Felicity Thomas is the former Associate Editorial Director of Pharmaceutical Technology, Pharmaceutical Technology Europe, BioPharm International.

Felicity Thomas

 On 14 May 2019, the council of the European Union adopted a new regulation, which introduces a manufacturing waiver for supplementary protection certificates (SPCs) that is aimed at removing the competitive disadvantages faced by EU-developers of generic or biosimilar therapies. However, the introduction of this new SPC waiver has prompted polarized opinions throughout the pharmaceutical industry.

	To learn more about the SPC waiver, and its implications for the European pharmaceutical industry, 

spoke with Ayesha Raghib, an associate at the law firm Powell Gilbert, which specializes in intellectual property (IP) and EU pharmaceutical regulatory law.

Could you outline the key aspects of the SPC manufacturing waiver, recently introduced by the EU council? 

On 1 July 2019, EU Regulation 2019/933 (the ‘Regulation’) came into force, amending the current SPC Regulation 469/2009, and introducing an SPC manufacturing waiver (SMW). The SMW now permits manufacturing and storing of generic drugs/biosimilars of SPC-protected products in the EU during the entire term of the SPC for products intended exclusively for export to countries where patent protection for the original medicine does not exist or has expired, and manufacture and storing during the final six months of the SPC term for post-expiry Day 1 EU launch. 

	Generics/biosimilar manufacturers must comply with certain legal obligations including notifying national IP offices and the SPC holder at least three months in advance of manufacture (or first related act); affixing a specific logo to products manufactured for export; and due diligence requirements in respect of parties in the supply chain. 

	The SMW will apply to SPCs applied for on or after 1 July 2019 but not to SPCs in force before that date. However, for SPC applications applied for before 1 July 2019 and taking effect on or after that date, the SMW will apply from 2 July 2022, allowing for a three-year transition period. 

How is the industry viewing the SMW differently?

On one hand, the aim of introducing the SMW is to create a level global playing field between generic drug/biosimilar manufacturers established in the EU and third-country manufacturers, thereby creating jobs and promoting the manufacturing industry in the EU. Thus, the SMW has understandably been welcomed by the generic drug and biosimilar industry. 

	Conversely, from the perspective of the originator industry, introduction of the SMW has been vehemently opposed and perceived as a significant step towards eroding the EU IP framework, raising concerns that the EU does not fully support innovation. The European Federation of Pharmaceutical Industries and Associations (EFPIA), the voice of the originator industry operating in Europe, is of the view that (1): ‘The adoption of the SPC manufacturing waiver will ultimately impact on our capacity to develop new treatments for patients living with diseases like cancer, diabetes, and dementia. It sends a negative signal to the world that Europe is devaluing its intellectual property framework, making Europe a less attractive location for research and development, impacting on jobs and investment.’

	The benefits are clearly in favour of the generic drug/biosimilar manufacturing industry as manufacturers of generic drugs or biosimilars can now compete on the global playing field. However, whether or not established global manufacturers will relocate their manufacturing facilities to the EU remains to be seen. Comparative cost of manufacture will of course be a driving factor. These factors could negate the intended effect of the SMW to some extent. The long-term impact of the SMW is very difficult to predict at this early stage.

Could you highlight the importance of IP in the pharma industry?

Drug development is very expensive and risky. Originator companies spend vast sums of money investing in the research and development of innovative medicines, and seeking marketing approval to place those medicines on the market. 

	In the pharmaceutical industry, IP rights-specifically patents and SPCs-are a means of protecting these new medicines by affording the patent/SPC owners a limited monopoly for the new medicines and preventing other companies from ‘copying’ the medicines. As a result of these monopolies, innovator companies are able to maintain the prices of their medicines and recoup the costs of development as well as invest in the development of new therapies. Thus, IP rights provide an incentive for innovation, which in turn means that patients have access to new therapies to meet their needs. 

What impact might the newly passed SPC waiver have on legal disputes concerning IP in pharma?

