The adoption of single-use systems in biopharmaceutical production originated with the desire to minimize cleaning and associated validation. Before long, bioprocessors recognized the value of flexibility, reduced capital costs, and faster campaign turnaround times attainable through disposable bioprocess equipment. Single-use technologies have evolved from a supporting role for upstream operations (e.g., media storage and cell harvest) to near-complete platform processes that include all downstream processing steps except chromatography. 

Numerous technical advances have contributed to the attractiveness of single-use equipment across the range of production scales, such as improvements in materials and quality of construction and connectivity. Another positive factor has been suppliers’ increased commitment to providing a robust regulatory package. Vendors have taken on the burden of documenting their products’ suitability and safety, which had previously fallen to a large extent on end-users. Moreover, the 5- to 10-fold improvement in product titers for monoclonal antibodies over the past 15 years has nearly eliminated arguments against single-use based on scale. Bioprocessors can now generate production-scale batches in what were once pilot-scale volumes.

With many technical hurdles overcome, the focus has shifted to maximizing the operational and economic advantages of single-use bioprocessing. As the case continues to build for end-to-end disposable bioprocessing, bioprocessors should be aware of the key factors associated with implementing single-use components or platforms. Potential adopters must focus on which criteria to consider as their plant’s manufacturing capacity evolves away from multi-use unit operations to disposable components and systems. 

Bioprocessors have successfully deployed single-use equipment at nearly every conceivable production scale. Yet, the decision to “go disposable” is significant. It involves considering the product, scale, process, and corporate objectives that include the value of legacy stainless-steel equipment. Decisions made early in process development and scaleup will affect the trajectory of manufacturing at successive production scales. Employing best practices when selecting single-use products during process development can ease the transition from clinical to commercial production, and maximize the overall benefit of single-use systems. Based on shared and observed experiences in single-use bioprocessing, the principal factors to consider when embarking on a new single-use deployment include materials of construction, components, system design, and vendor support. Neglecting these considerations can result in costly delays and an uncertain pathway to commercial scale-up.


  Materials of construction constitute a principal factor when specifying single-use bioprocess equipment. At the most basic level, materials must meet stringent biological and chemical guidelines. Bioprocessors should settle for nothing less than fully compliant materials from proven suppliers with established reputations. 

Consistency and availability of materials of construction directly affect the quality and supply of single-use products. End-users should expect availability of the same or equivalent materials of construction, of equal or higher quality, for the duration of a development and/or manufacturing campaign. To meet these requirements, suppliers should maintain a redundant or backup supply chain for their raw materials. Customers should inquire about a vendor’s contingency plan in the event of a supply interruption.

Frequent change notices from single-use system suppliers altering materials of construction carry potential quality implications that may delay program timelines and add to quality assurance costs. Consider the frequency of a vendor’s change notices, or whether the vendor is providing notifications at all. New materials introduced to existing systems can lead to validation issues and delay a product’s time-to-market.


  Components are the building blocks of single-use systems that are, in essence, only as robust as their weakest link. Users should specify components that are consistent with overall facility practices. For example, if commercial production relies on sterile welders and sealers, users should specify compatible tubing that meets material requirements. 

Proprietary components not generally available to the market at competitive prices will almost certainly add to cost and reduce customer choice. Bioprocessors can strengthen their long-term supply position by specifying components 
    from suppliers that trade freely with other single-use system assemblers. Only by evaluating both performance and marketing practices can a user make the best selection decisions. Also important is the component supplier’s 
    reliability and delivery history. 

Delaying a development program or manufacturing campaign due to an unreliable supplier can be a real issue. Delays occur when a crucial component of a single-use system has a very long lead time. Because the assembler cannot produce the system without this component, system delivery times will be dictated by the long-lead item. To avoid this pitfall, assess potential suppliers by the experience of users who have previously worked with them. 

Bioprocessors will commonly use any component on hand during preclinical development. By the time a product reaches clinical testing, processors will have a much better idea of what the process looks like. By Phase III, the process 
  becomes “locked.” As integral components of manufacturing processes, single-use components and systems should parallel biopharmaceutical development in terms of maturity. When processes become established, so should the components. 


  The decision to replace a multi-use unit operation with its single-use equivalent depends on the product, the process, and the organization’s business objectives. Some reasons include the need to eliminate a processing bottleneck (e.g., cleaning and cleaning validation), process issues such as frequent contamination failures, or to realize the calculated benefits that stem from cost, time, and product quality. 

Best-in-class single-use system suppliers possess expertise that customers can use to help select components and systems most suitable to their business needs. The best designs are often minimal in terms of materials and accessories. Products such as single-use bags with unused ports, tubing, and fittings, which are commonly found in off-the-shelf systems, add nothing but excess cost and waste, thus reducing the advantages of single-use systems. 

The vendor’s job is to produce and ship precisely the system a bioprocessor requires. The system should arrive with the required regulatory documentation, ready to use. At one time, bioprocessors made routine modifications to single-use systems at their facilities. These days, it is best to avoid changes at a facility, particularly those that might compromise sterility.


  Because the business relationship between supplier and customer may last many years, vendor qualification is crucial. Top vendors view customer interactions as opportunities to improve their products and services. Conversely, a vendor’s lack of expertise, poor communication, or reluctance to collaborate with or on behalf of the customer can limit the benefits of single-use bioprocesses. 

Suppliers should be prepared to play an ongoing support role for every single-use system sale, and for the product’s entire lifecycle. End-users should ask for specific examples of a proposed vendor’s ability and willingness to provide support, process expertise, and technical advice that add value to the business relationship. 

The customer-vendor relationship should be capable of feedback that results in system optimization, thus, creating significant value for both parties. Because flexibility and ease of modification are key advantages of single-use systems, end-users should seek vendors that are adept at maximizing these advantages.

Customers must assess the value associated with every aspect of vendor contact. This process need not be difficult. If the vendor consistently assists in problem-solving, improving processes, and educating their clients, then the time is well spent. Vendors should demonstrate competence in their clients’ bioprocessing niche, and be eager to point out how changes in equipment or operations will improve the process. Suppliers who do not provide this level of value should be avoided as time spent with them offers no tangible return. 


  Choices made when specifying single-use equipment can have cascading and lifecycle-long effects on process throughput, cost, efficiency, and risk. Adopting disposable bioprocessing by jumping on the bandwagon is not the way to go. Bioprocessors must first analyze benefits and risks for their specific situation, keeping in mind their expression system, process, product, and corporate objectives. 

After determining the suitability of single use, bioprocess organizations must consider the practical aspects of “going disposable.” Examining the impact of materials, components, systems, and the vendor relationship at each stage and at every level will help customers make good decisions. Value should always be the bottom line: Does deploying a single-use device improve your process, and thereby your product? Will adoption drive key business objectives such as improved quality, shorter timelines, and cost savings in a concrete, quantifiable way? Will your relationship with your supplier be a two-way street, supportive, and mutually value-driven, or does the vendor’s responsibility end with the sale? Bioprocessors who follow the decision process outlined here are in the best position to benefit from the considerable promise of single-use systems.


  John Schmitz, director of strategy and business development, life sciences, Saint-Gobain Performance Plastics.

Articles

Boot Camp Technical Guide

Key considerations for implementing single-use components or platforms when moving from research to process development.

