities are looking to cut costs. 

Fewer clients evaluate CMOs on their cost


  The data concerning cost-cutting changes specific to outsourcing is particularly interesting in light of separate results from BioPlan’s study where we evaluated the importance of various attributes that clients consider when they partner with CMOs. When companies were asked to rate the importance of 19 attributes when considering outsourcing to a CMO, only 22% this year said that cost was “very important.”  Essentially, CMOs are less expected to demonstrate the cost-effectiveness of their services. We found this attribute in the bottom quartile of the list. 

That is a significant departure from last year’s study in which this attribute was in the top third—cited by almost twice as many respondents (42%) as being very important. Additionally, it was one of only a few attributes that saw a decrease in perceived level of importance this year relative to last, and the 22% of respondents seeing it as such was the lowest share seen in BioPlan’s annual studies going back at least to 2006. 

What could be the cause of this change? It’s possible that the biopharmaceutical community is increasingly looking at CMOs as partners that can provide highly-specialized equipment, expert technical knowledge and flexibility beyond what is available in-house. That’s especially the case when companies look to outsource higher-value activities, such as process development or quality-by-design services. From that context, selecting a CMO is less about going with the lowest bidder and more about finding the right fit from a technical and relationship standpoint. 


  Another reason why biomanufacturers may be paying less attention to outsourcing from a cost standpoint is that their budgets for outsourcing are expanding relatively quickly. The BioPlan study asked respondents to consider the current economic situation and forecast to what degree their funding would change over the next 12 months for 12 separate areas. In comparison to areas such as new capital equipment (where budget increases are not keeping pace) or process development, budgets for outsourcing of biopharmaceutical manufacturing are growing, year-on-year, far more quickly than other areas (see 

Overall, the study reveals that more than one-third (37.3%) of respondents are projecting an increase in their funding of outsourced manufacturing to some degree. As a result, on average, it is estimated that budgets for outsourced manufacturing will increase by 3.9% this year. That’s a marked uptick from budget projections made over the prior 5 years, which ranged from a low of -1.3% to a high of 1.7%. 

Other results from the study confirm this shift. Separately, respondents were asked to indicate how their spending on outsourcing will change over the following 12 months in manufacturing or R&D. Budget enthusiasm was even more pronounced: Almost half (49.9%) of respondents (vs 43.7% last year) said that their spending on outsourcing of R&D or manufacturing would increase to some extent over the next 12 months 

Based on these data, BioPlan estimates that, on average, budgets for outsourcing at individual facilities will increase by 15.6% over the next 12 months, compared to estimated increases of 10.4% last year and 9.3% the year before. 


  A common complaint made by CMOs in past editions of BioPlan’s industry study has been that clients want to contain costs by doing limited development runs, but still expect successful full-scale manufacturing. Biomanufacturers’ focus on cost-cutting has no doubt affected their interactions with CMOs, and relationships can easily be strained when clients are looking to cut corners. 

Data indicate that these concerns might ease this year as clients take a more balanced approach to outsourcing that values benefits beyond simple cost containment. This is likely a reflection of financial situations, which have eased somewhat after the dire conditions seen a few years ago. It remains to be seen whether or not clients will retain this new approach to outsourcing when difficult times strike again.

 (Rockville, Md., April 2014).
  2.	BioPlan Associates, 

10th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production


  Eric Langer is president of BioPlan Associates, tel. 301.921.5979, elanger@bioplanassociates.com, and a periodic contributor to 

Perspectives on Outsourcing

With budgets growing, clients see CMOs' costs as less crucial.

Outsourcing No Longer Just for Cost-Cutting

The manufacture of biopharmaceuticals presents some unique challenges when ensuring product quality and patient safety. Analytical testing can provide the data needed to produce a safe and effective drug product. The development and validation of analytical methods is crucial in drug development. While the biopharmaceutical industry has had success in reducing risk by incorporating analytical platform technologies for monoclonal antibody products, new molecules and drug conjugates pose additional challenges, according to Stephan Krause, principal scientist, analytical biochemistry, at MedImmune. 

The extended drug development timeline of biopharmaceuticals also poses a challenge. According to Yong Guo, professor of pharmaceutical sciences at the School of Pharmacy, Fairleigh Dickinson University (FDU), analytical development and cross-functional development teams should work together to anticipate changes when creating the development timeline. Alice Krumenaker, manager, QC Stability Administration, at West-Ward Pharmaceuticals, suggests that an analytical method may need to be modified and/or updated during the drug-development process to minimize inaccurate data, and the modified method may need to be revalidated. 

Technologies, such as FDA’s quality-by-design (QbD) initiative, may have a positive impact on analytical method development and validation according to Paul Smith, EMEAI laboratory compliance productivity specialist at Agilent Technologies, because methodologies are identified early in the development process.
  
  

 spoke with Krumenaker, Krause, Guo, and Smith about the challenges involved in analytical method development and validation in biopharmaceutical manufacturing. 

