Novel expression systems are becoming practical alternatives to the more common mammalian cells (e.g., Chinese hamster ovary [CHO]) and microbial expression systems used for decades for biopharmaceutical manufacture. Vaccine production will likely be the area first and most affected by plant-based manufacturing platforms. With dramatically lower manufacturing costs expected, these will be the products most likely to radically impact bioprocessing in coming years. An increasing percentage of facilities involved in plant cell systems are outsourcing some of their production (1). Because of the relatively unique knowledge required in these systems, it could be a growing opportunity for CMOs that possess this specialized expertise over the coming years. And once the technology is more fully proven, with the benefits plant expression can offer, plant expression could become a common offering by CMOs, along with microbial and mammalian options.

A variety of vaccines are currently in development using different plant expression systems. Much of the push and initial funding for these technologies and products has come from the US biodefense R&D sector and its efforts to develop capabilities for rapid manufacture of tens, if not hundreds, of millions of doses of pandemic influenza and other vaccines. One such endeavor is Project Greenvax, a consortium of companies (Texas Plant-Expressed Vaccine Consortium), which is nearing completion of a tobacco plant-based modular facility in Texas that will be capable of producing more than 100 million doses of influenza vaccine/month (>1.2 billion doses/year) at a cost of pennies, not dollars, per dose. 

With plants able to make recombinant proteins often at much lower cost and in vast quantities, and with these technologies approaching commercialization, biopharmaceutical developers may be considering their use more than they currently do. Yet truly novel expression systems, such as plants and transgenic animals, have yet to make much of an impact on marketed products or even those in later-stage development. The industry remains satisfied with continued use of the proven and safe CHO, 

That satisfaction may shift as newer expression systems offer increased cost-effectiveness. Plant expression, with production costs considerably lower than conventional technologies, could well result in some companies adopting non-conventional (for biopharmaceuticals) pricing strategies, such as selling vaccines and other products for a few dollars per dose. This strategy may be feasible given production costs of pennies/dose, allowing acceptable profit margins even at very low prices, and enabling companies to emphasize high-volume markets, with this ideal for vaccines. 

Current state of plant cell capacity utilization


  BioPlan Associates looked at developers’ plant cell manufacturing capacity, as well as current and planned outsourcing of production in this area. Relatively few respondents to BioPlan’s study are involved in plant cell manufacturing. Over the past seven years of studies that the survey has asked about plant expression, only about 4% of respondents indicated participating in these production operations. By comparison, between two-thirds and four-fifths of respondents have been involved in mammalian cell culture during that same time frame. 

Given the small sample size for plant cell systems, the data can be susceptible to fairly large swings. Even so, the results give a window into this area and some data to compare with anecdotal evidence. 

In BioPlan’s 2012 study, respondents’ plant cell capacity utilization was at its highest level in several years, with biomanufacturers operating at 65% of total capacity. So, current plant expression-based capacity and facilities are actively being used.  Since 2006, capacity utilization rates in this area have grown at a compound annual rate of 4%, far outstripping comparable utilization rates for mammalian cell culture (-0.7%) and microbial fermentation (-0.8%).


  CMOs reported zero capacity use for plant cells for 2013, with few, if any, mainstream CMOs yet offering these newer systems (1). The data, however, represent capacity utilization, rather than capacity in general, and should not be confused with a lack of opportunity for CMOs to be involved in plant cell manufacturing.   

What is interesting to compare is planned plant cell capacity increases against planned outsourcing activity. And this is where the trends become interesting for CMOs. In 2012, on average, biomanufacturers involved in plant cell systems planned to increase their total production capacity by just 14% over the next five years. That compared to a 43% planned increase in mammalian cell culture capacity and a 30% increase in microbial fermentation capacity. 

Planned capacity expansions for plant cell systems appear to be trending downwards, with the 14% planning a capacity expansion down from highs of 29% and 34% in recent years. At the same time, separate data indicate that respondents are increasingly outsourcing plant cell manufacturing and will continue to do so over the next five years (1) (see 

In comparison with other manufacturing platforms, plant cell culture tends to be much less performed in-house, a fairly dramatic shift from years past. This past year, just 28.6% of plant cell culture users reported performing these activities 100% in-house, down from a range of 69-89% during the previous seven years. 

What’s more, an increasing percentage of respondents are projecting future outsourcing of plant cell production. BioPlan’s latest study found 57.1% of respondents involved with plant cell production planning to do some outsourcing over the next five years. 

Taken together, these trends suggest that rather than increase their plant cell capacity over the next few years, biomanufacturers are planning to look to CMOs for plant cell-based production.  


  Novel manufacturing systems for influenza and biodefense vaccines are becoming increasingly necessary and are being developed to meet the need for rapid manufacture of these vaccines. These new technologies must be scalable to exceptional levels that could reach as many as hundreds of millions of doses per week per facility or process line. These volumes are needed as population counts continue to grow in emerging markets, which will need coverage in the event of a serious threat.  And in case of a severe pandemic, everyone everywhere could even require immunization. The development of many plant expression systems is being driven by this requirement for rapid and disseminated production, and it seems unlikely that these efforts will be declining anytime soon. 

As plant-based influenza and other vaccines are developed, there is likely to be a shift in vaccine manufacture, and the current near-monopolies that exist among vaccine manufacturers will be challenged. With plant cell manufacture—both cultured cells and whole plants—generally offering significant cost-savings for large-scale manufacture, we can expect more established CMOs and also new specialty CMOs start to offer plant expression-based services. While data represent the views of only a small number of companies involved in plant cells, the results suggest that the industry will put CMOs’ plant cell service capacity to good use. 

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

Eric Langer is president of BioPlan Associates, tel. 301.921.5979, 

, and a periodic contributor to Outsourcing Outlook.

Articles

Perspectives on Outsourcing

CMOs may find opportunities in alternative expression services.

Novel Expression Systems Opening CMO Opportunities

Gaining access to Europe’s pharmaceutical markets has become a multi-stage operation. The licensing of a new drug is only a preliminary step in what can be a lengthy and complex procedure for some pharmaceutical companies, particularly those with innovative biopharmaceuticals. First a drug has to be approved for its quality, safety, and efficacy by one or more regulatory authorities. This approval can be either by the central London-based European Medicines Agency (EMA) for a product to be marketed across the region or by a national licensing authority for one to be sold only in specific European countries.

To be made available, however, in the public sector health services a medicine has to be approved for reimbursement. These reimbursement decisions are increasingly determined by separate investigations of medicines often called Health Technology Assessments (HTAs). While the regulatory agencies deal with benefit-risk aspects of medicines, HTA bodies gauge their effectiveness in relation to existing products, particularly on the basis of cost.

The role of these assessments in European national pharmaceutical markets has become so important that EMA has agreed to collaborate with EUnetHTA, a European Union assessment network, on involving HTA bodies in giving advice to manufacturers in the early stages of a drug’s development. 

The move has been welcomed by small- and medium-sized enterprises (SMEs), especially those developing advanced medicines. “SMEs are generally interested in receiving joint EMA/HTA advice, in particular when it comes to advanced therapies,” says Paolo Morgese, chairman of the HTA Topic Group of EuropaBio, a biotechnology trade association. “This is not necessarily linked to the development of new manufacturing processes per se, but more to the need of identifying a common set of data requirements,” he added. 

EUnetHTA has been stepping up its efforts to achieve more consistency in the methodology and content of HTAs among the European Union’s 28 member states. The assessments, carried out by a range of public agencies and advisory bodies comprising key opinion leaders (KOLs), mainly comprise appraisals of clinical trial data to ensure that a medicine gives added therapeutic value. But they are also starting to cover types of formulation and manufacturing processes particularly when gauging cost effectiveness. National bodies do not always carry out assessments but they are also conducted by regional clinical and funding organizations within countries. Their support can be necessary for a drug to be put on local formularies.  


  As a result of cuts in healthcare costs due to post-recession austerity programs, favorable HTAs are becoming crucial to the successful market launch of pharmaceuticals in Europe. HTAs backing reimbursement can be vital for new innovative medicines but can also be important to biosimilars. 