Best Practices in Adopting Single-Use Systems

The underlying question for any drug in development is: What price can (or will) patients pay for the needed therapy? Patient sticker shock, government austerity programs, blockbuster drugs falling off the patent cliff, and varying medical and regulatory practices worldwide continue to shape the financial outlook of biopharmaceutical companies.

The Global Use of Medicines: Outlook through 2017

, IMS Institute for Healthcare Informatics projects that in 2014 total global spending on medicines will, for the first time, exceed $1 trillion. In 2017, the total will reach almost $1.2 trillion. The report identifies challenges, but also indicates opportunities for the industry to improve the lives of patients. 

In 2012, developed markets (United States, Japan, Germany, France, Italy, Spain, United Kingdom, Canada, and South Korea) reduced spending for the first time due to patent expirations, austerity measures, and moves toward generic drugs. Annual growth in spending levels will reach a low point in 2013, the report says, but will rebound from negative $3 billion in 2012 to $20-25 billion by 2017. In emerging markets (more than 20 nations including China, Brazil, India, Russia, Mexico, and Turkey), growth will increase from $26 billion in 2012 to $30-50 billion in 2017, primarily due to increased access to medicines.

In 2017, spending on medicines for specific diseases will differ between mature and emerging markets, the report says. In developed markets, increasingly aged and obese populations drive spending in oncology and diabetes, a trend that is increasing in emerging markets. Specialty pharmaceuticals that treat complex, serious diseases will grow in importance in developed markets; traditional therapies will drive spending in emerging markets.

Biologics have a growing impact on the drug market and prices. In 2002, biologics represented 11% of total drug sales; in 2012, the number was 18%. IMS estimates biologic agents will continue to outpace overall pharma spending growth and will represent 19-20% of the total market value by 2017. Monoclonal antibodies and human insulin will spur this growth.

Competition generated through established biosimilar pathways will drive down the high costs of biologics in Europe, the US, and some emerging countries. However, in markets with less rigorous IP protection, IMS reports a surge in non-original biologics--copies of innovator brands that have not been approved a dedicated pathway. Biosimilars account for less than 0.5% of biologic spending in mature markets; in emerging markets, non-original biologics represent more than 10% of all biologics spending.

The mix of total global spending on medicines will shift toward generic drugs, rising from 27% to 36% of the total by 2017; however, brand drugs will account for more than two-thirds of spending in developed markets. In emerging markets, generic-drug products will represent 63% of spending.

Traditional pharmaceuticals used to treat chronic diseases in primary care will increasingly be dispensed as generics; total spending will only rise 5% by 2017 in developed countries. In emerging markets, total spending on traditional pharmaceuticals is expected to rise from $199 billion in 2012 to $336 billion in 2017 as patients gain access to affordable generic drugs.

IMS reports spending on specialty medicines in developed markets is expected to increase by 30% over the next five years; the use of specialty medicines in emerging markets is at very low levels, but the costs are expected to rise by nearly 90% between 2012 and 2017.


  1.	IMS Institute for Healthcare Informatics, 


Rita Peters is the editorial director of 

From the Editor

Generics, market dynamics, and global demand are changing drug spending patterns.

Drugs, At What Cost?

In a recent survey of current trends and practices in biopharmaceutical manufacturing, 

 readers revealed a preference for disposable systems, identified challenges for different therapies, and suggested technology enhancements to improve process efficiencies (1).

Survey respondents represented research, development, formulation, and process development functions, as well as quality control/assurance, validation, and laboratory management titles at branded and generic-drug companies in the United States, Europe, and Asia. More than half of the respondents work for organizations that produce both small- and large-molecule drugs; 25% added a biopharmaceutical-manufacturing program in the past year. 

The responses indicate some signs of manufacturing growth. More than half of all respondents said their companies increased biomanufacturing capacity in 2013. Less than 3% reported a drop in production. Only 8.1% said their companies decreased spending for biopharmaceutical equipment and services in 2013 compared to 2012; 37.8% reported increased spending; spending was flat for 33.8% of respondents. Nearly 40% expect spending to increase in 2014 compared to 2013; only 5.4% expect a decrease.

The top reasons for increased capacity were the addition of new products and increased production of existing products. More than 40% of respondents said that internal development of new products spurred the need for additional capacity. About 20% said capacity was added due to the acquisition of another company or in-licensing of a drug product

Almost 45% of the respondents reported that their organizations manufacture therapeutic monoclonal antibodies (mAbs); more than 55% of those surveyed said they manufacture protein-based drugs other than mAbs. More than 40% of the respondents said their companies manufacture vaccines; 17.7% develop cells for tissue and/or cell therapies; 10.4% manufacture nucleic-acid based drugs.

. Facilities that manufacture protein-based drugs reported use of multiple purification technologies. Ion-exchange chromatography was the most frequently reported (71.6%) followed by chromatography with other resins (62.7%), chromatography with Protein A (56.7%), and membrane-based filters (53.7%).

Protein stability led the list of reported technical challenges, cited by 53.7% of the respondents. Producing sufficiently high product yields was a close second at 52.2%; purifying high product yields was the third most frequently cited at 49.3%. Protein aggregates, formulation issues, creating/cloning cell lines, regulatory issues, and analytics were mentioned by more than 25% of the respondents (see 

. Although they represented a smaller subset of the respondents, those that manufacture nucleic-acid based drugs reported multiple manufacturing challenges. Purification (reported by 45.5%), stability (36.4%), drug delivery (31.8%), and scale up (27.3%) led the list (see 

. Process development for cells for tissue- or cell-based therapies created technical challenges for half of the respondents. Scale up (35.7%), cell viability (31%), and cell stability (28.6%) were also listed as top technical issues. Regulatory issues, costs, analytics, and inadequate bioreactor volumes were other challenges (see 


  More than half of the respondents reported using process analytical technology (PAT) in manufacturing operations. High-performance liquid chromatography was the clear leader, used by 71.7% of those that implement PAT. Use of Fourier transform infrared spectroscopy was reported by 41.3%; almost one-third use near-infrared spectroscopy. More than one-quarter use disposable sensors


  Hybrid manufacturing systems, which feature traditional stainless steel and disposable, single-use equipment, were used by almost three-quarters (74.4%) of the respondents; 16.3% use all stainless; 9.5% use all disposable.

All respondents, those who used and did not use disposables, shared perceptions about the value of single-use systems. Nearly 70% cited reduced contamination as an advantage. Time savings, ease of use, and flexibility for multiproduct manufacturing were identified as advantages by more that half of the respondents (

). While 38.1% said disposables offered cost savings, 55.6% said high cost was a challenge to using a disposable system. Other challenges were limitations on size and leaching of the disposable material into the product (


  Improved process understanding (68.4%), improved product quality (66.7%), and reduced variability in product quality (57.9%) were the top advantages identified by the respondents who have implemented quality-by-design (QbD) principles in their biopharmaceutical manufacturing operations. Nearly half reported improved manufacturing efficiency as a result. However, almost 32% of the respondents have not implemented QbD. Reasons cited for not implementing QbD include a lack of guidance and direction from regulatory agencies (46.2%), no process or quality advantage to be gained (30.8%), a lack of understanding of the QbD initiative (23.2%), or it is too costly.


  Respondents revealed insight about recent innovations that have improved process efficiencies.  Equipment and products related to disposable systems were most frequently mentioned. 