What are the challenges involved in analytical method development and validation for biopharmaceutical manufacturing? 

The use of analytical platform technologies for common product types, such as monoclonal antibodies, has lowered the uncertainty/risk for the manufacturer; therefore, less development, qualification, and/or validation work is required.  More challenges typically exist for new types of molecules and/or conjugate products. For example, patient-specific cancer vaccines require conceptually a different approach in that some (routine) test methods should test for non-patient-specific manufacturing (batch-to-batch) consistency versus those that are patient-specific critical quality attributes. A direct potency test method may not exist, and instead, several surrogate test methods may need to be used for potency. Obviously, whenever new test methods are to be used throughout product development, more focus should be on the development, optimization, and validation of these new methods.
  

As in any drug-development program, there are, of course, many challenges involved in analytical method development and validation for biopharmaceuticals.  I would like to mention one particular challenge to analytical development. Often times, the development timelines are compressed for various reasons; however, insufficient consideration is given to analytical method development and validation. People expect the analytical methods to be ready all the time regardless of timeline or resource changes. To handle this challenge, the analytical development team needs to be closely working with the cross-functional development team to anticipate the changes and actively participate in any relevant discussion regarding the timeline. It is also important for analytical development to educate and work with the project managers to be more visible during the development process.

 How is analytical method development incorporated into the long development timeline of biopharmaceuticals? Can methods be changed mid-stream? At what point should analytical methods be validated?

Method development and validation are typically done in the product development stage. While the method may go through various iterations during product development, as the final formulation emerges, the method will be optimized and ready for validation. The analytical method will become part of the submission package and, once a submission is approved, there is usually great resistance to submit changes to FDA. So at this point, it is important to have confidence in the method and its ability to be transferred from R&D to quality control or, in some cases, to a contract organization that will be doing the testing. A successful validation will provide this level of confidence. 

As far as making changes to the method goes, if the changes are necessary, they must be made. Sometimes, changes to a process, necessary reagents no longer being manufactured, improvements in technology or other circumstances out of the lab’s control may result in a method no longer being suitable as written and/or as validated. Depending on the extent of the changes, some form of revalidation is likely to be needed. This may range from a simple verification, demonstrating that the method still performs as intended, to a full-blown validation for significant changes. In addition, this may impact the regulatory submission; however, the bottom line is if the original method isn’t accomplishing what it needs to accomplish, it needs to be modified and updated to minimize the possibility of inaccurate results. In that event, all appropriate amendments must be made to the original submission. 

The timing and effort spent on method development correlates with the degree of novelty of the product type and manufacturing process.  The sooner methods are optimized, the lower the risk will be to overall product development.  

Methods can be changed any time during and/or after product development. The change to faster, more sensitive, accurate, and/or reliable test methods is encouraged by the regulators.  Besides providing sufficient qualification or validation results for the new method, method comparability results should also be provided. In some cases, product specifications may need to be re-evaluated and potentially adjusted to the established bias and/or increased sensitivity of the new method.  

Regulatory expectations vary a little bit depending on product type and/or quality attribute tested.  For example, for blood or blood plasma products, validation of potency and some safety test methods is expected much earlier (clinical Phase 1). For most other products, method validation is typically executed against the to-be-commercial specifications prior to process validation, which is typically initiated during the pivotal clinical phase. Method validation is usually completed one to two years prior to commercial license application(s), depending how much real-time stability data are desired following the process qualification runs (process validation stage 2).

The analytical methods need to be validated for any GMP activities. This is a GMP requirement as well as FDA expectation. The analytical methods should be properly validated even to support Phase I studies. Appropriate approaches should be considered to validate the analytical methods to support different clinical phases. The concept of ‘phase appropriate validation’ has been proposed and applied to method validation.

What regulatory parameters exist for analytical method development and validation for biopharmaceuticals?

A FDA draft guidance for development and validation was recently made available for public commenting (1). Biopharmaceuticals are in the scope of this draft guidance. A more comprehensive practical guidance, Technical Report 57, published by the Parenteral Drug Association (PDA) and specific for biopharmaceuticals, provides the best practical guidance currently available to industry and regulators (2). FDA used much of PDA TR 57’s content for their current draft guidance.

 The International Conference on Harmonization (ICH) has a general guidance on method validation, 

ICH Q2(R1): Validation of Analytical Procedures: Text and Methodology

 (3). This is the guidance that the industry generally follows in performing analytical method validation. The ICH guidance, however, does not specifically address method validation for biopharmaceuticals. Interestingly, FDA issued a new draft guidance for industry on 

Analytical Procedures and Method Validation for Drugs and Biologics

 early this year (1). The new draft guidance supersedes the 2000 draft guidance for industry on 

Analytical Procedures and Methods Validation

, and covers both small-molecule drugs and biologics. In comparison to the 2000 guidance, the new guidance specifically addresses analytical method development and suggests the parameters that should be evaluated during method development, including specificity, linearity, limits of detection (LOD) and quantitation limits (LOQ), range, accuracy, and precision. The robustness of methods is particularly discussed in the new draft guidance, and a systematic approach for method robustness study (e.g., design of experiments) should be adopted to ‘fully understand the effect of changes in method parameters on an analytical procedure’ (1). The new draft guidance refers to the ICH guidance Q2(R1) for more details on each method validation characteristics and also removes the recommended validation characteristics for various types of tests.