To date, the SPC regulation has led to the launch of many court cases to try to clarify its operation, and it is likely that the legislation underlying the SMW will add further questions over the scope and effect of SPCs. For example, ‘storing’ for the purpose of Day 1 EU Launch is expressly permitted; however, it appears that only ‘temporary storing’ is permissible as a ‘related act’ for the exclusive purpose of export. In fact, the term ‘related act’ is also not defined clearly in the regulation and could be the subject of litigation. 

	In addition, there may also be disputes in regard to alleged non-compliance with the notification obligations placed upon generics/biosimilar manufacturers. When commencing manufacture in the EU for export, issues may also arise as to whether, in fact, the product is not (or is no longer) protected by patents in the countries for intended export. It would not be surprising to see litigation as a result of the SMW and its operation.  

Supplementary to the potential IP disputes, could there be other legal ramifications of the SPC waiver for pharmaceutical companies?

In order to offset the narrowing of IP rights under the SMW, we may see an increase in applications for secondary patents (e.g., patents for manufacturing methods, formulations, and uses) by originator companies. This in turn may lead to increased litigation in respect of these patents.

	Also, as patent and SPC rights are no longer co-extensive (i.e., previously the same acts were prohibited under both patents and SPCs), we expect that national legislation will need to be amended to reflect the changes introduced by the SMW. 

Do you believe there are, or will be, any plans for legislation, such as the SMW, to be introduced globally?

With regard to possible globalization, the first point to note is that SPCs (or equivalent patent-term extensions) are not available globally and only exist in certain countries, such as the United States, Japan, Canada, and Australia. Whether or not a SMW is introduced in those countries would depend upon the size and influence of the local generic-drug/biosimilar industry. 

	For example, in Canada, as a result of strong lobbying by the genericâdrug industry, generic-drug companies are already permitted to manufacture for export while the Canadian equivalent of the SPC (Certificate of Supplementary Protection) is in force. Whereas, in Australia, the introduction of an export waiver with a similar scope to that in Europe has been considered in the past, these efforts appear to have been abandoned for the time being.  

Do you think there is the possibility that the SMW could discourage companies from basing pharma manufacturing in Europe?

It may be that the notification obligations placed upon genericâdrug/biosimilar manufacturers may discourage EU-based manufacturing from making use of the SMW opportunities as much of the information required to be disclosed to SPC-holders/national IP offices is considered to be sensitive and/or commercially confidential (for example, the name and address of manufacturers) and could also provide an indication regarding generic drug/biosimilar launch plans. However, Medicines for Europe-which represents the generic drug/biosimilar industry-while opposed to the notification requirements in the legislation, remains optimistic and considers that the SMW will ‘create manufacturing opportunity and jobs and to increase Europe’s capacity to manufacture and supply its own medicines’ (2). 

	Nevertheless, the envisaged stimulus to the EU manufacturing industry may be hampered by the prospect of genericâdrug/biosimilar manufacturers becoming embroiled in litigation with SPC-holders. There is unlikely to be any immediate impact on EU-based manufacturing from the perspective of originator companies, unless it transpires that the SMW has a negative impact on the research and development of new medicines in the long run as feared by EFPIA. 

Will the UK’s exit from the EU have any effect on the implementation of the SMW?

In preparation for a ‘no-deal’ exit, the UK has enacted legislation that will enable EU law relating to SPCs to be retained in UK law at the point of leaving the EU. The regulation underlying the SMW will, therefore, automatically become part of UK law. In addition, the UK IP Office has recently opened a consultation regarding proposed draft legislation amending certain parts of the regulation to ensure that the SMW operates correctly, for example, references to the ‘Union’ instead refer to the ‘United Kingdom’ (3). 

	Otherwise, the proposed legislation is intended to operate in an equivalent manner to the SMW such that manufacture of SPCâprotected medicines for export outside the UK, and stockpiling for sale on the UK market immediately after SPC expiry will be permitted. EU-based manufacturers will also be free to export to the UK as it will have third-country status after Brexit. 