There is, however, room for improvement. Better disposable systems, including bags that do not leak, reduced leaching, larger bioreactors and fermenters, and disposable sensors were identified as desired new technologies to improve process efficiencies. Better filtration, chromatography columns, automation, and process analytical technologies also were identified as ways to improve manufacturing efficiencies.
  an Alternative:


  Outsourced manufacturing plays a prominent role among the companies represented in the survey. Almost 40% reported outsourcing of some component of biopharmaceutical manufacturing. Leading outsourced function, are drug substance manufacturing, API and finished products manufacturing, and clinical trial-materials manufacturing. More than one-third of the companies outsource full-scale commercial manufacturing. Limited internal manufacturing capabilities and cost-effectiveness were the leading reasons cited for outsourcing different functions in biopharmaceutical manufacturing.

BioPharm International Manufacturing Trends 2014 Survey

Cover Story

Single-use systems and other technologies drive process efficiencies, but there is room for improvement.

2014 Manufacturing Trends and Outlook

Europe’s regulatory agencies have been stepping up their support for small- and medium-sized enterprises (SMEs), including micro companies, in an effort to stimulate more development of innovative medicines. The companies are being offered free or heavily discounted regulatory and administrative assistance, training sessions, evaluation, and above all, scientific advice that can raise the chances of a new medicine gaining marketing approval. 

The importance of early dialogue with regulators 


  Companies are encouraged to contact the agencies as early as possible during the drug-development stage to ensure that their manufacturing processes, in particular, will not lead to regulatory difficulties. It is, however, hard work for the regulatory agencies because many small companies (a high proportion of which are startups founded by individual specialists or small groups of scientists) tend to delay contacting the regulators until product development is close to the preclinical or clinical-trial stage. For the agencies themselves, the promotion of innovation can be a new role with which they need to become more familiar with to bridge any communication gaps with entrepreneurs.

 “The people running SMEs know the science and technology of the area in which they are doing business,” explains Hooshang Zavareh, founder of ChemPharmServe, Cambridge England, a company that specializes in drug-development services. “They often don’t see the need for help from regulators until they get to the point where they need to make a regulatory application. If they want outside help before then, they will go to a consultant.”

Nonetheless, the agencies believe that having much closer ties between themselves and entrepreneur innovators is essential. With Big Pharma cutting down on research activities, they believe much of the impetus behind innovation in medicines--at least in the early stages of development--will come from the companies with low turnovers and few employees, for example micro companies with less than 10 employees and annual revenues of less than €2 million (approximately $2.7 million), SMEs with not more than 50 personnel and annual turnovers not exceeding €10 million, and medium enterprises of less than 250 employees and an annual turnover of not more than €50 million.


  The European Union is currently calling SMEs “the powerhouse” of its Innovation Union, a concept behind its new seven-year €77 billion research program called Horizon 2020, which starts next year. Approximately 10% of the program’s budget will be allocated to healthcare, demographic change, and well-being. A major objective is the elimination of fragmentation in the fields of scientific research and innovation. Among the EU’s regulatory agencies in pharmaceuticals, the most active in championing innovation has been the European Medicines Agency (EMA), which is responsible for centralized approvals of medicines, including those based on novel technologies and orphan medicines.

SMEs make up a growing proportion of companies applying to the EMA for an initial evaluation of a new medicine, including its manufacturing process. Almost 30% of applicants in 2012 were SMEs, which also accounted for 68% of evaluation applications for orphan medicines compared to 27% in the previous year

In 2012, the number of SMEs registered with the agency rose 62% to 1098 mainly because of fees reductions and also because companies could make electronic submissions of product information. This year, the rate of increase has slowed down with the total registrations by late November going up only a further 12% to 1234. Of this total, 43% were micro-sized companies, 12% were academic spin-offs, and 5% of registrated SME companies had been created since January 2012. The registered SMEs had a total of approximately 3000 products under development.

Understanding the route to drug approval  


  “From a survey we did in 2011, we found that a lot of SMEs lack personnel with experience of bringing products through the regulatory system. As a result, they attach great importance to having early access to EMA,” says an agency official in an interview with 

 “Our figures show that those who do contact us early are more likely to be successful with a marketing-authorization application.”

One of the most highly valued services of the agency among SMEs has been the provision of scientific advice, available at a 90% discount, particularly on quality and process issues. “Quality documentation continues to be a problem area for many SMEs, particularly relating to manufacturing process validation, the setting of specifications, and stability data,” says the SME Office in its latest annual report (1). The agency’s SME Office has been disappointed with the low uptake of services from SMEs involved in the development of advanced therapy medicinal products (ATMPs)—such as gene therapy, somatic-cell therapy, and tissue engineering. SMEs have been accounting for 75% of EMA’s small number of ATMP approvals, for which there were only three applications last year.

To provide an incentive for more SMEs to develop advanced therapies, EMA has set up a scheme for the scientific evaluation and subsequent certification of the quality and, where necessary, non-clinical data of new products in the area. Certification should help ease the way to marketing authorization. Another attraction is that it should help companies obtain funding and license out their technologies. The quality data to be certified would include information on starting and raw materials, the manufacturing and characterization of the active substance, and the reproducibility of the manufacturing process for the medicinal product.


    By early this year, however, there had been only two applicants for certification, which prompted the EMA to conduct a survey to find out the reasons for the apparent lack of interest. Survey results showed that nearly two thirds of SMEs who answered questions on certification—amounting to only 21% of those surveyed—considered it to have positive advantages. EMA concluded from the study that participants seemed confused about the scope of certification, which indicated a need for the agency to clarify its role in the regulatory process. 

From EMA’s viewpoint, one big success story with its new relationship with SMEs has been the way it worked closely, from an early stage, with the Dutch start-up uniQure to enable its product alipogene tiparvovec (Glybera) to gain the EU’s first marketing approval for a gene therapy in late 2012. The product was developed for the treatment of lipoprotein lipase (LPL) deficiency. For the agency, the approval process was scientifically challenging because alipogene tiparvovec was a new type of medicine without any regulatory history. “The lessons learned from this case are multiple and it paved the way for approval of similarly complex medicines in the future,” said Christian Schneider, chair of the EMA’s committee for advanced therapies (CAT) and Hans-Georg Eichler, senior medical officer, in a joint article in the agency’s latest annual report (2)

EMA stresses that in its efforts to forge closer ties with SMEs, it is not trying to undermine the position of consultants and other experts in the pharmaceutical industry. Its role is not “to substitute the industry’s responsibility for the development of their products,” says the agency

A similar point is being made by national agencies for medicines regulation, which have been setting up innovation offices to liaise with SMEs. So far, out of the 28 EU member states, agencies in Germany, France, UK, Sweden, and Finland have created SME units that have already formed themselves into a network to exchange experiences and best practices.

In the UK, an Innovation Office, opened in March by the country’s Medicines and Healthcare Products Regulatory Agency (MHRA), is providing multi-disciplinary guidance on “novel development methodologies, manufacturing processes, or new materials that have not been previously used in either the pharmaceutical or device industries,” says Ian Hudson, the MHRA’s licensing division director.