For the qualification of new reference standards, the new draft guidance recommends a two-tiered approach, which involves ‘a comparison of each new working reference standard with a primary reference standard so that it is linked to clinical trial material and the current manufacturing process’ (1). The new draft guidance does not address specific method validation recommendations for biological and immunochemical assays.

How can method validation benefit from a QbD approach?

For a QbD approach for analytical methods, the analytical method lifecycle stages should consider the desired method performance at each product development stage. Starting with the appropriate, pre-established method performance expectations (Analytical Target Profile), the selection of the test methodology and instrumentation are the first step. Depending on the intended use of the method, typical performance criteria (accuracy, reliability, specificity, sensitivity, range, robustness/maintenance, as well as speed and throughput capacity, but possibly also costs and ease of operations) should be established early and re-evaluated as needed throughout product development.

The use of analytical platform technology (APT) methods can also greatly reduce the selection, development, and validation effort and lower the uncertainty/risk(s) for unsuitable method performance during product development. Since method performance criteria are well established, only product-specific suitability would need to be confirmed.  

The QbD approach is more applied to method development than method validation since method validation is the process of demonstrating that a well-developed analytical method is suitable for its intended purpose. The QbD concept is often narrowly interpreted in literature for analytical method development, and the method robustness study is often used as an example of the application of the QbD approach.  Well, the QbD approach is certainly applicable to the robustness studies and also consistent with the FDA expectation. The results of the robustness studies do not actually define the design space. A broader design space can be evaluated using the QbD approach during analytical method development. This would mean a significant more upfront effort in method development.

 QbD is not always employed in method development and validation because it is often considered to build time into the process. Often, labs are working under aggressive timelines to develop and validate methods. Using a pre-established set of parameters for method validation may be helpful in expediting the validation process, but it doesn’t necessarily provide relevant information about how the method will perform in ‘real world’ conditions.  When QbD is utilized in the development stages, critical product attributes are identified. Why not utilize this information in regards to analytical methods? For small molecule products, light or humidity may affect the product, so there may be potential problems related to these conditions that could arise during testing. The same is true for biopharmaceuticals. 

Until the method has been stressed to the point where a problem occurs, you don’t know what the true boundaries are. Don’t just assume that by modifying a mobile phase by 2% or varying a pump speed by 0.1 mL that you have demonstrated that the method is sufficiently robust. Issues may not develop until a larger change has been made. The goal in method validation isn’t just to document that the method is suitable for use. It should be to truly confirm that it would work as expected, regardless of the analyst, the lab, and within reason, the environmental conditions. It may take longer to incorporate ‘real-world’ parameters into the process, but there will be more value in the day-to-day use after validation is complete and the method is transferred. 

The application of QbD principles to analytical method development and validation will have a significant positive impact. Fundamentally, analytical QbD requires that the analytical target profile (ATP) is identified before the analytical technology is considered. This means that fundamental requirements of the methodology are identified ‘up front.’ For HPLC methods, systems are available that integrate experimental design software with the chromatography data system (CDS) and analytical instrument, so that analytical method screening can be performed in an automated manner to identify a lead column, gradient, and mobile phase combination. This ‘Lead’ system is then subject to additional optimization experiments and final verification of the analytical design space.  This approach has the potential to significantly speed up analytical method development and result in HPLC methods that are more robust, and therefore, analytical transfer problems are reduced.

 What deviations or variations may occur during analytical method validation in biopharmaceutical manufacturing? 

We should distinguish a validation deviation from a validation failure. Both can occur, but the frequency of these events should be low so that the manufacturer can maintain a state of control. In general, an unplanned deviation is typically easier to resolve and may not hold up the completion of the validation study. For example, the use of an incorrect analyte spike or a confirmed sample mix-up may require an execution repetition but may not need to be treated like a (potential) validation failure once the deviation has been confirmed.  Often, a lack of clarity in the validation protocol, poor preparation and planning, and/or insufficient analyst training can result in unplanned deviations.

A validation failure results from failing to pass the protocol acceptance criteria (or test method performance specifications).  Validation failures are always serious and difficult to deal with under current interpretation of regulatory compliance. PDA TR 57 provides good ideas and suggestions on how to systematically deal with such events and to set up an appropriate quality system (similar to dealing with out-of-specification results).

What are the challenges in method transfer?

Like method validation deviations and failures (see ‘Deviations and Variations’), similar root causes may also exist for method transfers (validation extensions). The readiness of the receiving laboratory should be evaluated. Consideration should be given to the availability of required analytical and supporting equipment, software, critical reagents, standards, controls, and analysts who are skilled in the relevant analytical techniques as well as the qualification status of all materials, equipment, and analysts.  