	In the event that there is a deal between the UK and the EU, under the current withdrawal agreement-which has not been ratified by the UK Parliament-EU law would continue to apply to the UK during the transition period, which currently ends on 31 December 2020, so the SMW would apply during the transition period. 

	1. EFPIA, “Redressing Europe’s Innovation Balance,” Press Release, 17 April 2019.

	2. Medicines for Europe, “Medicines for Europe Applauds Compromise on SPC Manufacturing Waiver,” Press Release, 14 Feb. 2019.

	3. UK IPO, “Supplementary Protection Certificate Manufacturing Waiver in a ‘No Deal’ Outcome,” Draft Legislation, 


	Vol. 31, No. 8
	July 2019
	Pages: 55–57

	When referring to this article, please cite it as F. Thomas, “The Great Divide? Implications of the SPC Waiver,” 

Articles

Europe has introduced a waiver for supplementary protection certificates, polarizing opinions throughout the industry. In this interview, the SPC waiver and its implications for European pharma are discussed.


The Great Divide? Implications of the SPC Waiver

Protein therapeutics are a promising class of drugs that are increasingly coming to the fore in development pipelines as a result of their utility in difficult-to-treat diseases. Yet, these complex, large-molecule drugs pose significant challenges for developers and regulatory bodies alike.

	An important issue for protein-based drug developers is that of structural characterization, which can help in understanding whether or not a drug product will be stable and of sufficient quality to be launched at all. “It is critically important that the complete and in-depth characterization of therapeutic proteins is performed throughout all stages of the drug discovery and development process,” says Simon Cubbon, senior global marketing manager, chromatography and mass spectrometry, Thermo Fisher Scientific. “Ultimately, this facilitates the transfer of appropriate knowledge throughout the pipeline, ensuring product consistency, safety, and efficacy.”

	In agreement, Jeff Zonderman, chief commercial officer of RedShiftBio, highlights the concerns around how stress conditions, manufacturing processes, and storage may affect protein structure and adds that drug developers are limited by current analytical tools that do not offer sufficient insight. “This is especially the case with stability and aggregation, where earlier detection and monitoring in the development and manufacturing processes result in better, more effective drugs,” he notes. “Promising biological drug candidates, those that exhibit therapeutic activity and inherent stability, as assessed via simple screening techniques, become subject to increasing levels of structural elucidation as they progress through the pharmaceutical pipeline.” 

	Protein characterization, along with demonstrating a product’s consistency and reproducibility, are imperative for developers aiming for a quality product, states Brian R. Berquist, director of process development and technology transfer at iBio. “Obtaining information on your protein early on is a way to examine the quality and consistency throughout the drug development cycle,” he asserts. “Protein characterization is critical even in the earliest stages of process and drug development. The process development phase is when one can get a first glimpse at the potential product molecule and how the purification processes applied affect it.”

	During early development stages, it is possible to make changes easily to address any potential purity issues. “In some instances, the product may be susceptible to proteolytic activity, either during expression or during purification, so being able to identify small changes in product mass and the amino acid location of that proteolytic activity allow one to address the issue in a logical manner early on instead of having a process where each step must be re-examined due to changes made,” Berquist says. “Additionally, as purification steps are developed, one is selecting based upon properties of the protein molecule itself. Sometimes a purification step or steps can enrich a certain proteoform, multimer, or glycoform that is not highly desirable. Again, it is easier early on to obtain this knowledge and change the purification accordingly.”

	Going beyond the early stages of development, and as processes start to become more fixed in nature, Berquist explains that protein characterization can be used to examine the vigor of the purification process on the whole, as well as for each unit operation. “Protein characterization provides critical information regarding the reproducibility and robustness of each step in the process and the purification in its entirety,” he says. “When the process is locked, and manufacturing begins, it is essential to characterize each lot produced to ensure that a quality product is being generated routinely and consistently.”

	“In today’s challenging pharmaceutical environment, research scientists are pushed to screen drug candidates as quickly as possible to increase chances of reaching clinical trials,” says Bill Barrett, product specialist at W.L. Gore’s PharmBIO Products business. “Using monoclonal antibodies as an example, speeding the purification process leads to more candidates and higher productivity.”