The agencies are emphasizing that their units can be used by any SMEs developing an innovative product or process whatever regulatory procedure or route they intend to use. “We want to encourage SMEs to come to a regulator as soon as possible in the development work so that they get the production process right from a regulatory perspective early on,” explains Lars Dagerholt, head of the Innovation Office at the Swedish Medicinal Products Agency, Stockholm. “We also don’t mind them shopping around other agencies. These agencies can have different procedures and experts with different approaches.” The idea is to persuade SMEs that the regulatory mechanism is not as complex as it looks and above all, that the regulators are there to assist rather than hamper innovation.


 Sean Milmo is a freelance writer based in Essex, UK, 

European Beat

With SMEs gaining wider recognition as the powerhouse of research and innovation in Europe, regulatory agencies are urging companies to engage with regulators early in the drug-development process.

Early Communication with Regulators is Essential for SMEs

Monoclonal antibodies (mAbs) are an important class of therapeutics. In recent years, however, increased attention has been devoted to the development of next-generation biologic therapeutics and manufacturing platforms. This shift is being driven by several factors, including the need to develop more efficient and cost-effective processes, reduce side effects, and increase specificity to enhance efficacy. 

Fragment antigen-binding (Fab) antibody fragments are one example of these next-generation biologics that are emerging as credible alternatives to the widely accepted mAbs (see 

). Abciximab (ReoPro, Eli Lilly), ranibizumab (Lucentis, Genentech), and certolizumab pegol (Cimzia, UCB) are Fab products currently on the market. There are several more in development by other companies.  

Fab fragments can be produced using more economical expression systems (e.g., microbial) while retaining the target specificity of a mAb. Other advantages include elimination of non-specific binding between the fragment crystallizable (Fc) portions of antibodies and the Fc receptor on cells, and the ability to engineer the Fab region for improved specificity and therapeutic efficiency. 

Antibody fragments are commonly produced as intracellular products in microbial expression systems such as E. coli. Following fermentation, cell harvest, and disruption, the downstream purification process typically consists of clarification, capture chromatography, polishing chromatography, and ultrafiltration/diafiltration. Final formulation and sterile filtration take place prior to the fill-finish (see 

). In this article, the authors outline some of the unique requirements and challenges posed by antibody fragments in terms of recovery, purification, and formulation. 


  Fab molecules are typically expressed in E. coli or yeast microbial systems. To achieve higher final titers, the amount of solids in these expression systems is increasing, making clarification more challenging. For soluble Fab proteins, the use of normal flow filtration clarification methods post-lysis is not common. Instead, tangential flow filtration (TFF) is often used in place of normal flow filtration. 

If normal flow filtration is desired, it is typically incorporated post-centrifugation, using a disk-stack or tubular centrifuge. Because particle size distribution in the batch is small (the particle size is quite homogenous and has small particle-size impurities), a tight depth filter is often employed. Microfiltration can also be used without centrifugation.

With secreted proteins, whole cells are separated from the fermentation broth and the particle size is larger. As a result, microfiltration (using TFF), centrifugation, and normal flow filtration are viable options. Endonuclease agents can also be added prior to clarification to digest DNA and RNA and to aid in the efficiency of the clarification process.

Protein A, followed by cation exchange and anion exchange, can be successfully used for the purification of Fabs containing the Fc region. For a Fab that does not contain an Fc region, capture is typically achieved via cation exchangers or mixed mode resins in bind/elute mode. The advantage of mixed mode is a wider operating window for binding in various conductivity and pH settings. With mixed mode, however, a longer period of process development may be required to develop an elution strategy. To overcome this disadvantage, single mode resins are being developed that operate in higher conductivities with high binding capacities and easy elution processes. A subsequent polishing step, typically using a smaller bead size, generally follows the capture step with the aim of improving the resolution. This polishing step could be ion exchange (IEX) or hydrophobic interaction chromatography (HIC) depending on the previous step and the protein of interest. If further purification is required, a third step could be implemented with HIC or IEX depending on previous steps. A typical purification for non-Fc containing fragments would therefore be cation exchange > HIC > anion exchange.

Fab molecules are typically small in comparison to mAbs (approximately 10 kD-80 kD), and as a result, tighter tangential flow devices are commonly used for the ultrafiltration/diafiltration (UF/DF) step. The membrane molecular weight cut-off recommended for adequate retention is one fifth the size of the product of interest. 1 kD-10 kD molecular weight cut-offs are typically used for Fab molecules. These smaller cut-offs can lead to lower permeate fluxes.

Additionally, some Fab molecules are PEGylated, which leads to higher viscosities as concentration increases. Because of this, the influence of the type of screen (screens help create turbulence and promote mass transfer) in flat-sheet devices is paramount and should be taken into account in small-scale optimization studies. A final consideration for 

i-expressed Fab molecules is downstream endotoxin removal, which is often achieved through anionic membrane adsorbers or charged membrane filters.


The primary consideration in formulation design for Fab protein therapeutics is stability. Protection of the protein from chemical degradation during storage as well as degradation from proteolytic influences in vivo is crucial in the formulation for all protein-based therapeutics. A second and equally important function of formulation design related to protein activity is to inhibit aggregation tendency. Aggregation may result in decreased efficacy due to inactivation associated with intermolecular interaction and the effect of steric inhibition.

When considering bioavailability, Fabs present both opportunities and challenges. Being significantly smaller than native monoclonal antibody molecules, Fabs designed as cancer therapeutics have the ability to provide better tumor penetration as compared to larger molecular species. Conversely, Fabs suffer the disadvantage of significantly shorter circulation and half-life in vivo, two characteristics that can be addressed in formulation.

PEGylation, the chemical modification of a protein by conjugation to poly(ethylene glycol) (PEG) polymers offers, in many cases, an appropriate solution to the challenge associated with shortened circulation and half-life of the Fab therapeutic. The most common chemical modification of Fabs with PEGs involves reaction of an activated PEG-maleimide with the cysteine residues on the Fab. PEGylated proteins offer the advantage of appearing 5-10 times larger in volume than proteins of comparative molecular weight because of the hydration effects associated with PEG. PEGylation results in improved stability and solubility while maintaining efficacy associated with the antibody fragment. Additional benefits of PEGylation include reduced toxicity, decreased clearance rates and proteolysis effect, and reduced immunogenicity and antigenicity although recent understanding of PEGylation effects has indicated an immune response associated with PEG that must be considered and monitored (1, 2). 

Another effect of PEGylation is the tendency of a PEGylated protein to demonstrate slower sedimentation behavior, even with larger volume as compared to the native Fab. In fact, the protein-associated PEG tends to dominate hydrodynamic properties associated with the bound protein and may also contribute to a decrease in aggregation-related effects.

Despite the aforementioned benefits, however, PEGlyation is not the only methodology that can yield stabilizing effects on therapeutic Fab protein formulations. Non-ionic detergents, such as polysorbates, are commonly employed in Fab formulation strategies as additional means of promoting solubility and reducing aggregation. Careful constructs of excipient ingredients (salts and buffers) may also be considered in terms of viscosity-related effects. Salt-enhanced repulsive protein-interaction effects based on the Debye theory of diffusive charge indicates a correlation to improved repulsive effects associated with monovalent salts (e.g., NaCl) as opposed to divalent (e.g., MgCl2) ionic ratios (3). Monovalent versus divalent cations and/or anions may also be considered as stabilizing species in formulation. F


Fab molecules are generating increased interest as the demand for target therapeutics with improved efficacy continues to grow. These molecules present unique challenges for the “traditional” processes used for the development and manufacture of monoclonal antibodies. 