If gaps are identified (e.g., the receiving laboratory has a similar analytical instrument), a risk assessment should be performed before execution of the formal transfer studies. Shipment and receiving procedures are needed to allow transfer of critical reagents, standards, and samples between laboratories. Incorporation of the test method procedure into the receiving laboratories quality system is also part of the transfer process.

The sending laboratory should provide hands-on training in the specific test method to analysts at the receiving laboratory, if needed. The type and amount of training needed will vary depending on the analytical method transferred and the existing experience of the receiving lab and its personnel.  The evaluation of the capability of the receiving laboratory to execute the system suitability requirements of the method successfully during the training is recommended. It is important that all responsibilities between sending and receiving laboratories are established. PDA TR 57 provides additional practical tips on what to consider for analytical method transfers.

Method transfer is a very challenging exercise and can become more challenging if the receiving laboratories are in different countries/continents. Planning is definitely the key to a successful method transfer. The timelines for method transfer need to be discussed and clearly understood by both the originating and receiving labs. The methods transfer protocols should be carefully reviewed and approved by both labs. Logistics should be well coordinated especially when shipping the reference standards and samples overseas. Proper documentation including import licenses, if necessary, should be obtained in advance. To ensure the success of method transfer, the analytical methods should be evaluated by the receiving laboratory before the initiation of method transfer. The samples for method transfer should also be carefully selected. The new FDA draft guidance recommends that forced degradation samples or samples containing pertinent product-related impurities should be analyzed at both labs for a stability indicating method. For biopharmaceuticals, it might be difficult to ship the forced degradation samples to the receiving lab. A suitable protocol may be needed to guide the receiving labs to generate similar forced degradation samples themselves.

 When transfers are being done between sites, or with contract facilities, issues may arise with different makes of instruments. While an HPLC has the same basic components, regardless of manufacturer, there may be subtle differences in performance. For example, the construction of the optics in the detector may be just different enough to present unexpected issues with linearity or sensitivity. In some cases, older models from the same manufacturer may have differences in construction that will challenge a method in ways that will prevent a successful transfer. Some exploratory testing may be valuable, prior to transfer, to determine the compatibility of the instruments in the receiving lab and the method. 

 Method transfer can go smoothly or can result in significant problems. The robustness of the analytical methodology being transferred and the experience of the ‘receiving’ laboratory have a significant impact on the success of the transfer. Differences in instrumentation, culture, and ways of working also contribute to possible challenges, and therefore, need to be considered. The analytical technology transfer (ATT) usually follows an analytical protocol, where results obtained between the ‘receiving’ laboratory are compared to those obtained by the ‘donor’ laboratory transferring the technology.  Subjective tests such as color and appearance can cause disproportionate problems unless the specification is carefully worded. Tests with low level impurities require careful evaluation using % absolute differences, rather than student’s t-tests in the ATT protocol. In part, cultural and ‘ways of working’ differences can be overcome through training and short-term secondment, so that analysts in the ‘receiving’ laboratory acquire as much knowledge of the methodology as possible.

For transfer of HPLC methods, generally, fewer problems are experienced where both laboratories have the same analytical equipment. However, this is not always possible and ‘hidden’ differences in instrumentation (such as gradient formation, delay volume, or signal to noise) can contribute towards problems with transfer. Testing or qualifying the equipment in the same way can help identify differences in performance between analytical equipment.

Analytical Procedures and Methods Validation for Drugs and Biologics

, Draft Guidance (CDER, Rockville, Md.,  February 2014).
  2. 	PDA, Technical Report 57 (PDA, 2012).
3.	ICH, Q2(R1): 

Validation of Analytical Procedures: Text and Methodology

Cover Story

Experts give insight on method transfer, QbD, and regulations for analytical method development and validation for biopharmaceuticals.

Challenges in Analytical Method Development and Validation

Though the Bruneian pharmaceutical sector is at its infancy, it possesses huge potential for growth and development. According to Business Monitor International (BMI), the sector is valued at US$100 million in 2013 with a compound annual growth rate (CAGR) of 9% between 2013 and 2018, reaching a value of US$150 million by 2018.  

The country is located in a region with great biodiversity, which offers an excellent platform for the exploitation of rich sources of medicinal plants and other useful forest products. Though this has attracted the attention of international scientists, more efforts will have to be put into inviting experts to the country and conducting more studies in collaboration with universities and relevant departments to explore what is out there and how they can be used to the industry’s advantage, Jason Paul Bin Rumpun, key accounts manager of Simpor Pharma says.


In addition, the country is also tapping into the growing halal market. In 2010, the Ministry of Religious Affairs issued the guidelines for the manufacturing and handling of halal medicinal products, thus setting the stage for the establishment of halal pharmaceutical companies in the country, and Brunei to be the hub for halal pharmaceutical exports, Cher Boon Piang, analyst of BMI Asia says.  