	Adding to the requirements of reduced time and cost, and improved research productivity, Scott Peterman, senior global marketing manager, chromatography and mass spectrometry, Thermo Fisher Scientific, emphasizes that scientists are also now developing advanced protein drugs that are more extensively engineered and, as such, more complex. “As complexity increases, there are opportunities for greater levels of post-translational modifications and molecular heterogeneity,” he says. “Understanding the complexity of a protein-based therapeutic, and being able to control these modifications and variations, is becoming increasingly more difficult but remains critically important.”

	Therefore, bio/pharma scientists are required to employ a cornucopia of various analytical technologies and strategies to adequately characterize protein-based therapeutics, continues Peterman. “Each analytical technology or strategy presents its own knowledge requirements and challenges, which scientists and vendors alike must address,” he notes.

	Looking at analytical techniques in particular, Richard Moseley, chief technologist at Microsaic Systems, highlights that most can be categorized into two facets-either on-line with low specificity or off-line with high specificity-potentially challenging the ability to obtain answers fast. “In addition, the protein products are in complex chemical cell media making exact identification difficult. Therefore, some critical quality attributes are difficult to characterize and require complex workflows,” he says. 

	Furthermore, Moseley stresses that established analytical techniques suited to smallâmolecule drugs have been found to be unsuitable for bioprocessing. “So, biopharma scientists need to look to new techniques, specifically developed for their challenges and workflows to meet the complex needs of bioprocessing,” he asserts.

	For Zonderman, challenges lie in the structure of drug studies, irrespective of dosage requirements. “Defining an optimal formulation and manufacturing route relies on assessing the impact of variables such as processing conditions including temperature and applied shear stress, and storage,” he says. “Stress-induced structural changes may have significant consequences including a loss of efficacy, and in the worst case present a safety risk, so demonstrating comparability (that successive stages of formulation, manufacture, and storage do not materially impact the structure of the drug) and stability up to the point of administration is essential.”

	Protein aggregation: An undesirable process

	Aggregation-where proteins bind together and form undesirable impurities-can be detrimental to protein-based therapeutic development. These clusters of molecules can result in an incorrect drug dosage, or unwanted and even fatal immune responses to the drug, stresses Peterman.

	“Consequently, monitoring protein aggregates is important for safety and quality assurance,” he adds. “Complete characterization and in-depth structural insights allow scientists to better understand what factors can lead to aggregation and undesirable outcomes, aiding clone selection and the delivery of a robust biotherapeutic that will not aggregate undesirably.”

	This common indicator of protein instability, aggregation, can occur both upstream and downstream and can result in a product being deemed unfit for launch, continues Zonderman. “Characterization can help by giving insight into the onset of aggregation under certain conditions and help developers better formulate drugs to minimize aggregation or eliminate bad drug candidates earlier in the process,” he notes. “Being able to predict aggregation and resolve when you have true aggregation or self-association is critical.” 

	“Protein aggregation typically has been observed to be detrimental for both product activity and stability, as well as leading to the formation of higher levels of anti-product neutralizing antibodies in-vivo,” summarizes Berquist. “By applying rigorous size-exclusion chromatography (SEC) methodologies, we monitor even low levels of protein aggregation and use these data to optimize drug formulation to prevent aggregate formation.”

	The rise, and difficulties, of biosimilars

	It is well-known that many branded protein-based biopharmaceutical products are facing patent expiration in the coming years, and so, the growth of the biosimilars market is an inevitability. As specified by regulatory bodies around the world, in some form or another, biosimilar products must be proven to be highly similar to its reference product with no clinically meaningful differences in terms of safety, purity, and potency. 

	“Determining similarity for biologics is much more challenging than with small molecules due to their larger size and greater structural complexity,” explains Zonderman. “Along with functional comparisons, measurement and analysis of the structural similarity between proteins are an effective method of demonstrating bioequivalence. I believe for biosimilar work it is important that testing is done at the same concentration and formulation conditions. Today, for complete structural analysis including secondary structure, current techniques are not sensitive to achieve this challenge.”