Each of the steps in recovery and purification must be optimized based on the process requirements and the molecule characteristics to ensure a robust, stable, and scalable Fab production process.

The same careful consideration must be applied to development of the optimum formulation. As with all complex protein formulation development efforts, the goal of Fab formulation is preservation of product stability and efficacy. All potential technologies can and should be considered to assure a robust formulation. Care should also be exercised to ensure the appropriate level of both purity and consistency in all materials used in formulation to minimize the impact of impurities and the possible effect on stability and activity. 


The authors would like to thank Robert Blanck, market research & analytics manager at EMD Millipore, for his contributions to this article.

 97 (6) 2062-2079 (2008). 
  2.		R.P. Garay and J.P. Labaune,

, 2, 104-107 (2011). 
  3.		A.S. Parmar and M. Muschol, 

 97 (2) 590-598 (2009).
  4.		Chugai Seiyaku Kabushiki Kaisha, “Stabilizer for protein preparation comprising meglumine and use thereof,” EP 1908482 A1, April 2008.


  Claire Scanlan is process development scientist; Jeffrey Shumway is associate director, bioavailability enhancement; Juan Castano is process development scientist; Mark Wagner is purification technology and business development specialist; and Ruta Waghmare, PhD, is associate director, emerging biotechnology, 

, all at EMD Millipore, 290 Concord Road, Billerica, MA 01821. 

Feature

Antibody fragments pose unique challenges in recovery, purification, and formulation.

Downstream Purification and Formulation of Fab Antibody Fragments

By any measure, 2013 was a stellar year for life-science company IPOs on United States stock exchanges. A total of 50 companies completed initial public offerings through the first week of December and collectively raised $6.8 billion in capital.   

But after a busy summer, the fever pitch of life-sciences IPOs cooled in autumn even as companies continued to be added to the queue. Only four companies completed IPOs in November 2013 while seven companies postponed their offerings and one company withdrew its deal.    


    The slowdown reflects profit-taking by fund managers, who moved to book their gains to ensure their year-end bonuses, and it became difficult to get investors focused on new issues when they were more concerned about locking in profits as the year-end approached. The drop in the first day jump in prices reflects this, according to Burrill & Company analysis. Momentum drove the first day trading activity as investors chased first day IPO premiums generated within the sector. 

Between Epizyme’s IPO at the end of May and Foundation Medicine’s initial public offering on Sept. 24, 2013, 18 life-sciences companies debuted on US public markets. The share price of seven of these 20 companies rose more than 50% in the first day of trading. The volume of trading for these companies shows the first day frenzy that sent these issues soaring, which peaked with Foundation Medicine’s 96.4% return on its first trading day when 7.7 million shares were traded.

Of the 14 subsequent IPOs since Foundation Medicine’s initial public offering, MacroGenics’ Oct. 9, 2013 IPO was the only one to price in the target range and deliver more than a 50% return on the first trading day. By comparison, the two drug developers to complete initial public offerings in November, Relypsa and Karyopharm, rose modestly, or not at all in their first day of trading (see 

US life-sciences initial public offerings (IPOs) in 2013: First day of return and IPO-to-date performance.  

Burrill on Biotech

Frenzy cools as investors seeking a quick return turn elsewhere.

Life-Sciences IPOs Take a Breather

The pharmaceutical market in Brazil has been growing steadily and strongly in the past five years. The country is eying production and innovation, relying on the key support of the government. According to industry associations, biopharmaceutical innovation in Brazil will increase in the future as the country starts facing its challenges and participating globally with local and multinational companies.According to Abifina, Brazil’s Fine Chemicals, Biotechnology, and Specialty Industries Association, Latin American countries are still planting seeds to develop and compete in the global biopharmaceutical market. Brazil, however, plays a key role in the sector with a series of universities and institutes promoting research and development actions, a public health system capable of sustaining part of the local pharmaceutical industry, and a large consumer population at more than 200 million Brazilians. 


  “Brazil has definitely been growing steadily over the 10% mark in the past five years and we should continue seeing sales increasing annually by 12-15%,” Nelson Mussolini, executive president of Sindusfarma, told 

Sindusfarma is the São Paulo state pharmaceutical products industry association.

According to Mussolini, citing IMS Health figures for the past five years, Brazil’s growth is considered relevant if compared with data for Europe and the US, whose growth levels were around 1%. Mussolini says that the increase is especially due to the fact that the local job market has heated in the past five years and has been transforming the lower classes into active consumers of goods and services.  The country’s impact, however, within the biological products market is limited to production and sales and does not necessarily include relevant innovations, according to Reinaldo Guimarães, intellectual property director of Abifina.

“Our increase of the biopharmaceuticals market share is mainly a result of public investments for the government’s health system,” Guimarães told 

. The government is acting as a main buyer, currently responsible for as much as 25% to 30%, with an estimated participation reaching as much as 50% in the next few years, according to Guimarães. The government is establishing key partnerships with local and multinational, private, and public companies, and is funding local production capacity expansions for public and private companies. Brazil is also controlling the efficacy and safety of pharmaceutical products through market regulation. 

According to Guimarães, considering government expenditures with health products, biopharmaceuticals correspond to only 5% in terms of units sold and as much as 40% in terms of sales.

“The government is definitely investing on the development of the local biopharmaceutical sector through partnerships and by obtaining technology,” says Mussolini.

According to Sindusfarma’s executive president, however, companies face challenges to become profitable and still lack large production scales. It seems that Brazil-based companies seek to increase production scales and profitability, while government seeks less dependence on imports and a stronger local market, establishing a strong pharmaceutical partnership intended for growth.

With the economy still under control and regulation equivalent to those of developed nations, challenges now lie within high taxes and costs as well as strict price controls, according to Mussolini. Such issues impact profitability and affect future R&D actions as well as most companies’ long-term survival.

“Brazil competes globally in terms of market share …however, to become a global competitor in terms of new products or better products we still have issues to face,” adds the official. In terms of R&D, for example, challenges are attributed to the country’s lack of technological capacity to produce relevant innovation, says Guimarães.  

The Brazilian government through its Health Ministry has the goal of substituting imports for locally produced products by establishing technology transfer deals. Such measures have traditionally been used for vaccines and most recently for synthetic drugs as well as biopharmaceutical products.       


  The global financial crisis, which started in 2008, did not affect Brazil as severely as it affected the US and Europe; therefore, the local pharmaceutical market faced a smaller impact as well. During the past eight years (2003-2011), Brazil went from occupying the tenth position among global pharmaceutical companies to being the sixth largest pharmaceutical market, according to IMS Health. By 2016, a year in which the country will host the Olympic Games, IMS estimates Brazil will occupy the fourth position, with an estimated growth between 1415%, only behind the US, China, and Japan and surpassing the highly established markets of France and Germany. 

“I personally believe those estimates to be very optimistic, however, we are indeed heading in the direction of a 12% growth, which would include Brazil in the fifth position …,” says Mussolini.  

Nonetheless, “I am sure IMS Health knows exactly what they are saying,” adds Mussolini. “I believe there are no reasons for serious concerns,” concludes Guimarães.

Hellen Berger is a business news correspondent based in São Paulo, Brazil.

Global News

Brazil is the first Latin American country to emerge as a global biopharmaceutical collaborator.