Despite Europe being a pioneer in the development and commercialization of biosimilars, it has a fragmented market for the products with national health authorities following different policies for their reimbursement and inclusion in formularies. While the EMA has the central responsibility for licensing biosimilars in the EU, many states have been carrying out their own assessment of them.

Biosimilar filgrastim, for the treatment of neutropenia with a granulocyte colony-stimulating factor (G-CSF) analog, had by last year built up market shares ranging from 90–100% in the UK and Sweden and several Eastern European countries. In Belgium, however, the penetration was only 1%, Netherlands 27%, and Switzerland 40%, according to figures from IMS (1). 

An European Generic Medicines Association (EGA) survey of national regulators in the EU pinpointed a number of obstacles to the uptake of biosimilars, including a perception of biosimilars being inferior to the originals, with doctors being reluctant to prescribe them because of  “hypothetical uncertainties in their safety and effectiveness” (2). 

Assessments have done a lot to boost biosimilars prescribing in some countries. “Prior to a pro-biosimilar initiative in Hungary the country had the lowest uptake of biosimilars in Central and Eastern Europe,” Ken Walsh, responsible for global pricing and market access at Sandoz Pharmaceuticals, the leading biosimilars unit of Novartis, said at an EGA biosimilars symposium in 2013 (1). “(Then) payers in Hungary recognized the need to incentivize biosimilars uptake.”

Prominent KOLs were recruited to endorse a pro-biosimilars white paper, which after being submitted to the country’s Ministry of Health, led to a reimbursement agreement for the products. By early 2013, the share of biosimilars in the Hungarian filgrastim market was 99%.


  In Denmark, selection of biosimilars available in the country’s health service is based not just on price but quality issues like stability, according to Walsh. This has helped to overcome concerns among Danish patients about production standards with biosimilars. Within less than two years, biosimilar somatropin, the growth hormone, rose from a market share of around 20% in the country to over half.

In Germany, one of Europe’s biggest markets for biosimilars, there have also been worries among patients and doctors about the quality and safety of biosimilars. As a result, doctors’ organizations, and insurers or sickness funds have combined to raise the uptake of biosimilars through assessment work and the introduction of prescription quotas. “In some regions the physicians’ associations together with the sickness funds have been very committed to making sure that doctors are fully aware of the quality and benefits of biosimilars,” says Bork Bretthauser, managing director of Pro Generika, Berlin, the German generic-drug and biosimilars association. There are still big differences in the penetration of biosimilars between German regions. With biosimilar erythropoietin (EPO), for example, in regions like Saxony and Bremen it has shares of over half while in Saarland the proportion is only around 15%, according to Pro Generika. “There is still a concern about variations in product characteristics between batches of the same biosimilar, whereas doctors need to be made aware that there are variations between batches of the original as well,” says Bretthauser. 

Across Europe, HTA bodies are taking different approaches to the issue of quality in products like biosimilars and other biopharmaceuticals. “The matter of quality is a new area for HTAs,” says Paul Catchpole, director of values and access at the Association of the British Pharmaceutical Industry (ABPI), London.  “Once a product like a biosimilar is assumed to have clinical equivalence to the original, there is a question about what evidence should be assessed for matters like comparative quality.”

The difficulties facing biosimilar producers with reimbursement and market access are those confronting all producers of new medicines in Europe with innovative production processes. Gaining positive HTAs and reimbursement decisions can be especially hard for SMEs with advanced therapy products and orphan medicines. Unlike with large drug companies, they do not have the resources to lobby assessment organizations.

Three years after its cartilage regeneration, biopharmaceutical ChondoCelect was approved by the EMA, TiGenix NV, Leuven,. Belgium, a cell-therapy specialist, has achieved reimbursement deals for the product in only three European countries--Belgium, Netherlands, and Spain, all of which it has facilities in.

“Gaining reimbursement approval has been much slower than expected,” says Maria Pascual Martinez, vice-president, regulatory affairs at TiGenix. “In the current economic conditions, securing access to health service patients is difficult, particularly when your technology can be regarded as being disruptive.”

Uniqure BV (Amsterdam) formed a partnership last summer with Chiesi Farmaceutici SpA (Parma, Italy), a specialist in the development and commercialization of rare disease therapies, to help gain reimbursement approvals for its Glybera medicine, an expensive orphan treatment for lipoprotein lipase deficiency (LPLD).  It was the first gene therapy to be approved by the EMA in November 2012. Important to the success of Glybera will be the efficiency of the process at its Amsterdam plant for producing gene cassettes for delivering transgenes into the body’s cells.  Chiesi’s strategy is to obtain reimbursement deals this year in the large European markets on a single price, likely to be a six-digit US-dollar figure, for a one-time treatment.

The big challenge for Chiesi and Uniqure is obtaining consistent HTAs on an orphan medicine, particularly on its cost effectiveness when there is little comparative evidence on issues like manufacturing efficiencies.

Producers of new drugs need more uniformity in assessments across Europe if they are to obtain the reimbursement prices they need to recoup their development costs.


  1. 	K. Walsh, “Biosimilars’ utilisation and the role payers do play in driving uptake in Europe: an industry perspective,” Biosimilar Medicines, 11th EGA International Symposium, London, Apr. 25-26, 2013.
  2.	EGA,

Survey to Members States and EEA Countries on Biosimilars

(EGA, September 2012) accessed Jan. 16, 2014. 

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

European Beat

High technology assessments are having an impact on biosimilars development in Europe.

EMA Collaborates with HTA Assessment Networks

Although proteins are known to be linear chains of amino acids that can form three-dimensional (3D) structures and enable binding interactions with specific biologic molecules, only recently have biopharmaceutical companies begun to pay more attention to their higher-order structure (HOS) of proteins. The previous lack of attention to this area is surprising given the crucial role this specific attribute plays with respect to the stability, efficacy, and safety (e.g., immunogenicity) of protein-based drugs. While there are numerous effective analytical methods for determining the chemical composition of proteins, such as the order of amino acids in linear chains, the weakest link in the development of protein therapeutics, according to Steven Berkowitz, a senior principle scientist at Biogen Idec, is HOS characterization. Much of this problem is a result of the limited capability of biophysical methods to obtain this information for routine purposes.

 While there are several types of analyses that can give low-resolution structural information (e.g., fluorescence, ultraviolet-visible [UV-VIS] analysis, circular dichroism [CD], etc.) and even more detailed data (e.g., differential scanning calorimetry [DSC], vibrational circular dichroism [VCD], and analytical ultracentrifugation [AUC]), only two methods to date—hydrogen deuterium exchange-mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) spectroscopy—are capable of providing detailed high-resolution fingerprint data on the HOS of proteins. Of the two, HDX-MS (which provides this high-resolution fingerprint indirectly) is becoming increasingly adopted as advances in instrumentation, methods, and software for data analysis are making this technique more accessible. NMR, while capable of determining the 3D structure of a protein directly, is slowly finding more frequent use due to the need to overcome several hurdles. 

A breakdown on higher-order protein structure


   Proteins have multiple layers of structure. The primary structure consists of the linear order of amino acids. When this linear structure folds on itself, it can take the form of an α-helix, β-sheet, or other shape, which comprises the secondary structure. The secondary structure can also fold to different degrees, and this level of folding is considered to be the tertiary structure of the protein. Although this folding can to some extent be stabilized via the formation of permanent interactions, (e.g., covalent disulfide bonds) folded protein structures are predominantly stabilized through weak interactions, such as hydrogen bonding and van de Waals interactions, which can be easily disrupted with changes in temperature, pH, protein concentration, and other conditions. HOS includes everything but primary structure, with some emphasis on tertiary structure. 