In October 2013, Simpor Pharma was established after acquiring the full halal certification from the government. In a $26 million joint venture between Canadian-based Viva Pharmaceutical and private equity fund Aureos (Brunei) Capital, Brunei’s first drug manufacturing plant produced 25 million Syariah-compliant capsules for exports by February 2014, aiming to ship them in three to five months. 

Rumpun adds, “The halal brand will gain strong acceptance amongst the Muslim community be it in Europe, Africa, or America. Hopefully, the Bruneians will take advantage of this opportunity to expand and reach out to the global market.” This is echoed by Edward Ko, managing director of Simpor Pharma, who commented that halal sales are strong and growing in countries such as Azerbaijan, Iran, Yemen, and United Arab Emirates (UAE).


  The government has identified the sultanate’s rich biodiversity and educated workforce as means for attracting overseas investors to partner in specific projects and use Brunei as a strategic location for supporting their regional expansion plans, Abhijit Ghosh, pharmaceutical and healthcare leader of PricewaterhouseCoopers (PwC) Singapore says. Recognized as the world’s most stable macroeconomy by the World Economic Forum (GCI 2010/11), Brunei possesses ‘pull’ factors such as political stability and low corporate income tax. The corporate income tax rate has been reduced to the current rate of 22% compared to 30% in 2008. There are no personal income tax, as well as export, sale, payroll, and manufacturing taxes (1).

Ghosh adds, “The country offers research and development (R&D) and training grants, as well as tax incentives and exemption from import duties on raw materials and machinery for qualifying projects.” Currently, Japanese-based National Institute of Technology and Evaluation (NITE) and Bruneian scientists are researching the possibilities of using microbes for pharmaceutical purposes. Germany is also exploring the idea of partnering with the country for developing pharmaceutical products culled from the Sultanate’s diverse plant field. China is also looking to partner with Brunei for the supply of raw ingredients for Chinese traditional medicines.

However, Brunei will need to improve on areas including starting a business, enforcing contracts, and property registration. Based on the World’s Bank Doing Business Report 2014, the country was ranked 59 out of 189 countries and behind neighboring countries Singapore (first), Malaysia (sixth), and Thailand (18th) (2). Bureaucratic red tape is one of the issues for companies. For example, it took Viva Pharmaceutical six years to build its facilities due to bureaucratic slowdowns. The country also does not possess high quality infrastructure to facilitate the expansion of manufacturing facilities, Ko says.
  
  


  Ghosh shares a similar view that the lack of adequate infrastructure facilities for R&D and manufacturing could be stumbling blocks for the Bruneian pharmaceutical industry. He says, “Given the shortage of a skilled workforce, the government should start investing on training facilities for developing pharmaceutical experts and biotech researchers if it wants to succeed in the industry. It should also place its emphasis on liberalizing its policies to eliminate certain red tape and make it easier to do business in Brunei.” The country may want to explore the idea of seeking help from established neighbors such as Singapore to develop its infrastructure and move up the value chain to develop its pharmaceutical sector.   

Rumpun maintains a positive outlook for the Bruneian pharmaceutical industry. “Though Brunei is still in the experimental stages of manufacturing, the government has been willing to review and streamline existing processes to make it easier for companies to do business. Once the processes and systems are in place, there is a market for more companies to open manufacturing facility in the country. Moreover, the government is on a constant lookout for potential foreign direct investment that is essential to bring about development,” he says.

, The World Bank,  accessed May 9, 2014. 
  
  


  Jane Wan is a freelance writer based in Singapore.

Global News

Brunei harnesses its rich biodiversity and the growing halal market in a bid to develop its pharmaceutical sector.

Brunei's Potential Pharmaceutical Market Growth

The biopharmaceutical industry is advancing at an unprecedented rate as new cells and viruses are being used to generate biological therapeutics in greater yields with shorter production cycles and more efficacious properties, according to Audrey Chang, executive director of global services at BioReliance.

“New cell substrates (e.g., transformed cell lines, insect cells), new product types (e.g., gene and cell therapy, tissue engineering), new production process, and the discovery/detection of new contaminants have contributed to an elevated complexity of product testing to evaluate safety and efficacy,” Chang explains. Newer techniques for lot-release testing can offer numerous advantages in this situation, but there are challenges to assuring equivalence to standard methods. 


  New test methods can improve quality and promote efficiency, according to Niall Dinwoodie, global coordinator of analytical testing in the Biologics Testing Solutions business of Charles River Laboratories. “New methods or improved versions of traditional methods allow quality control laboratories to provide a greater level of information about the finished product, whether through improved detection limits, discriminating power, or quantification,” he observes. “In addition, newer techniques tend to be more automated, allowing faster sample throughput in larger numbers.” 

The use of newer analytical techniques can also help mitigate risks, according to Chang. “The cost and safety impact of contamination has led the industry to embrace quality-by-design principles with respect to building quality into a product and process. As a result, biopharmaceutical companies are starting off with knowledge of the risks involved and how best to mitigate those risks, which includes the use of the most effective analytical methods,” she says. 