Innovation and Market Growth in Brazil

 analysis and the evaluation of formulability, aided by new analytical tools such as hydrogen deuterium exchange mass spectrometry (HDX MS), are enabling the improved design, production, and formulation of protein-based drugs. As the development of biologic drugs based on antibodies and other protein/peptide molecules continues apace, the control and prevention of aggregation during manufacturing, downstream processing, formulation, storage, shipping, and administration remains an important issue. As a result, significant efforts have been invested in understanding the mechanisms behind aggregation and the establishment of predictive models and more advanced analytical techniques, particularly high-throughput methods, to enable the design and formulation of stable, protein-based therapeutics.


  Because aggregation can occur at any point during manufacturing, processing, formulation, and use of protein-based drugs, the consequences of this phenomenon can be significant. “Cells may stop secreting a protein if it takes on a new, aggregated form, leading to low titers and reduced yields and productivity. In addition, the formation of larger precipitates can cause the physical clogging of equipment and handling of the product during downstream processing,” says Jesus Zurdo, head of innovation with Lonza Custom Manufacturing’s development services team.

If aggregation occurs during formulation, which is of particular concern for protein-based products that require high concentration, the performance and safety of the product can be affected. “There is evidence to suggest that the presence of aggregates increases the immunogenicity of biologic drugs. How aggregates trigger immune responses, and which aggregates are responsible, isn’t known at this point, but it is thought that aggregates are more easily recognized than the parent protein,” Zurdo explains. As a result, there is increasingly regulatory pressure for biopharmaceutical manufacturers to reduce the risk of immunogenicity by not only controlling, but also preventing, aggregate formation.


  Aggregation involves the undesired unfolding or misfolding of proteins, which then enables protein-protein interactions and the formation of a possible range of larger molecules, from oligomers to very high molecular weight species. The most well understood aggregates are amyloid fibrils, which are composed of beta sheets stabilized by hydrogen bonds.

Typically, short polypeptide segments of 5-15 amino acids in length within folded protein structures that are affected by changes in the protein environment are responsible for nucleating the aggregation of the entire protein. Aggregates are classified as soluble or insoluble, as well as covalent (involves the formation of a covalent bond, often a disulfide linkage) or hydrogen-bonded (weaker interactions). Self-association of therapeutic proteins via covalent bonds is typically irreversible while aggregates formed via weaker interactions may be reversible upon changes in protein concentration, temperature, and pH, for example. As a result, aggregates can range in size from minute, invisible, non-filterable particles to large precipitates that are visible with the naked eye. In addition, some aggregates may be static while others may be dynamic. 

External factors do not alone determine when protein aggregation will occur. The physicochemical characteristics of the short polypeptide segments that comprise protein therapeutics also determine the propensity of each segment to undergo aggregation. While much remains unknown about the actual mechanism(s) of aggregation, a great deal has been learned about the influence of these physicochemical factors on the likelihood of an amino-acid sequence to serve as an aggregation nucleating site, according to Zurdo. 


  This knowledge about the relationship between the structural characteristics of peptide segments and the likelihood of aggregate formation has been used to develop predictive models. Other factors that have been used to develop these models include the unfolding kinetics and the thermal and colloidal stability of native proteins. The properties of the peptide segments of interest generally include the polarity, charge, dipole moment, hydrophilicity and hydrophobicity patterns, accessible surface area, charge, and number and location of aromatic residues.

 “The idea is to match specific amino acid sequences (and structural features) within proteins to the probability of aggregation by comparing their physicochemical and structural attributes to those of sequences with known aggregation behavior using predictive algorithms generated based on actual experimental data,” notes Zurdo. “It is very difficult to predict aggregation 

,” he adds. Thus, efforts have been invested in synthesizing analogs and studying their behavior to expand the database of information on known proteins and antibodies. Lonza’s database currently includes information on the aggregation behavior and properties of more than 1000 individual antibody molecules. 

Potential protein-based drug candidates are screened against the database to identify the potential for aggregation. If the results indicate that any amino acid sequences within the molecule might have the propensity for aggregation, then the protein can be modified, or if necessary, redesigned. “We have been successful to date in using such 

 analyses to address the issue of protein aggregation much earlier in the drug development process, which helps avoid potential problems throughout the scale-up, manufacturing, and formulation stages,” asserts Zurdo. 

New analytical tools for aggregate prediction


  Because the success of these predictive models depends in large part on the accuracy of the physicochemical property data of the peptide segments, effort has also been directed at developing more advanced analytical methods for obtaining more detailed structural protein information. One such method is hydrogen deuterium exchange mass spectrometry (HDX MS), which enables the detection of potential sites of aggregation because they are systematically protected from solvent exposure (HD exchange) when in the aggregate state, but not protected when in the free protein (monomer) state. “By knowing the specific site or sites prone to aggregation, targeted formulations or mutagenesis studies can be devised,” says Asish Chakraborty, business development manager with Waters Corporation.


  In a peptide-level HDX MS study, HD exchange for labeled soluble protein and aggregated protein samples is evaluated. The regions of a peptide involved in aggregate formation will show a different level of deuterium uptake from those regions in the free protein. Targeted electron-transfer dissociation (ETD) fragmentation of the peptides that exhibit deuterium uptake provides further detailed information. “Additional information on the dynamics of these interaction sites can be obtained by looking at uptake over a series of time points, where more tightly bound regions (less dynamic) will have a lower deuterium uptake profile than more transiently interacting sites that have more average solvent exposure,” observes Chakraborty.

HDX MS complements other technologies (e.g., nuclear magnetic resonance and X-ray crystallography) and has the advantages of requiring a minimal sample size, tolerance for many formulation components, and simplified sample preparation. It can also be used for larger proteins (hundreds of kilodaltons) and protein mixtures, and the presence of certain protein impurities is tolerated. The most important recent advances in this technology, however, have been in the development of informatics tools, such as Waters’ DynamX software, that enable much more rapid screening of the data produced during HDX MS analyses to determine the key regions of interaction. In addition, Waters has developed an integrated HDX MS system that has made HDX more routinely accessible and enabled automated data acquisition. 

“With these two advances, it is now possible to rapidly reduce a very large quantity of analytical data very quickly to interpretable results,” Chakraborty states. “What used to take weeks can now be accomplished in a couple of days.” He also points to the use of the liquid chromatography/mass spectrometry technique LC/MSE for data-independent fragmentation and identification of hundreds of peptic peptides evaluated in sub-10-min HDX-MS runs and ion mobility LC/MSE (LC/HDMSE) for gas-phase separation of ions to achieve cleaner ion detection and better quantitation of deuterium uptake for individual peptides as being additional important developments. He would, however, like to see ETD fragmentation more widely used. To achieve that goal, Waters is working on peptide-specific optimizations, looking to improve HDX MS structural data generated for larger and more complex protein systems to develop new informatics tools to facilitate more efficient acquisition and interpretation of ETD fragmentation data for HDX MS experiments.

Evaluating the formulability of protein-based drugs


  There have also been significant advances in analytical methods for the determination of protein aggregation, including high throughput capillary electrophoresis, immunoassays, and the optical imaging of aggregates for both quantitative and qualitative evaluations, according to Zurdo. Many of these methods, however, do not meet the requirements for very high-throughput analysis of potential drug formulations. 