   Higher-order protein structure is crucial for the performance of protein-based drugs. Because some aspects of the HOS of proteins can change in response to changes in the protein environment, it is important that the behavior of the protein structure under different conditions and the actual structure of the protein in the formulated product be as well-defined as possible. This sensitivity of the HOS of proteins to the environment can cause problems with some biophysical characterization techniques. These technologies require the use of a different solvent, high dilution, high-salt concentrations, and/or the removal of interfering excipients, or require other conditions that are different from the environment of the formulated product, says Berkowitz. As a result, the structure being analyzed may not be the structure of the protein in the drug product. Therefore, biophysical characterization techniques that do not significantly alter the sample environment are highly valued. The results from analytical methods that do require changes to the formulation matrix of a therapeutic protein must be carefully designed so that meaningful comparisons can be made and the actual presence of real differences detected. 


   Although common and simple routine biophysical characterization techniques such as calorimetry, absorbance, and fluorescence provide low-resolution information on protein structure, collectively this information provides a coarse fingerprint picture of the HOS of protein drugs. 

Because different types of protein structural elements are known to melt at different temperatures, observing the melting behavior of a protein using DSC can provide some basic information about these structural elements in the protein, which generally correspond to higher-order structural elements, such as domains or possibly super-secondary structures, explains Berkowitz. Analysis of the UV–VIS absorption and fluorescence spectra of a protein provides general information about the local environments of the aromatic amino acids. Information about the amides in the peptide bond present along the polypeptide backbone of the protein can be obtained using fourier transform infrared spectroscopy. 

 “The overriding problem here, unfortunately, is the limited resolution. For example, the signals from all of the aromatic acids in the UV–VIS or fluorescence spectra just overlap too much to see the individual peaks. In fact, it is this low-resolution that initially lulled researchers into not paying attention to the HOS of these drugs,” Berkowitz observes.
 

Determination of secondary structure with VCD


  Another technique based on the vibrational motion of atoms in the protein is VCD spectroscopy, which is useful for determining the secondary structure of proteins, notes Wasfi Al-Azzam, a scientist in the Bioanalytical Sciences department at GlaxoSmithKline. VCD is sensitive to the interactions of dipoles, and the information in VCD spectra (e.g., peak frequencies, signs, patterns, and band shapes) makes it possible to distinguish between different secondary structures.

“This technique has the advantages of being effective at high concentrations and in various liquids, including water and buffer solutions,” Al-Azzam notes. “It is, in fact, specific for high concentrations and is being increasingly used as a result, because growing numbers of protein therapeutics are being formulated at high concentration for subcutaneous delivery. The minimal level of sample manipulation is a real benefit,” he continues.

VCD is also being used more frequently for comparability studies (e.g., under varying temperature and pH conditions). The spectrum obtained provides a fingerprint of the secondary structure of the protein, and can indicate the formation of fibrils and other folded forms. Conformational changes can also be seen to some extent, according to Al-Azzam.  

The only real limitation for this method is the concentration, which has a lower limit of < 100 mg/mL, although taking longer scans can extend that level somewhat. As protein-based drugs have increased in size, the fact that there is no limit on the size of the proteins that can be analyzed has become important. Even insoluble proteins and aggregates can be evaluated. Al-Azzam also points out that advances in instrumentation have contributed to the increased use of this analytical method, including improvements in the laser and photomultiplier, as well as the detectors, which have led to improvements in the quality of the spectra and the sensitivity.


    The use of AUC by the biopharmaceutical industry has been growing since the 1980s and early 1990s when its use as a complement to size-exclusion chromatography (SEC) was shown to be invaluable for the detection of aggregates that often are not observed using the former method, comments Tom Laue, a professor at the University of New Hampshire and cofounder of Spin Analytical, a developer of recently commercialized next-generation AUC instrumentation.  

AUC can provide two types of data—the sedimentation velocity (most common) and the sedimentation equilibrium. In both experiments, the material is placed in the centrifuge and its movement from the top to the bottom of the centrifuge tube is monitored with various optics systems/detectors, including UV–VIS and fluorescence detectors and refractometers. A chromatogram-like graph is produced with a baseline and peaks. The x-axis, rather than time, is the sedimentation coefficient, which is directly related to the ratio of the material’s molar mass and its size, and thus its state of aggregation. Sedimentation coefficients for monomeric monoclonal antibodies (mAbs) typically range from 6.1-6.9, while the value is around 9 for mAb dimers and 12 for mAb trimers. Thus, the data can be used to quantify the amount of dimer and trimer present, although it is not accurate at very low levels (<1%).

With sedimentation equilibrium, the data are directly proportional to the material’s molar mass. For either velocity or equilibrium sedimentation, samples with different protein concentrations are analyzed to determine if the fractional amount of aggregate increases or remains constant with protein concentration. A lack of change in the amount of aggregate formation indicates the presence of irreversible bonding, while an increase in aggregate formation with concentration proves the presence of reversible interactions. 

The advantage of AUC over SEC is that aggregates can be unknowingly retained on the column, and even if present at just 1–2%, these protein structures can be a real problem in the formulated drug, Laue points out. In addition, the high salt concentrations used with SEC have the potential to influence the protein HOS, as does the significant dilution of the sample that passes through the column. “AUC can be carried out in any liquid and does not suffer from these issues. As a result, it has become an excellent orthogonal method for confirmation of SEC results,” Laue states.

There is also growing interest in using AUC to evaluate the behavior of protein therapeutics in different media, such as serum. “In some cases, proteins are designed to interact with components like albumen to influence the pharmokinetics, while in other cases interactions are not desired. AUC can be used to determine if these types of interactions are occurring,” Laue explains.

The biggest limitations are the complexity of the data workup and the length of time for each analysis. Laue, however, notes that there are numerous workshops offered on a regular basis that provide training on data analysis, and he also expects that the software will continue to improve. “Throughput is the biggest issue. Only 7-8 samples can be done per day,” he says. John R. Engen, professor of bioanalytical chemistry at Northeastern University, notes that multi-angle light scattering is also being increasingly used along with SEC to obtain information similar to that available from AUC, but more rapidly.


Unfortunately, methods that provide high-resolution structural information suffer from lengthy analysis times and require training in data interpretation. According to Berkowitz, this situation, however, is changing for proteins that crystallize, x-ray crystallography can provide the most information, and depending on the protein, the analysis can be achieved rapidly. For proteins that do not crystallize, advances are being made particularly for HDX-MS, and to some extent, NMR. 

“NMR can provide massive amounts of information,” says Berkowitz. For some large and complex proteins, however, too much overlapping information is obtained such that it is difficult to deconvolute the spectral data. “Advances in analytical tools that make NMR more amenable for larger molecules and more complex structures are needed,” he adds. “Despite the high cost of NMR instruments, the high level of training required, and the complex data analysis, NMR plays a useful role in characterizing the HOS of protein biopharmaceuticals in certain cases. In the future, however, NMR should eventually be a dominant and valuable tool in the biopharmaceutical industry for the characterization of the HOS of proteins. We just need more innovation.”

HDX–MS has seen wider adoption than NMR due to significant advances in both the instrumentation and software tools, observed Engen. “The introduction by Waters Corporation of an instrument available for HDX-MS that includes the instrumentation, additional specialized equipment, and software tools for data analysis has greatly increased HDX-MS use in the marketplace.  Better processing power and software for data analysis have also made the experiment more accessible to people,” he notes. 

In addition, as the number of biotherapeutic proteins has increased, so has the use of HDX-MS because it provides useful information that is hard to obtain using other methods in a relatively short period of time for a wide variety of proteins, says Engen. “For example, proteins, and even specific conformational states of proteins that won’t crystallize can be studied under conditions that mimic physiological conditions. Furthermore, the location of effects in proteins that are hard to see with other techniques, such as the allosteric effects of binding, can be obtained because HDX-MS has higher spatial resolution than most classical biophysical tools (except NMR and x-ray crystallography),” he explains.  

There are some limitations, though. In HDX-MS, backbone amide hydrogens are detected through exchange of hydrogen and deuterium, and some sites involved in complex formation are in side-chain hydrogen’s, which are generally not detected by MS. Not all interactions (such as binding) alter the environment of backbone amide hydrogens, particularly in folded proteins, and a good understanding of the theory is essential to make sense of the data. In addition, analysis of large proteins (>500 kDa) is challenging because large numbers of peptides are produced during the analysis, and the data processing can be difficult or the spectrum can be compromised due to suppression of some signals as a result of complex mixes of peptides.  