  The adoption of alternative methods into a regulatory program can be based on several factors, such as the desire to reduce/eliminate the need for animal testing, shorten the turnaround times for testing, and/or provide a more comprehensive safety profile through either increased sensitivity or specificity, according to Chang. Some alternative test methods are in fact improvements over existing techniques rather than true alternatives and rely on the same properties of the molecules to perform the analyses, according to Dinwoodie. He points to capillary electrophoretic methods as substitutes for gels to obtain greater resolution of closely related proteins within the product and provide more robust quantification of these components. In addition, he suggests the use of ultra high-pressure liquid chromatography (UHPLC) methods instead of high-performance liquid chromatography (HPLC) to reduce run times and increase sample throughput as an example. New technological methods for rapid detection, identification, and quantification of adventitious agents have also emerged, according to Chang. “Many of these techniques are molecular-based, such as polymerase chain reaction (PCR) for mycoplasma, next-generation sequencing for virus screening, and digital PCR for residual DNA quantification,” she comments. Charles River is also seeing growing interest in rapid microbiology methods for sterility testing of autologous or allogeneic cell therapies where cells need to be infused into patients before traditional sterility results would be available, according to Dinwoodie. He also believes that PCR coupled with mass spectroscopy (Plex-ID) and massively parallel sequencing may provide advantages over current techniques for detecting adventitious agents in cell substrates and biological products.  “New techniques for determining sub-visible particulates that enable the detection and evaluation of particles of much smaller sizes are also providing conformational data that goes beyond traditional light obscuration methods,” adds Dinwoodie.


  To prove equivalence statistically, a large data set is needed. “Generating a data set to show equivalence requires testing multiple samples that cover all potential degradation routes and impurity profiles. Therefore, in order to show equivalence of a new method, testing must be conducted on a bank of retain samples from historical production runs and new stress and stability samples,” Dinwoodie states. 

Beyond this issue, companies often face the challenge of correlating the conclusions generated from the old and new methods when there are apparent differences between the data sets, according to Chang. “A good example is the use of PCR, where the presence of the nucleic acid is used as a substitute to monitor the presence of live/infectious organisms. Frequently, the term ‘false positive’ is used when a positive result is seen with a PCR assay and a negative result is obtained with the corresponding cell culture assay (e.g., mycoplasma PCR and mycoplasma cell culture assays). In actuality, the PCR result was not a false positive; the presence of the DNA produced the PCR positive result as the assay was intended.” 

The alternative method may also identify new or increased levels of known impurities, according to Dinwoodie. “If batches used for safety and clinical assessments are still available for testing and can be shown to contain similar levels, the impact of these impurities can be more readily assessed.” In addition, in the case of quantitative assays, he notes that the alternative methods may be concordant (i.e., show similar dose-response curves) but not truly equivalent. “Understanding the relationship between data sets derived from the two assays is critical, particularly in the case of potency assays,” Dinwoodie stresses.

Another key challenge for Chang is correlating the limit of detection (LOD) of the two assays. “Again, using the mycoplasma example, the PCR LOD is reported as genomic copies (GC), whereas the cell-based method is reported in colony-forming units (CFUs). The lack of an industry standard mycoplasma stock can make it difficult to compare results because the GC/CFU ratio can vary depending on the species, storage, handling, and processing parameters,” she explains. 


  “The best way to show equivalence is through statistical comparison of the results for the same samples tested under standard and alternative methods; the greater the range of samples, the more convincing the statistical argument,” asserts Dinwoodie. He adds that it is important to recognize that the process will be a long one, and that alternative methods should be introduced to stability programs when there is typically sufficient sample available and they can be run alongside the current method to provide an initial assessment of equivalence. 

If an alternative method has been used from the start of product development, it is possible to justify using that method rather than the standard approach, according to Dinwoodie, but ultimately it is necessary to provide data to show that the method is fit for purpose. “In general, though, traditional techniques are commonly used in the early stages of product development and newer, more sensitive, technologies are introduced as the product moves through clinical trials; this approach gives time to run the stability studies and gather data on multiple batches before the final submission,” he notes.

The key message from Chang is the need to have well-executed validation strategies. “Good science requires well-planned, well-executed, well-documented assays that generate meaningful interpretation of the results. The overall validation approach needs to be risk-based, with the level of validation required for each step established. The goal is to define the risk and validation strategies employed, as well as outline the steps in the validation lifecycle. If this approach is adopted, industry/regulators will be encouraged to move away from old techniques and embrace the next generation of novel platforms that are far more sensitive and rapid,” 
  she asserts.