“The formulation readiness of protein-based drugs--or their formulability--can be an issue, particularly for biotherapeutics that must be administered in high concentrations. Because there are too many variables, there as yet have been no models developed to predict protein aggregation in a product formulation. Therefore, manufacturers must rely on physical analyses. As a consequence there is a significant need for inexpensive, simple, highly robust, and very high-throughput techniques for the determination of protein aggregation that give either yes/no or better/worse-type information that can be used to map the regions of experimental space where products appear to behave well,” says Zurdo. 

The development of such methods is in the early stages. Some examples include modified immunoassays, self-interaction chromatography, and visual viscosity determination based on the diffusion of latex beads. “Unfortunately, there have been very few such techniques commercialized to date, and those that have been have not yet been fully validated. But progress is being made, and we expect to see some new methods being implemented in the not too distant future,” Zurdo notes. 

The behavior of the formulated product once administered to patients must also be considered, because protein aggregation can occur in the human body as well (e.g., Alzheimer’s disease is caused by protein aggregation). There are ongoing studies aimed at developing sophisticated cell-based assays for the evaluation of the immune response to protein aggregates, but despite some preliminary links between in vitro data and clinical outcomes, to date there is not yet a definitive or absolute direct correlation between results obtained through this type of assay and the actual responses observed in patients.

It is a challenge to do this type of analysis because the final formulation of a therapeutic protein often does not contain the exact same composition that was used during clinical testing, and even the minor changes that are acceptable during final formulation can lead to protein modification or aggregation and thus promote immunogenic responses,” observes Zurdo. 

Techniques to enable the design and formulation of stable, protein-based therapeutics.

Progress in the Prediction of Protein Aggregation

A main goal for FDA officials in 2014 is to establish a sounder basis for ensuring that drugs and biotech therapies meet high quality standards throughout the product lifecycle. The ongoing crisis in drug shortages has heightened public awareness to the importance of manufacturing operations in maintaining reliable access to needed therapies. Deaths and sickness from substandard products made by less-regulated pharmacy compounders persuaded Congress to bolster FDA authority to prevent such failings, and to require a more effective electronic tracking system to keep unauthorized and counterfeit drugs out of the global supply chain. 

Janet Woodcock, director of the Center for Drug Evaluation and Research (CDER), has made drug quality a top priority for 2014. To meet legislative requirements to improve systems for preventing drug shortages, CDER has been discussing options for more comprehensive collection of quality data at numerous industry meetings this past year (1). The process for establishing informative drug-quality metrics was a main topic at the Parenteral Drug Association (PDA)/FDA Regulatory Conference in September 2013, the Generic Pharmaceutical Association’s (GPhA) October Technical Conference, and the annual meeting in November 2013 of the International Society for Pharmaceutical Engineers (ISPE). PDA sponsored a Pharmaceutical Quality Metrics conference in December 2013 specifically to reach agreement with FDA officials on a “points to consider” document on the subject.


  FDA officials cite the FDA Safety and Innovation Act (FDASIA) of 2012 for providing a firm legal basis for setting clearer standards and for collecting data to assess manufacturing operations. Section 705 of FDASIA requires FDA to establish a risk-based schedule for inspecting drug manufacturing sites, which requires access to more detailed information on facilities and quality controls. Section 706 of the bill authorizes the agency to obtain information on drug production sites and operations in advance of an inspection (2). Together, these provisions support FDA’s strategy for improving how it assesses the ability of a manufacturing site to consistently produce medicines fit for intended use.

CDER officials emphasize that they are not asking industry for new and unknown information, but for access to system performance data already collected by manufacturers for internal use. Routine company metrics include number of lot release tests, out-of-specification results, and lots attempted, rejected, reworked, and reprocessed. Such manufacturing information generally appears in company annual product reviews that are made available to site investigators when requested. But that is too late to support CDER planning for inspections and postmarketing oversight, noted Russell Wesdyk, scientific coordinator in CDER’s Office of Strategic Programs, at the GPhA conference. And each company tends to collect data in its own way, using a range of definitions and sampling plans. FDA anticipates that standard metrics would reduce differences in how firms define “batch,” “lot,” and even “rejection.”

CDER now, consequently, has to assess the risk level of a manufacturing site based primarily on its compliance history, as seen in warning letters and field reports, plus records on recalls and quality problems. But these are not the most informative measures, said Wesdyk. More relevant data provided in advance would better determine the need for an actual site visit, as well as risk factors that might predict future drug shortages and other quality problems. The aim is not to issue “restaurant-style grades,” Wesdyk emphasized, but to provide broad results of industry performance so that companies can see where they stand relative to their peers. 

To establish this more informative drug quality data analysis system, FDA has embarked on a broad initiative to define the “right” metrics to collect and audit. Agency officials and industry executives involved with plant management, quality control, and regulatory compliance are examining how to analyze different quality measures and how this information should be provided to regulators to support FDA risk assessment and to help firms improve quality. There is interest in examining how company culture can shape these efforts, plus concerns about the unintended consequences of selecting the wrong metrics. A company might try to speed up lot closeout, for example, if there’s more focus on measuring batch cycle time. Review of complaint trending might lead firms to resolve problems superficially without getting to root causes.

These discussions support FDA efforts to develop guidance on quality metrics, which will be open for comment and feedback and should spur development of further consensus documents. Based on all this input, FDA hopes to define initial metrics by the end of 2014, setting the stage for collecting quality metrics from industry in 2015. 

Clear incentives for manufacturers to achieve higher quality production also are part of the discussion. Industry has been disappointed with past FDA quality initiatives partly because the agency granted little regulatory relief to companies that invested in often costly quality improvement efforts. FDA failed to curb requirements for regulating postmarketing changes or to reduce inspections. Now CDER leaders indicate that more complete information on establishments could lead to less frequent field inspections at firms that can document a site’s quality achievement, and there could be reduced supplement-filing requirements for low-risk manufacturing changes. 


These initiatives will be supported and managed by a new Office of Product Quality (OPQ) in CDER, which Woodcock expects to become operational this year under her personal direction. It’s “not a done deal,” Woodcock cautioned at the ISPE meeting, but plans are moving forward, and she anticipates that the new organization will “become real” in the coming months. 

A goal for OPQ is to establish “one voice for how FDA regulates drug quality”--for new molecules, generic drugs, over-the-counter products, biotech therapies, and small molecules alike. A main theme is to unify review and inspection so they can work together to ensure quality production through a product’s lifecycle. A review and inspection unit, for example, will oversee APIs. Specialists for new drugs may be grouped by clinical indications, while those monitoring generics may be organized according to dosage forms. Microbiologists for all products will be combined to provide a unified approach to microbiology. 

An Office of Surveillance in OPQ will manage inspections based on a complete inventory of facilities, plus metrics to assess performance and to identify quality operations--all supported by extensive information systems. And a central Policy Office will issue guidance and regulations and strive for more consistency in CDER actions. 

“We have to be predictable and consistent,” Woodcock emphasized at the ISPE meeting. She noted that more modern regulation of manufacturing is crucial for FDA to meet new user-fee goals, to accelerate the development of breakthrough therapies and biosimilars, to prevent drug shortages, and to further harmonize regulatory requirements around the world. At the same time, FDA looks to provide industry with flexibility in achieving standards for review and inspection. 