Obtaining single-amino acid resolution is also a challenge. “Methods like electron-capture dissociation and electron-transfer dissociation fragmentation inside the mass spectrometer try to address this issue but they are not without problems. Most importantly, there is yet very little software available to evaluate the data obtained from these analyses,” Engen says. He also notes that creating many overlapping peptides, a technique that has been known for many years, can be used to increase resolution. By altering the digestion conditions, the mix of peptides produced can be slightly altered, and then subtractive analysis can be used to extract the data. This method, however, has some downsides and does not totally solve the problem.

Despite these issues, both Engen and Berkowitz see a bright future for HDX–MS. “This method is already leaps and bounds ahead of where it was 10 years ago, and great strides have been made in just the last 5 years as well.  We used to be afraid of proteins larger than 30 kDa, and now we are routinely working with analytes that are 200 kDa, and HDX-MS is now a common analytical technique,” Engen asserts.

In fact, HDX-MS data are starting to be used more extensively in the biopharmaceutical industry. “HDX–MS data appear in filings with FDA and are used to check formulation and comparability, to look at small molecule binding, and to map epitopes,” Engen observes. He goes on to add that academic research groups continue to further advance the limits of the method by expanding into the membrane protein space, enabling the analysis of ever larger complexes and improving the analytical aspects at every step. 

Features

Advances in instrumentation, software, and methodologies provide more information than ever on the higher-order structure of proteins.

Tackling the Challenge of HOS Determination

The Affordable Care Act (ACA) has elicited intense national debate and sent biopharmaceutical companies scrambling to understand how their business model, including its ability to innovate, will change as healthcare reform moves forward. Within this uncertain market, what is obvious is that the industry is in the midst of significant transition. Critical drivers of this change are largely macroeconomic; policy is simply accelerating change. Understanding this backdrop is crucial as the biopharmaceutical industry contemplates product innovation within the changing landscape of healthcare in the United States. 


  Costs associated with healthcare in the US account for a significant piece of the economic pie. A few key facts:  In 2010, the US spent $2.6 trillion on healthcare or an average of $8402 per person; the share of gross domestic product (GDP) devoted to healthcare has increased from 7.2% in 1970 to 17.9% in 2009 and 2010; and healthcare costs per capita have grown on average 2.4 percentage points faster than the GDP since 1970 (1). Despite this spending increase, Americans’ average life expectancy and the efficiency of the US healthcare system are ranked near the bottom of countries with advanced economies (2). These cost increases have a significant effect on individuals, households, and businesses. 
  
  The problem is not entirely a healthcare one; it’s a much bigger economic problem. According to a Rand study, increased family contributions to healthcare absorbed almost half of the growth in US before-tax income from 1999 to 2009 (3). Howard Shultz, Starbucks CEO, has been quoted as saying that his company spends more on healthcare than it does on coffee beans.  The problem is clear: healthcare spending is affecting the economy of the US and there must be a solution.

The Patient Protection and Affordable Care Act (PPACA), also known as the Affordable Care Act (ACA) or “Obamacare”, has a number of provisions baked in that propose to address cost and efficiency in healthcare. The ACA, for example, includes demonstration and implementation of new payment and care delivery models for Medicare patients (e.g., accountable care organizations [ACOs]), provisions to better coordinate care for people dually eligible for Medicare and Medicaid, reductions in Medicare payments, and new rules (e.g., disclosure and transparency) and new institutions (i.e., exchanges). Despite these attempts at cost-reduction, healthcare expenditure in the US is still projected to increase faster than national income for the foreseeable future (1).

In the pre-healthcare reform environment, drugs and devices were generally priced at a premium based on modest, incremental improvement in features and benefits (i.e., length of drug action). A fundamental belief within the industry has been that premium pricing equals healthcare innovation. Ultimately in today’s cost constrained world, a premium price without demonstrated economic and clinical value is just a cost driver, and cost drivers are being targeted with increasing intensity by payers, providers, and patients. As risk is shifted from payers to provider systems and consumers (patients), the need to show cost-effectiveness of products becomes a core requirement for market access today and in the future. 
  
  


  With the ACA’s endorsement of ACOs, there is a clear emphasis on shifting risk associated with the quality and costs of healthcare to the provider. The focus on the ACO model of coordinated and accountable care is largely based on the belief that rising healthcare costs are, to a large degree, driven by uncoordinated care focused on treating disease rather than on maintaining health. The goals of ACOs are laudable—reduce costs and improve quality of care through coordination among providers. In the current environment, and especially in locations where hospital systems already control significant market share, moving quickly will enable them to lead in creating and controlling ACOs. The result will be that more physicians will likely become employees of systems and have less influence and control over how healthcare is provided.  
  
  Another equally significant trend is healthcare consolidation. There has been a spike in the formation and growth of hospital systems and the employment of physicians. In addition, payers are also becoming more involved by creating partnerships with hospitals.  Data suggests that the percentage of physicians working in practices owned by a hospital or integrated delivery system increased from 24% in 2004 to 49% in 2011 (4). It’s no surprise that consolidation brings greater purchasing power. The consolidation of hospitals into fewer, larger systems increases the leverage of buyers with all suppliers, including pharmaceutical companies. Beyond that, the integration of insurers with delivery organizations potentially increases the power of administrative perspectives in purchasing decisions relative to clinical perspectives. The likely consequence is accelerated pricing pressure. 

With the consolidation of provider organizations and the employment of physicians has come a profound shift in power. Treatment decisions that historically were made at the physician level are being institutionalized, with management’s role growing at the expense of clinicians. Enterprise-level senior executives and hospital administrators increasingly are making decisions about what drugs will be on the formulary, and which will not. Not only have clinicians been forced to share decision making power with management, but administrators are increasingly restricting the access of sales reps to individual physicians. The upside is that biopharmaceutical companies will no longer need as many sales representatives to promote their products. The downside is if they want to educate physicians about the value of their products, manufacturers will need to explore new avenues for doing so.  

Even more important is that influencing decisions like what choices go on the formulary will require an entirely new approach to business development that doesn’t begin to resemble physician detailing. Finally, as healthcare systems continue to develop standardized, evidence-based care paths as a means of controlling clinical quality and costs, biopharmaceutical companies must understand the clinical and economic data these decision makers value and how this information can be communicated to them in an effective manner. In a world of new payment models and changing reimbursement values, demonstrated economic and clinical value is becoming paramount.



  Payers are already demanding data from manufacturers that demonstrates a product’s economic and clinical value in directing reimbursement decisions. Moving forward, hospital administrators with decision-making power will rely on published evidence as well as the experience of their clinical teams to determine a product’s value.  To this end, clinical studies must continue to become more comparative by design, not just to the “gold standard” treatment, but also to non-pharmaceutical comparators and watchful waiting. 
  
  

Similarly, while randomized controlled trials (RCTs) are still essential for regulatory approval and market entry, payers and healthcare administrators are becoming more focused on measuring outcomes in the broader population. Biopharmaceutical manufacturers will need to place greater emphasis on post-market surveillance studies and real world data to ensure their product’s claims of effectiveness are being met. If biopharmaceutical manufacturers fail to plan for and conduct this research, be assured that decision-makers will gather the evidence to determine how competitive products at different price points actually compare. Today, true innovation (things that make healthcare better and cheaper) that addresses broader stakeholder requirements is able to command premium pricing, but marginal improvements focused on incremental features and benefits are suffering.

As healthcare consolidation continues, payers and delivery institution management increasingly share the physician’s once exclusive role in treatment decisions. With cost as a significant driver, for example, payers and provider systems already place pressure on physicians to use generics. In fact, this pressure is built into computerized physician order entry (CPOE) systems, making branded drugs an exception requiring additional justification. Clear differentiation in terms of clinical outcomes and economic value will be a primary way for manufacturers to take on this issue. 