  It is crucial to include degraded or historical production samples in the equivalence test when changing a method, according to Dinwoodie. In addition, if the alternative is to be used from the start of the development program, the validation of the method must be considered. Chang adds that it is important to assess an alternative assay’s readiness for use in a release program, which requires planning and accounts for performing the validation and documentation of the information. “Companies need to ensure that resources are dedicated to a validation quality program to ensure that the performance characteristics of the alternative method meet the requirement for the intended application. In addition, for all methods, but particularly for new/alternative methods, plans need to be in place to monitor assay performance over time,” she states.

Finally, Dinwoodie recommends that regulatory authorities be consulted whether considering an alternative for an existing method used for an approved product or when developing the specification for a new product at the investigational new drug stage.


  Cynthia A. Challener is a contributing editor to 

Features

New test methods can provide improved quality and efficiency, but they must be validated to demonstrate equivalency.

Assuring Equivalency of Alternative Lot-Release Test Methods

Rigaku Raman Technologies’ handheld ramen analyzer, the Rigaku Progeny, is designed to deliver accurate materials identification and improve ease of use, adaptability, and reliability. The Progeny has a 1064 nm high-power excitation laser, which increases speed and sensitivity of analysis and minimizes fluorescence interferences. A 512-pixel detector delivers improved resolution, analytics, and signal-to-noise ratio for handling challenging mixture analyses. The sealed system (IP-68) guards against moisture and dust, enabling rugged warehouse use and easy cleaning while minimizing cross-contamination risk. 

The ergonomic design and shape provide single-hand entry into industrial barrels/drums, allowing users to view the screen while the instrument is in a downward orientation. The Progeny’s customizable workflow software is 21 

 Part 11 compliant and has a smart phone-inspired user interface allowing quick implementation of material ID methods.

Handheld Analyzer Improves Accuracy

Recombinant protein production in the biopharmaceutical industry mostly relies on fed-batch mammalian cell culture (1, 2). Often in fed-batch processes, the feed volume can be more than 1000 L for a 5000-L bioreactor with 30 to 40% (v/v) feed. To alleviate workload in media preparation and simplify the manufacturing process, a reduction in feed volume is greatly desired. Feed-volume reduction can also reduce the cost of consumables, such as disposable bags and medium storage space. Because the feed dilutes the culture and reduces product concentration, the use of lower feed volumes can improve overall process productivity as well.

One approach of decreasing feed volumes but still stoichiometrically supplying nutrients to cells is to concentrate the feed media. The preparation of highly concentrated feed media is, however, challenging because of the limited solubility of medium components. The solubility of many amino acids can be increased by adjusting the pH (3). Recently, surfactants have been used to increase the solubility of chemicals in the feed media (4). In this study, the authors compared strategies including raising the pH and adding surfactant(s) in the preparation of concentrated feed media. In the experiment, the authors fed less volume of concentrated feeds to the cell cultures, as opposed to the constant volume feeding strategy used by Hossler et al. (4). Among the surfactants evaluated, Poloxamer 188 (P188) (i.e., Pluronic F68) is a nonionic surfactant that is an additive in mammalian cell culture media and drug substance formulation (5-7). It was found that the feed volume could be reduced by 2.5-fold with the concentrated media supplemented with higher levels of P188, while volumetric productivity was improved by 10-50%. The improvement of productivities was likely due to the enhancement of protein folding and secretion machinery suggested by quantitative polymerase chain reaction (qPCR) array assay. Supplementing surfactant and raising pH approaches were successfully used in the preparation of concentrated feed media for 7-L and 5000-L bioreactor runs, respectively. These straightforward approaches can be applied to large-scale production of recombinant proteins. 

 Three recombinant Chinese hamster ovary (CHO) cell lines, developed in-house to produce three different monoclonal antibodies, were used in this study. Cell lines A and B are dihydrofolate reductase-deficient (DHFR-deficient) DG44 cell lines. Cell line C is a CHO glutamine synthetase (GS) cell line. Frozen vials of the cell lines were thawed and grown in shake flasks containing Bristol-Myers Squibb’s proprietary basal media. The flasks were shaken in incubators at 36.5 °C with 5% CO

 To prepare feed media for cell lines A and B, the formulated powder was added into water for injection (WFI) or cell-culture-grade water at 37°C, followed by addition of Polysorbate 80 (PS80) or P188. 10 N sodium hydroxide (NaOH) was used to raise the pH until the solution was clear. After the chemicals were completely dissolved, the medium was made up to the required volume by adding WFI or cell-culture-grade water and filtered through a 0.22-µm filter (Millipore). The medium pH was measured by a pH meter (Thermo Scientific), and the medium osmolality was measured by an osmometer (Advanced Instruments). This medium is referenced as M1 in 

To prepare the feed medium for cell line C, the formulated powders were added into WFI or cell-culture-grade water sequentially at room temperature. 10 N NaOH was used to raise the pH until the solution was clear. After mixing well, the solution was made up to the required volume and filtered through a 0.22 µm filter (Millipore). The pH and osmolality of the medium were measured. This medium is referenced as M2 in 

Fed-batch studies in shake flasks and bioreactors. 