Regulators don’t make drugs, Woodcock observed, but can set the goal posts, encourage improvement, and hold industry accountable. In addition to establishing performance metrics that FDA can use to assess risk, the agency wants to encourage continuous improvement by industry. Manufacturers, Woodcock noted, have to recognize that there is a “cost to poor quality.” 

, 37 (10) (2013). 
  2. 	FDASIA, available at 

Regulatory Beat

Regulators work with manufacturers to improve systems for measuring product performance and manufacturing reliability.

Metrics Sought to Ensure Drug Quality

The most recent FDA (1) and International Conference on Harmonization (ICH) (2-4) guidance documents advocate a new paradigm of process validation based on process understanding and control of parameters and less on product testing. Consequently, the means of determining criticality has come under greater scrutiny. The FDA guidance points to a lifecycle approach to process validation (see

In Part I of this series, the author introduced the concept of continuum of criticality and applied it to the concepts of critical quality attributes (CQAs) and critical process parameters (CPPs). In the initial phase, the CQAs had their criticality risk level assigned according to the severity of risk to the patient. Applying a cause-and-effect matrix approach, the potential impact of each unit operation on the final product CQAs was assessed and each unit operation was thoroughly analyzed for its directly controllable inputs and outputs. Finally, a qualitative risk analysis or a formal failure mode effects and criticality analysis (FMECA) was conducted for each of the identified process parameters. The purpose of this assessment is to provide a focus for the downstream process characterization work required to complete process validation Stage 1 (process design).

This initial risk assessment is performed prior to the baseline characterization work and can be used as the primary means of determining the criticality of process parameters under the following conditions:
  
  •	When a platform process that possesses similar properties and process to another commercial product (e.g., new strength or new dosage form)
  •	When there is a significant body of published data on the process 
  •	When experimental studies and commercial data are available, such as when the process validation lifecycle is applied to a legacy product to substantiate the initial assessment. 
  
  In these cases, this initial assessment can be further bolstered through the addition of an uncertainty component to the traditional risk score. For example, a high-risk critical parameter with low uncertainty (due to substantial supporting data) may not require further study, but a medium-risk parameter with high uncertainty may require further experimentation to quantify the risk to product performance. 

The challenge facing most organizations is how to effectively evaluate the impact of potentially hundreds of process parameters on product performance to determine what is truly critical. Few companies have the time or resources to design experimental studies around all potentially critical process parameters. The initial risk assessment provides a screening tool to sort out the parameters that have low or no risk. 
  
  


  The goal is to increase process knowledge by providing a mechanistic understanding of the relationship between process parameters, raw material attributes, and CQAs. This is defined as both the demonstration of impact and the quantification of the contribution of each parameter to the product’s performance. Through this exercise, it will be possible to identify the process design space. The ICH guidance defines three elements--knowledge space, design space, and control space--to establish a process understanding (see 

ICH Q8 defines design space as, “The multidimensional combination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality.”

The design space is part of an enhanced process development approach referred to as quality by design (QbD). Prior to QbD, pharmaceutical development did not require the establishment of functional relationships between CPPs and CQAs. Consequently, process characterization experiments were primarily univariate (one factor at a time [OFAT]), showing that, for a given range of a process parameter (referred to as proven acceptable range or PAR), the CQAs meet acceptance criteria. While univariate experiments can provide some limited knowledge, a compilation of OFAT studies cannot typically define a design space because it cannot substantiate the importance or contribution of the parameter to the product CQA being evaluated. To do this, multivariate studies must be performed to account for the complexities of interactions when several CPPs vary across their control ranges. 

Design spaces can be developed for each unit operation or across several or all unit operations. Although it may be simpler to develop for each unit operation, downstream unit operations may need to be included to sample and test the appropriate CQAs. For example, to perform a multivariate study on a fermentation unit operation, additional processing through cell lysis and purification unit operations is needed so that CQAs may be sampled and tested. The challenge faced by most development programs is how to efficiently and cost-effectively derive maximum process understanding in the fewest number of studies. To do this, a staged approach using multiple studies is most efficient. 
  
  


  The following is an example of a simple staged design of experiment (DOE) approach. More complex DOE designs and strategies may be required, but these designs are typical:
  
  •	

Screening (fractional factorial, Plackett-Burman)

. To identify or screen out process parameters that have no significant impact on a CQA. Screening designs can test main (individual impact and contribution) effects of each parameter being evaluated.
  •	

. Having dropped out parameters, which do not impact the product CQAs, the refining step tests both main effects and interactions between the remaining parameters and generates first-order (linear) relationships between process parameters and CQAs. The criticality level of a CPP is determined from the quantitative impact on the CQA shown in the modeled relationship.
  •	 

Optimization (central composite, Box-Behnken).

 To generate response surfaces and illustrate second-order (quadratic) relationships between process parameters and CQA. This analysis allows optimal set points for the design space or control space to be identified to target desired CQA (or performance attributes) values.
  
  The DOE design assists in determining which parameters are studied and what set point value is used for each experimental run. The initial risk assessment, using prior knowledge and scientific principles, provides an expected relationship as to which CQAs and their related in-process controls will be affected by the given process parameters. Although the focus is on quality impact, process performance attributes (no quality impact) should also be sampled and measured as appropriate. This step is especially important during the optimization stage because a trade-off may be required in terms of optimizing quality and performance attributes. 

For process validation Stage 1 process characterization studies, analytical methods for measuring CQAs may not yet be fully validated, but still, must be scientifically sound. 
  The level of accuracy and precision of the analytical method or measurement system must be well understood because they directly impact the quantitative decision process when interpreting study results early in the process-design stage. Techniques of measurement system analysis such as Gage repeatability and reproducibility (Gage R&R) studies are recommended because they provide information on the variability of the measurement system. The Gage R&R study provides a quantitative measurement of the measurement tools contribution to variation for any measurement made. Typically, a percent contribution from R&R variability must be < 20% and demonstrate at least five distinct categories for the method result to be meaningful. The distinct categories are the number of discernable groups of measurements that can reliably span the range of the CQA. 

Including replicate runs in addition to the study experimental design provides crucial data for estimating the underlying variability of the study. This is because, during each run, small unmeasured and uncontrolled variations always occur and may influence the result. Two otherwise identically configured runs may produce slightly different responses due to changes in environment, equipment, measurement, sampling, and operators, among others. Even deliberately fixed parameters (those not under study) may not be exactly identical from run to run. Together, these are called “noise” factors and are important in discerning true responses (i.e., “signal”) caused by the changing parameters from the inherent variability. Differences between sets of replicate runs allow for the quantification of this variability. Large changes in the responses between replicates may indicate either an unstable experimental platform (such as poor run-to-run control) or that a low-risk CPP or non-CPP, may have a higher impact on the CQAs than originally assessed. 

Where a raw material has a critical material attribute (CMA) of medium-risk to high-risk impact to CQAs, it should be included as a parameter of the study where possible. Multiple lots or lots with extreme variation in CMAs may not always be available during early development or characterization studies. This limitation frequently is one of the primary drivers of establishing the continuous process verification (CPV) program in Stage 3 to monitor the future impact from this raw material variation. For large studies, multiple lots of raw materials may be required. Consideration should be given to either proportional mixing of the raw material lots for each run or use of a statistical technique called blocking, which incorporates change of material lots into the experimental design. 

Criticality is used as a risk-based tool to drive control strategies.

BioPharm International-01-01-2014