Within this context, the concept of bundled pricing is gaining momentum as a reimbursement model designed to address demands for better health outcomes at lower cost. Promising to deliver a standardized set of services for a fixed price with specific quality commitments will create incentives—lacking in the current fee-for-service payment system—for providers to align their efforts to contain underlying costs. Providers adopting bundled pricing must focus on evidence-based care paths that allow them to define the crucial actions, decision points, and products used in treatment. When optimally formulated, such care paths will provide the much-needed link between clinical activities and associated costs. They offer a tool that hospitals can use to gain control of their core production process, using data to collaboratively lower costs and improve outcomes. Being unable to produce a compelling economic and clinical value narrative may set the stage for a product being left out of a provider’s care path, and that will have a significant impact on market access.
  
  


  There is still an urgent need to innovate. With the decision maker (stakeholder) set expanding and the criteria for purchasing and defining innovation changing, business model change is required for success. The reality of the new world is that resistance to new products by decision makers is increasing while access and physician influence decline. Healthcare providers (and payers) are focused on the cost and quality of the care they provide and are increasingly risk-averse about new technology and products. Overall, this scenario does not augur well for innovation and, in fact, suggests the need for change in what products are developed and how they are commercialized.
  
  To be successful in this new environment, biopharmaceutical companies must adopt business strategies that consider the interests of all key stakeholders who can influence medical plan design, treatment, and other related decisions. This includes physicians, patients, payers, and policymakers. Every functional team within an organization, from business development and R&D to market access, pricing and reimbursement, has a role to play in making sure the company is set for success in this evolving environment:
  
  • The business development department will need to broaden the competitive set as they look at potential opportunities. More than ever, they will need to make sure that prospective assets address unmet needs, and offer meaningful differentiation that is sustainable over the product’s lifecycle.
  • R&D will need to determine the requirements for clinical differentiation and build studies to incorporate these data points, based on identification of what’s really important to stakeholders.
  • Sales and marketing departments will have to develop messages, including economic messages, targeted to the specific needs of stakeholders. They’ll also need a broader group of strategic marketing capabilities as they interface in new ways with a broader group of internal and external constituents.
  • Quality and regulatory functions will need to be prepared to meet new hurdles both before products get approved and once they’re in the market. With an increasing emphasis on post-market safety surveillance and real-world evidence, companies must have the appropriate measures put in place to capture this information. 
  • Clinical groups will need to broaden their approach to collecting data and how they look at research design. They’ll need to work closely with health economics, market access, and managed market teams. As observational and non-traditional studies play a more prominent role in defining what the specific comparators should be, companies will need to look at how they use in-market data to influence new study design and provide insights to guide future product development.
  • Market access teams will become increasingly important as they’re called upon to translate the implications of what’s happening in the external environment for their internal partners. They’ll need to take on a more strategic role as they interpret market dynamics and sort out where new thinking needs to be integrated within their current structure.
  • 	Pricing and reimbursement teams will need to clearly demonstrate how they established their price, specifically the comparators used, the data that supports it, and the particular patient population that will benefit from their product.

Specific strategies that may be used include:
  
  • Develop “service wraps” around products/portfolios that provide more complete solutions across the continuum of care.
  • Create strategic sales teams directed at evolving healthcare delivery models (ACOs, bundled pricing, population health) and payers. Ensure this new sales team is capable of strategic conversations and joint problem solving (versus feature and benefit discussions).
  • Consider developing at-risk payment models with employers and payers based on achieving a certain outcome.
  • Ensure the early development of a strong economic and clinical value message relevant to all stakeholders for all new products. 
  • 	Ensure that the comparative value proposition for a new product is as clear as possible.
  • Consider alternative clinical data sources in addition to RCTs such as post-market longitudinal studies, patient reported outcomes, and other sources of real world evidence. 
  • Generate evidence that delivers the net benefit of biopharmaceuticals in the broadest “real world” context (i.e., clinical, economic, quality of life, and productivity measures) in addition to generating evidence that satisfies the needs of regulatory agencies.
  • 	Invest in targeted therapeutics/personalized medicine based on biomarkers with the goal of drug-diagnostic companions with the ability to demonstrate high value in small subsets of the population. 
  
  

The implications for the industry are clear: incremental innovations will be closely scrutinized. Unless there is clearly demonstrated value, new products are unlikely to command premium pricing. As “generics as standard-of-care” firmly settles in, biopharmaceutical companies will need a higher degree of clinical and economic differentiation to be successful. In today’s market, differentiation is more important than ever. Key stakeholders include payers, large employers, and increasingly, patients who care about health outcomes and affordability. In addition, it’s wise to remember that it’s a much more transparent marketplace. Government agencies are funding cost-effectiveness studies; payers and large providers are investing in health technology assessments and forming alliances to analyze real world data; and most information is available on the Internet.

This new reality has a number of implications for innovation in biopharma companies, most of which point to the need to produce cost-effective, tangible health improvements. The business reality is that close to 11% of the US healthcare spend is on branded pharmaceuticals (5). However, the perception is that it’s drug prices that are driving healthcare costs. If generics were good before, they are even better now with multiple players in the decision-making process, including patients facing escalating co-pays and deductibles. The incentive is to select a generic over branded unless there is clear and compelling economic and clinical value.  

Despite these challenges, manufacturers must continue to innovate; significant unmet medical needs remain. Changes already taking place in healthcare are resulting in market access challenges for new technologies that, in turn, can be expected to impact innovation investment decisions. The exact magnitude of the impact remains hard to predict. 
  
  

The U.S. Health System in Perspective: A Comparison of Twelve Industrialized Nations

 (Commonwealth Fund, July 2011).
  3.	D.I. Auerbach and A.L. Kellermann, 

, 30 (9) (September 2011).
  4.	MGMA 2012 report based on 2011 data. Englewood, CO Medical Group Management Association (2012).
5. 	R.E. Numerof, 

, is president ofNumerof & Associates, Inc. (NAI).   

Global Reports

The biopharmaceutical industry contemplates product innovation within the changing landscape of healthcare.

The Affordable Care Act's Impact on Innovation in Biopharma

In addition to comprehensively characterizing the physicochemical/structural properties of a biosimilar protein/glycoprotein (APIs and formulated therapeutics), manufacturers of biosimilars must demonstrate comparability to the innovator biologic. Biosimilarity is achieved by tweaking the production process until it yields a product that is similar to the innovator drug as determined through extensive characterization. Initially, batches of the innovator molecule are evaluated to determine the exact amino acid sequence, any post-translational modifications, and the level of structural attribute variability over time. Once manufactured, the structure of the biosimilar must then be confirmed and the data compared directly with that of the innovator protein. “Demonstrating a high level of similarity between the biosimilar and the branded drug can have a big impact on the approval process for the biosimilar; fewer differences translate to lower risk and the opportunity for reduction of clinical experience needed for approval,” notes Scott Berger, senior manager for the Pharmaceutical and Life Science Team at Waters Corporation.

As a result, tests must be carried out to determine the molecular weight and size, amino acid sequence and composition, the peptide map, including the location of sulfhydryl group(s) and disulfide bridges, other post-translational modifications, such as deamidation and oxidation, the degree of heterogeneity, the carbohydrate structure, the pegylation pattern, and impurity and degradation profiles. Various analytical fingerprints must also be obtained, including the electrophoretic and liquid chromatographic patterns and various spectroscopic (e.g., ultraviolet-visible, circular dichroism) profiles. Numerous orthogonal techniques are used for these analyses to more definitively detect any differences in the biosimilar and branded biologic, and many of them involve separation via high-performance liquid chromatography (HPLC) or, more frequently today, ultra high-pressure liquid chromatography (UHPLC).


  One of the biggest challenges that biosimilar manufacturers face is the need to do extensive monitoring of product attributes using multiple modes of liquid chromatography throughout the development process, including peptide mapping, charge variant assays, ion exchange (IEX), and size exclusion chromatography (SEC) to name a few, according to Berger.