Fed-batch studies in shake flasks were carried out in 250-mL Erlenmeyer flasks in incubators at 36.5 oC with 5% CO2. Applikon 7-L benchtop bioreactors were used for scaling up shake-flask studies. The process for cell line C was scaled up to the 5000-L production bioreactor. 

Cell counts, antibody titer, protein quality analysis, and qPCR array.

 Cell-culture samples were counted by a Vi-CELL (Beckman-Coulter) using the trypan blue exclusion method. The pH and metabolites in spent media were measured by a NOVA FLEX instrument (Nova Biomedical). Antibody titer was measured using high-performance liquid chromatography (HPLC) (Waters Corporation) equipped with a protein A column (Applied Biosystems). The protein charge profile was analyzed by imaged capillary isoelectric focusing (iCIEF) (ProteinSimple, Santa Clara, California). Antibody N-glycosylated forms were analyzed by PNGase F digestion, reductive amination labeling, followed by separation using ultra-performance liquid chromatography (UPLC).

 cells of day 9 and day 11 from each treatment were used to extract messenger ribonucleic acid (mRNA) following the protocol of RNeasy Mini Kit (Qiagen). 400 ng of total RNA for each sample was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using RT2 First Strand Kit (Qiagen). Four experimental cocktail solutions composed of cDNA synthesis reaction and SABiosciences RT2 qPCR master Mix (Qiagen) were dispensed into 4 × 96 wells (10 µL/well) in 384-well PCR unfolded protein response array (Qiagen). The array was analyzed by ViiA 7 real-time PCR system (Life Technologies). The raw CT values were further analyzed by free web-based RT2 profiler PCR array data analysis version 3.5 (Qiagen). The ratios of gene levels between day 9 and day 11 for each treatment were reported. The gene functions were obtained from the website of the National Center for Biotechnology Information (

Concentrated feed medium M1 test in shake flasks.

 In the concentrated feed medium study, the original concentration is designated as 1X. The 1X medium was used as a control. Feed media with concentrations of 1X, 1.30X, 1.60X, 1.75X, 1.90X, and 2.50X were prepared by adding 10 N NaOH to increase the pH and dissolve all of the components into the solutions. The higher the feed concentration, the more the base needed for solubility, resulting in an increase of pH from pH 8.58 for 1X medium to pH 10.41 for 2.50X medium, as shown in 

 The osmolality of the concentrated feed media was increased from 835 mOsm/kg of 1X medium to 2432 mOsm/kg for 2.50X medium. To test if a surfactant can increase the solubility of medium components without increasing the medium pH, 2.50X concentrated feed media with various percentages of PS80 or higher levels of P188 were made. The data show that these media required less NaOH compared to the 2.50X concentrated medium, resulting in a lower final medium pH and lower osmolality (see 

The concentrated feed media described previously were used in the fed-batch culture of cell line A. The feed volumes are shown in 

 Nutrients were added to the cell culture for the concentrated feed media at the same quantities for the 1X feed medium. The productivities on the last day of the process (day 14) showed that the 1.60X medium led to 12% and 20% increase of the titer and specific productivity respectively, which is significantly higher than the 1X medium control (p < 0.05). The 2.50X medium had the lowest productivity (see 

), suggesting that there was an upper concentration limit of feed media prepared by raising the pH, which was due to the lower cell viability at higher cell culture pH and osmolality (data not shown). Among the surfactants evaluated, the addition of 0.5% (w/v) P188 in 2.50X medium increased the titer and specific productivity by 15% and 25%, respectively, while the feed volume could be reduced by 2.5-fold. More favorable culture pH and osmolality profiles from the 2.50X + 0.5% P188 feed medium have been observed as these are closer to the 1X control condition (data not shown). The 2.50X + 0.5% P188 feed medium also led to 42% and 116% increase in the final titer and specific productivity of cell line B respectively (see 

Antibody production in 7-L bioreactors using concentrated feed media of M1.

 To test the scalability as well as to remove the negative effects from the high pH feed media, 7-L bioreactors were used to run cell line A process with pH control. The profiles of viable cell density and viability are shown in 

 The 2.50X + 0.5% P188 medium was tested in parallel to the 1X medium that was used as a control. The viability for the control decreased from day 8, whereas the concentrated feed maintained higher viability for a longer period of time. The productivity profiles shown in 

 indicate that use of the concentrated feed medium improved the productivity by 33% towards the end of the process. The use of the concentrated feed medium also led to a significantly higher (p < 0.05) specific productivity than the control (data not shown). These results suggest that productivity can be improved when feed volume is reduced, which is consistent with the shake-flask studies. The product qualities, including charge heterogeneity and N-glycan profiling, were not affected by the increase of P188 concentration in the feed (see 

). There were no changes in antibody monomer percentages and protein purity with addition of P188 (data not shown). 

Antibody production in 5000-L bioreactor using concentrated feed media of M2.

Different approaches to prepare highly concentrated feed media for fed-batch Chinese hamster ovary cell culture are evaluated.

BioPharm International-06-01-2014