	The primary goal of protein characterization for biosimilars is to provide sufficient evidence of similarity to the originator product, adds Berquist. “However, the problems with this are multifaceted,” he continues. “First, there are difficulties in obtaining sufficient supplies of the innovative drug product for comparison. Second, there is technological gap between original drug product characterization results and the instrumentation available for characterization of the biosimilar. The challenge is to prove that any observed differences are not significant and do not have clinical relevance.”

	Concurring with Berquist, Cubbon further explains that biopharmaceutical companies exhaustively characterize therapeutics and associated manufacturing processes to improve the specificity of the patents, and these specifics may not be accessible to the biosimilar developers. “Consequently, the biosimilar developers are required to perform characterization to the same exhaustive levels,” he continues, “for example, to determine protein drug glycosylation, aggregation, and charge variant profile.”

	As a result of this level of specificity that may be required when approaching biosimilar development, costs may not be reduced as significantly as is possible when approaching a small-molecule generic drug, for example. “The use of biosimilars, with such closely comparable performance to an original drug that can be used interchangeably, has the potential to drive down healthcare costs but is highly dependent on the rigorous demonstration of similarity in a wide range of attributes including protein content, activity, and stability,” asserts Zonderman.

	Currently, there are many methods available that look at different attributes of the protein molecule, however, these methods are not without limitations. “Many of the techniques and tools available are inherited from pharmaceutical’s roots in smallâmolecule drugs, which are normally unsuited to the complex bioprocessing workflows and cell media used,” says Moseley. 

	Traditional assays, such as capillary electrophoresis (CE) or liquid chromatography coupled with ultraviolet (LC–UV), are robust and reliable techniques; however, they can only provide limited information on the critical quality attributes (CQAs) that need to be measured, stresses Cubbon. “This means that numerous assays are required to confidently cover these CQAs and ensure the correct information is obtained,” he adds.

	According to Zonderman, there is a dependency on what technology is used and how it is applied, but in general, key limitations of current techniques include dynamic range, sensitivity, and automation (both analysis and data processing). “Many of the currently available technologies may meet some of the needs for research, but are a challenge for biopharma to move into more downstream, QA/QC [quality assurance/quality control], and process monitoring applications,” he notes.

	The detailed definition of the structure of a drug molecule can provide a basis for scientists to identify structure-function relationships that enable an understanding of how a drug is efficacious, explains Zonderman, but beyond this the structural characterization of proteins plays a critical role through the drug development lifecycle. “In particular, investigating structural changes is the key to understanding and controlling the factors and mechanisms associated with stability and aggregation,” he says.

	“Characterizing proteins during drug development is essential in reducing drug development times and manufacturing costs, and is critical for safety reasons,” agrees Moseley. “Furthermore, through protein characterization, industry is now capable of creating personalized medicine for patients.”

	Yet, challenges and limitations remain in the ability to characterize these complex molecules sufficiently, and industry needs to be fully capable of managing the complexity of the information that is obtained through characterization, says Berquist. “As technology advances, industry will gain an ability to address increasingly intricate questions about protein products,” he summarizes. “Today, we have the capabilities to analyze intact macromolecules to the detail of detecting microheterogeneities leading to difficulties associated with data interpretation and refinement.” 


	Vol. 43, No. 8
	August 2019
	Pages: 36–38

	When referring to this article, please cite it as F. Thomas, “Accepting the Challenge of Protein Characterization,” 

Protein characterization is a critical part of drug development, but as there are still limitations with available techniques, industry needs to look at technological advances to meet the specific requirements of complex molecule characterization.

Accepting the Challenge of Protein Characterization

As the numbers of poorly soluble and complex molecules, such as biologics, entering drug development are increasing, industry is also witnessing a positive impact within the parenteral formulation market. The advantages of non-oral administration techniques for these complex and poorly soluble molecules, where absorption of the active ingredient is simplified and the first pass the metabolism effect is avoided, are well-documented.