The first hurdle is accurately deformulating the innovator drug without affecting the structure of the protein, which often is very sensitive to the environment. Many analytical methods cannot be carried out directly using the formulated drug because the presence of surfactants, carriers, and other ingredients can influence the results. “The chemist needs to be aware of these potential issues, particularly if the analyses will be carried out at different pH and ionic strength levels than the formulated product. Orthogonal methods are particularly valuable at this stage,” Berger notes.

For Daniel Peckman, manager of the Biochemistry Department at Eurofins Lancaster Laboratories, the major question that must first be answered prior to developing an HPLC method is ‘What attributes of the biologic need to be evaluated?’  “The biosimilar may have unique properties that should be considered, such as undergoing undesired changes or modifications during production that might impact how it functions as a therapeutic. Therefore, the goal is to develop a method for monitoring that change in comparison to a standard over time. A more in-depth method, like liquid chromatography followed by mass spectrometry, may be warranted in such a case.”


  Ensuring consistent sample preparation is crucial for protein analyses for the same reason. Even slight variations in environmental conditions or sample properties may affect the results, both during method development and biosimilar analyses. Perturbations such as freeze-thaw cycles can lead to protein aggregation, for example, according to Peckman. Control of pH, salt content, and concentration is also important. One way to address this issue, according to Berger, is to use automated mobile phase mixing methodologies/approaches. “Not only does a properly designed, automated system enable robust method development with consistent and reproducible results, it can lead to increased productivity.”

For biotherapeutic analysis, Waters’ Auto-BlendPlus Technology uses pure solutions and concentrated stocks and calculates the percentage of each to achieve the desired system conditions over a wide range of pH and salt concentrations and gradients so that variations in the buffer solution can be minimized. The system, according to Berger, ernables researchers to efficiently explore separation spaces and is effective for the optimization of IEX methods for charge variant separations. 

Using columns designed for higher resolution protein separation


  Higher resolution is often needed for biosimilar analysis due to the high complexity of many biotherapeutics and the need to separate closely related product variants. “UHPLC provides superior resolution of variants and greater sensitivity for more in-depth protein characterization,” says Berger. He stresses, though, that combining UHPLC with columns designed specifically for protein analysis is important for obtaining the maximum resolution and ideal selectivity for the analysis.


Column selection can be tricky, however, depending on the nature of the biologic. “The key is to select a column that provides adequate separation and performs consistently over time,” notes Peckman. “One question to ask is ‘What column chemistry will give acceptable separation of the protein impurities and still yield a good mass balance of loaded sample?’  In other words, several HPLC columns may successfully bind and elute the protein of interest, but the best choice will be the column that repeatedly elutes all the protein injected, the same way each time it is used.”  

Carryover of residual protein on the HPLC column can also be an issue and typically results because the interaction between the protein and the column packing is too strong. Several things can be attempted to manage this problem, according to Peckman, including selection of a less-aggressive column packing, adjustment of the initial concentration of the mobile phase, and using more aggressive gradient elution percentages.        

“HPLC column manufacturers have really begun to focus on protein-related HPLC and are specifically designing columns that address the particular challenges associated with the analysis of biologics,” says Peckman. Examples include column packings that are stable at elevated pH, which has historically not been the case in protein chromatography. He also notes that new column packings have been developed that provide good separation while limiting non-specific binding, and columns that have historically been used for traditional chromatography are now available in versions that allow higher resolution separations and faster methods. 

In fact, there are application-specific columns available for ion exchange, size exclusion, reversed phase, affinity, and hydrophilic-interaction chromatography (HILIC). The Protein separation technology (PrST) columns from Waters, for example, are based on a core ethylene-bridged hybrid (BEH) particle designed to separate immunoglobulins and other recombinant therapeutics with high resolution while minimizing unwanted secondary interactions that can lead to column carryover, according to Berger.


  Making minor adjustments to methodologies once an effective system has been established and not introducing more variables into the equation through sample mishandling are two key factors for success in biosimilar analysis, according to Peckman.  He also recommends trending the data closely so sample stability can be established early. Using traditional methods, such as reversed-phase and size-exclusion chromatography, for purity assessment, but applying more in-depth techniques, particularly mass spectrometry (MS)-driven approaches, for the determination of slight modifications has also been effective for him. Peckman also notes that the more data points the better, and significant information can be gained by performing degradation studies on the biologic and then evaluating which methods show the resulting changes with reproducibility. 

Tackling the challenge of glycan analysis


  Glycan analyses can be particularly challenging given their diversity and complexity, but recent UHPLC/mass spectrometry solutions developed by professor Pauline M. Rudd at the National Institute for Bioprocessing Research and Training (NIBRT) in Dublin, Ireland are changing that.  “UHPLC is ideal for biosimilar analysis and confirmation of biosimilarity because it is high resolution and very sensitive (to 0.1 to 0.5% of total glycans). It is also robust and highly reliable, provides quantitative data, and is much less expensive than MS. The system we developed was also purposely designed to be easy to use and can be operated in a manufacturing environment,” Rudd notes. The flow-through from the UHPLC can also be analyzed directly using on-line MS and MS2 to give confirmation of the LC data.” She also notes that the latest Waters software enables these orthogonal sets of data to be conveniently compared in the same screen.

Detailed analysis of glycan pools involves the identification of the oligosaccharides present, including their monosaccharide sequences and linkage characteristics and the stereochemistry and level of branching. Rudd’s team has shown that HILIC chromatography enables the most complete glycan separation of both charged and neutral glycans in a single run, including structural isomers, while weak anion-exchange (WAX) and reversed-phase (RP) chromatography provide important specific information because they resolve the glycans on a different basis. Rudd notes that amplification of the signal by means of stoichiometric derivatization with the fluorescent tag 2-aminobenzamide for fluorescence detection prior to UHPLC analysis is advantageous because the relative amounts of the glycans can be determined. When a similarly labeled dextran ladder (poly-glycose) external standard is used, glucose units (GU) can be derived from the calibrated retention times. GU values for glycans are stable from day-to-day and allow accurate comparisons of HILIC data derived from different instruments. Furthermore, structural information can be obtained from the shift in the GU value if UHPLC analysis is performed after digestions of aliquots of the glycan pools with exoglycosidase arrays. These enzymes specifically cleave the glycosidic bonds of individual terminal monosaccharide units.

Rudd and her colleagues at NIBRT developed GlycoBase, a database that includes HILIC and MS data on more than 400 2-aminobenzamide-labeled N-linked glycans and 75 O-glycan structures, and GlycoExtractor, a web-based tool for the extraction of large volumes of processed glycan profile data. A low-cost, automated N-glycan analysis platform for the high-throughput glycoprofiling of all glycoproteins including immunoglobulin G antibodies (IgG) has also recently been described (1). This platform enables rapid IgG affinity purification and sample concentration, protein denaturation, glycan release, and fluorescent labeling on a 96-or 384-multiwell filtration device. Glycan purification is carried out on solid-supported hydrazide, and the released and labeled glycans are quantified by UHPLC. “Using this high-throughput workflow, it is now possible to profile immunoglobulin glycans as well as glycans attached to other biopharmaceuticals efficiently, rapidly, and relatively cheaply,” Rudd asserts.
  

Waters has incorporated the UHPLC glycan GU database and automated analysis platform developed by NIBRT into its suite of tools for glycan characterization and detailed analysis. “With the new Glycan Application Solution with UNIFI biosimilar manufacturers can achieve automated, high-confidence glycan structural assignments,” notes Berger. Adds Rudd: “This system will also be compliant with the guidelines of the MIRAGE (Minimum Information Required for a Glycomics Experiment) project that is being developed by glyco-analytics and bioinformatics experts from around the world.”

Troubleshooting

Automated sample handling, advanced glycan analysis and specially designed columns are helping biosimilar manufacturers speed up confirmation of the biosimilarity of their products.

Advances in UPLC Techniques and Column Chemistry Aid the Confirmation of Biosimilarity

Disposable biocontainers used for media preparation and storage, and for single-use bioreactors, have become well-established equipment for research, development, and production of preclinical and clinical batches of bioprocess drug APIs and vaccines. They are now increasingly being implemented for full-scale approved product manufacturing. Numerous studies have been published or presented at conferences demonstrating comparable performance characteristics for cell-line growth and productivity relative to media stored in traditional stainless steel or glass tanks and cultured in stainless steel or glass bioreactors. 

In the shadow of these broad successes, however, there have been several reports of diminished cell growth properties associated with some biocontainer or bioreactor films, generally during initial qualification, but on rare occasion, attributed to a change in film formulation or manufacturing/transport conditions with a previously qualified and historically suitable film. One of the earliest reported cases of reduced cell growth in a single-use bioreactor was associated with adsorption of cholesterol in a defined culture medium supplemented with a cholesterol-lipid complex that was coupled to methyl-β-cyclodextrin.  In this case, the bioreactor film had previously been qualified for culture of the same NSO cell line in the media supplemented with serum. Reduced cell growth in the bioreactor with the defined culture media was attributed to extraction of cholesterol from the cell membranes in the presence of excess methyl-β-cyclodextrin and subsequent adsorption to the low density polyethylene (LDPE) film contact layer surface. The problem was resolved by modifying the formulation of the culture medium to remove the excess methyl-β-cyclodextrin (1). 

More recently, Eibl et al. reported a study of six different cell lines cultured in different media formulated with water that had been stored in nine different polymer film biocontainers from various suppliers (2).  In this study, unidentified aqueous extractable compounds (cytotoxic leachables) from two of the nine biocontainers tested appeared to reduce growth properties of some cell line/media combinations, while others were not affected. 

Water extraction can result in solubilization of polar (ionic) compounds from the biocontainer film. Storage of culture media supplemented with serum or a lipid-based additive can be expected to leach these polar compounds (salts), but also extract (leach) moderately non-polar (non-ionic) compounds due to the non-polar solubilization properties of these more non-polar additives. Further study of cell line growth in supplemented media previously stored in single-use biocontainers is underway.

A related study using culture media as the extractant was reported by Wood et al., who described a survey of 13 different film biocontainers from eight different suppliers using different cell culture media warmed to 37 ˚C as the contact fluid, prior to culture of different Chinese hamster ovary (CHO) cell lines (3). Among all the biocontainers tested, only one was reported to show some cytotoxic effect under these conditions, for a single cell line.  Root-cause investigation of the problematic biocontainer suggested a volatile or air-quenched compound was associated with the observed negative growth parameters (cytotoxicity).  This volatile leachable was associated with water or media and concentration dependent on temperature and exposure time (as all leachables are). The cytotoxic effect could be eliminated by aeration, allowing the volatile compound to dissipate prior to filling with culture media. Growth rates of a model cell line in non-irradiated (non-sterile) bags and glass bottles were comparable and higher, suggesting the volatile leachable was a degradant generated by gamma irradiation. Based on their test data, the authors suggested cell-culture specific screening of single-use components for cytotoxic effect in addition to extractables studies with model solvents (generally assessed versus potential leachables levels and potential systemic toxicity effects in the final dosage).


The most controversial case was reported by Hammond et al., published in the 

PDA Journal of Pharmaceutical Science and Technology 

(4). This study has garnered an extraordinary amount of attention due to its investigation into a root cause of observed biocontainer-associated cytotoxicity.  The Parenteral Drug Association (PDA) has anecdotally reported this paper to be its most downloaded in history. The authors reported a cytotoxic effect in two sensitive cell lines with a particular single-use biocontainer, attributable to a degradation product of tris[2,4-di-tertbutylphenyl] phosphite (Irgafos 168, BASF), a commonly used antioxidant compound formulated into many biocontainer films and other plastics approved for use in drug and food manufacturing.  

This study has led to calls by some users for biocontainer suppliers to reformulate their products either with a different antioxidant or even no antioxidant at all.  Such suggestions, however, are ill advised. For one, other biocontainers formulated with Irgafos 168 have shown no cytotoxic effect in numerous studies. Being a widely used antioxidant in LDPE films, as well as other single-use plastic components—perhaps the most widely used—experience with potential cytotoxicity of degradants of alternative antioxidants is far more limited. Changing to a different antioxidant, therefore, provides no assurance that similar cytotoxicity effects will not be seen, and perhaps even to a greater degree.  Avoiding all antioxidants is also undesirable as many plastics will be rapidly degraded by oxidation under the heat of processing or the energy of gamma irradiation.  Antioxidants, often a component of extractables and leachables, are required to enable plastics to maintain their functional properties throughout shelf life.  They protect plastics polymers from oxidative degradation under the heat of processing (formulation and extrusion or molding) and prolong shelf life. Antioxidants preferentially degrade by scavenging free radicals that would otherwise break covalent polymer bonds, causing the plastic to become brittle and generate particles or potentially more undesirable leachables.

While the reported cytotoxicity was attributed to a gamma radiation-induced degradant of Irgafos 168, the absence of cytotoxic effect in other biocontainers also formulated with Irgafos 168 suggests it is not the Irgafos 168 or its primary degradant, Oxidized Irgafos 168, that was the problem per se. Furthermore, while radiation dosage was also shown to be a contributory factor in the levels of the cytotoxic degradant present, as well as its effect, the other biocontainers containing Irgafos 168 were also subjected to similar high radiation doses without showing cytotoxic effect. Clearly, some factor other than radiation dose and the mere presence of Irgafos 168 is at play here.   

Following publication of the Hammond et al. paper, the supplier of the implicated biocontainer came forward and presented a joint explanation during a webinar and at the BPI Conference in Boston (5). They disclosed that the problem was associated with an excess amount of Irgafos 168 and that the solution, arrived jointly with the user, was to reformulate the film with a reduced level of Irgafos 168. Studies subsequently showed that the cytotoxic degradant and its effects on select cell lines was eliminated by reducing the Irgafos 168 concentration in the polymer formulation, presumably without adversely affecting polymer properties (though impact on shelf life was not reported).  That solution also provides a possible explanation why other films with Irgafos 168, receiving similar radiation dosages, did not demonstrate cytotoxic effects. They likely already had lower, optimized levels of Irgafos 168 that do not generate sufficient levels of the degradant leachable to cause cytotoxicity.


  As suggested by some of these authors, end users and suppliers might collaborate to develop an industry acceptable standardized test by identifying a cell line and culture medium formulation that would serve as a representative worst case and satisfy a majority of the end-user requirements.  As with the current screening test, 

 (MEM Cytotoxicity), this new “worst case” cytotoxicity test could be conducted by biocontainer suppliers (or their contract testing laboratories), and results could be published in the supplier’s bio-container validation data packages,potentially reducing the need for user-specific qualification tests.  Efforts are underway to achieve this, however at present, no user has come forward with a sufficiently sensitive cell line they are willing to declassify. It, therefore, remains prudent for end users to screen all new cell lines or media formulations with their intended or potential biocontainer(s) and bioreactor film(s) prior to initiating production. Suppliers may provide evaluation kits to facilitate such testing.

Other key factors in avoiding cytotoxicity events are the change control, change notification, and traceability practices of the biocontainer supplier, as well as their understanding of the materials science. The last case discussed here purportedly did not occur in a new biocontainer screening, but rather was the result of an unannounced formulation change (increasing the Irgafos 168 concentration to excess). As seen here, supply chain-related changes in formulation, processing, component manufacturing, or transport and storage that do not impact form, fit, or general function--and even perhaps improve productivity of the film--could still potentially be harmful to cell-culture processes. Biocontainer and single-use system suppliers need to apply best efforts to avoid changes, and, where imposed in the absence of a suitably sensitive generic bioreactor cytotoxicity screening test, need to communicate change notifications with biocontainer/bioreactor customers in advance of any anticipated changes to enable users to qualify and accommodate any unavoidable changes.

Cholesterol Delivery to NSO Cells: Challenges and Solutions in Disposable Linear Low-Density Polyethylene-Based Bioreactors

, 103, No. 1, 50-59 (2007).
  2.	N. Steiger and R. Eibl, “

Interlaboratory Test for Detection of Cytotoxic Leachables arising from Single-Use Bags

 2013, 85, No. 1-2, 26-28 (2012).
  3.	J. Wood et al., “

Strategy for Selecting Disposable Bags for Cell Culture Media Applications Based on a Root-Cause Investigation

, 29, No. 6, 1535-1549 (November/December  2013).
  4.	M. Hammond et al., “

Identification of a Leachable Compound Detrimental to Cell Growth in Single-Use Bioprocess Containers

, 67, No. 2, 123-134 (March-April 2013).
  5.	S. Klein and M. Goodwin, “New Approaches to Managing Single-Use Systems,” presented at BioProcess International Conference, Boston, MA, October, 2013. 

Jerold Martin is Sr. VP Global Scientific Affairs at Pall Life Sciences, Port Washington, NY and Chairman of the Board and Technology Committee Bio-Process Systems Alliance (BPSA) jerold_martin@pall.com.

The Disposables Advisor

Studies show diminished cell growth properties associated with some biocontainer or bioreactor films.

Cytotoxicity Risks in Single-use Media and Bioreactor Containers

In its Strategic Plan for Preventing and Mitigating Drug Shortages, FDA reiterated its position that shortages of drugs and biologics pose a significant public health threat and preventing drug shortages remains a top priority. The agency also identified two central goals: improving mitigation response to shortages and implementing strategies for the long-term prevention of shortages by focusing on the root causes of the shortages.

FDA cites some recent success in working with manufacturers to mitigate shortages and restore lost production. A six-fold increase in early notification of possible shortages, combined with allocation of additional FDA resources, resulted in a decrease in the number of new shortages from 251 in 2011 to 117 in 2012, FDA reports. Despite this improvement, there is still much work to be done, particularly to address manufacturing and quality issues that are the root cause of drug shortages.

FDA strategies to move toward long-term prevention include the establishment of a proposed Office of Pharmaceutical Quality within the Center for Drug Evaluation and Research (CDER) and the establishment of risk-based approaches to identify early warning signs for manufacturing and quality problems.  In response to FDA calls for input on a third strategy, the broader use of manufacturing metrics to assist in the evaluation of manufacturing quality, two industry associations have offered proposals for potential metrics programs.

In December 2013, the International Society of Pharmaceutical Engineers (ISPE) released 

ISPE Proposals for Quality Metrics Program - Whitepaper

, in which the organization proposed “an initial list of quality metrics which are reportable to FDA to support a risk-based inspection program as given in sections 704 to 706 of US Food and Drug Administration Safety Act (FDASIA) and assist industry in moving towards the ‘desired state’.”

The ISPE team, with representatives from pharmaceutical organizations, identified metrics that are mostly site-based. The intention, ISPE reports, is to start with indicator metrics and consider refinement in subsequent phases to better link to FDA’s six systems used in the inspection program and to products.

The initial six metrics, all lagging indicators, are: batch rejection rate, rework and reprocessing rate, confirmed out-of-specification (OOS) rate, unconfirmed OOS rate, critical complaints rate, and % annual product quality reviews complete on time. ISPE also offered definitions for these metrics, suggested alternative metrics, an implementation program, and options for next steps. 

The Parenteral Drug Association (PDA) developed a “

” document, which puts forth the current thinking of the organization’s membership.  The document details the organization’s position on metrics and its approach to developing its recommendations. PDA recommends that FDA collect and assess both product and site quality metrics. Trend metrics collected per product include confirmed product quality complaint rate by product; batch reject rate by product; and confirmed OOS rate, (drug substance and drug product) by product. Suggested trend metrics collected by site are confirmed OOS rate, (drug substance and drug product) by site and batch reject rate by site. 

These initial efforts of ISPE and PDA to provide some guidance to FDA should be commended. However, there is much work to be done. We will be monitoring developments in future issues of BioPharm International and on our 


Rita Peters is the editorial director of 

From the Editor

ISPE and PDA take on the challenge of recommending quality metrics.

Defining Quality Metrics is No Easy Task

A widely used method for determining the concentration of purified proteins in solution is by absorption at 280 nm using commercially available ultraviolet/visible (UV/VIS) spectrophotometers. The concentration is determined according to the Beer-Lambert Law (1) calculation adapted for proteins of known absorptivity as shown below:

The absorbance at 320 nm is subtracted to account for light scattering by the test sample due to aggregation and should be low relative to the absorbance at 280 nm. According to the equation, the measured concentration is inversely proportional to the path length of the absorption cell (cuvette) used for the absorption measurement. As an example, for bovine serum albumin (BSA) with an absorptivity value of 0.667 mL mg

, a concentration range of 0.3 to 1.8 mg/mL can be measured using a standard spectrophotometer with a 1-cm path length cell, assuming the maximum absorbance of approximately 1.2 in the spectrophotometer. The absorption limits or linear dynamic range of a spectrophotometer is limited by noise and stray light and is influenced by the type of detector in use. It should be noted that absorbance by the cuvette and buffer solution used for blanking the spectrophotometer is part of the total absorption and must be considered. 

For the BSA described, if the path length of the cell is decreased by a factor of five to 0.2 cm, the measurable range can be increased by a factor of five to 1.5 to 9.0 mg/mL. If the path length is decreased by a factor of 100 to 0.01 cm, then the measurable range will be 30 to 180 mg/mL. A standard spectrophotometer can, therefore, be used with commercially available shorter path length cuvettes to measure higher protein concentrations without dilution. Spectrophotometers and spectrophotometer accessories (such as fiber optic probes) tailored for high protein concentration measurement are also available and are based on the same principle of reduced path lengths. 

The biopharmaceutical industry manufactures recombinant proteins at various protein concentration levels for therapeutic applications. Using the previously described BSA concept, for example, on purified recombinant transforming growth factor beta superfamily proteins with an absorptivity of 1.36 mL mg

or a monoclonal antibody (mAb) with a similar absorptivity, the measureable range is approximately 0.15 to 0.9 mg/mL with a 1 cm path length cell. However, if the cuvette path length is decreased by a factor of ten to 0.1 cm, the protein concentration range changes to approximately 1.5 to 9 mg/mL. For a cuvette with a path length 100 times shorter, 0.01 cm, the protein concentration range is approximately 15 to 90 mg/mL.

Therefore, according to the calculated results shown in 

, it is possible to measure a wide range of protein concentrations without performing any dilution of the samples. Protein concentrations for biopharmaceutical drug substances and drug products can be determined directly without prior dilution by selecting the appropriate path length absorption of the cuvette according to the absorptivity of the protein and the expected concentration. 

 Measureable concentration ranges by cuvette path length.

Bovine serum albumin (BSA) with absorptivity 0.667 mL mg-1   cm-1

Protein with absorptivity   1.36 mL mg-1 cm-1


  UV absorption of protein solutions was measured at 280 and 320 nm using an Agilent 8453 UV/VIS spectrophotometer, with various 1 cm path length quartz absorption cells. Quartz cuvettes with path lengths of 0.01, 0.1, and 0.2 cm from Agilent were used for high protein concentration measurements. The dimensional tolerances were ± 0.001 cm (0.1%) for the 1-cm path length cell, 0.001 cm (1%) for the 0.1-cm cell, 0.001 cm (0.5%) for the 0.2-cm cell, and 0.0005 cm (5%) for the 0.01-cm cell (2). For comparison, the acceptable systematic plus random error for mechanical pipettes are 1.4% for 10 μL, 1.0% for 100 μL, and 0.8% for 1000 μL. In addition, the viscosity of the solution, the goodness of fit of the tips used, and the technique of the operator can introduce error. Thus the dilution error introduced by one dilution can be larger than the dimensional error for all but the shortest path length cuvette (0.01 cm) and for multiple dilutions the error increases. 

Peer-Reviewed Research

Accurate protein concentration results can be obtained using standard spectrophotometers and commercially available short path length absorption cells.

Dilution-Free Protein Concentration Measurement for High Protein Concentration Samples

Issue PDF

Click the title above to open the BioPharm International February 2014 issue in an interactive PDF format.


BioPharm International-02-01-2014