	Parenteral formulations are dosage forms to be administered non-orally, via a subcutaneous, intravenous, intraosseous, or intramuscular route. According to market research, rising approval rates of new parenteral drugs worldwide should drive significant growth in the parenteral drug market revenue in the near future (1). Specifically looking at the global sterile injectable drug market, for example, Transparency Market Research anticipates the compound annual growth rate to exceed 11% between 2017 and 2025 (2).

	However, compared with oral-solid dosage forms, parenteral formulations require very specific and important considerations, and raise certain challenges and limitations, such as drug stability issues. Primarily, solutions, suspensions, or emulsions that are developed for administration by injection or implantation are directly entered into a human’s systemic circulation system and so must be sterile and safe for use. Furthermore, sterile parenteral formulations need to be pyrogen-free and clear of visible and sub-visible particles, to name a few compendia-specified criteria.

	To learn more about the intricacies of parenteral formulation, 

 spoke with Fabio Stevanon, director of Global Injectables Platform at CordenPharma International.

	Important considerations for parenterals

 What are some of the most important considerations for parenteral drug formulation?

 All considerations related to parenteral drug formulation begin with assuring the successful, targeted delivery of the API to treat the target condition effectively. Formulation must support the desired therapeutic effects of the API after administration.  

	So much of any given injectable drug’s therapeutic value is related to its specific API formulation in solution or suspension. Bioavailability and other desired pharmacokinetic effects are all dependent on the finished drug’s eventual, commercial formulation.

	Parenteral formulations are going places they’ve never been before, where we are seeing many sterile injectables being increasingly paired with new delivery technologies to assure convenient and timely, prescribed dosing of drugs over time. 

	For combination device-drug products, formulations must be tailored to achieve specific attributes related to both the device and drug product, like stability during targeted shelf life, compatibility with the primary packaging, viscosity and similar parameters, plus other aspects of drug administration relative to the specific medical device. Only proven expertise gained through testing and efficient analytical method development, as well as experience in combining device and drug products, will deliver a successful formulation in this space.

	Beyond that, parenteral formulation chemistries must serve commercial and manufacturing interests as best as possible. Formulation has direct influence on scale-up and process variability, stability and other processing, and manufacturing-oriented aspects related to the drug’s successful commercial development.

 Could you elaborate on the importance of excipients in parenteral drug formulation?

 Two things are important regarding excipients. First, whatever the API, excipients must be selected based on their performance and utility in service to the particular formulation and therapeutic function in question. The earlier this selection is established in the development phase, the better.

	The second priority is to evaluate excipients for specific attributes that help sustain the drug product’s commercial manufacture including compliance, sterility profiles, availability, reliability of supply, stability in process, and more. 

	Benefits and challenges specific to parenterals

In your opinion, what are the major benefits of a parenteral drug formulation over other forms?

Parenteral drugs deliver faster therapeutic action compared to slower-acting dosage forms such as oral-solid dosage (OSD). Parenteral drug formations also allow for higher dosage levels in circumstances when critical therapies must enter the blood stream right away, such as emergency surgery, heart attacks or infections, and so on. 

	There are also other benefits related to dosing accuracy such as how a proper, strictly validated injectable formulation will, in most cases, grant better bioavailability at the desired dosage level. 

 And the biggest challenges in parenteral drug formulation?

 The validation of a parenteral formulation is much more challenging compared to OSD. The fast-acting nature of this administration route means that parenteral formulations are subject to stricter compliance regulations to ensure patient safety and must stay within specific, and usually very narrow, parameters to obtain approval from regulatory bodies.

	Processing and manufacturing sterile parenteral dosage forms therefore requires a comprehensive approach, including a mastery of sterile containment, fill/finish and data-driven manufacturing and control processes. As this is a challenge that not all pharma companies can or are willing to address in-house, many are turning to strategic, full-service outsourcing partners to support their business models and drug development strategies as a result.

Could you discuss the major differences that need to be considered when formulating a small molecule or a biologic as a parenteral drug?

 There is an extra level of expertise, technology, and skill that is required to formulate a biologic as a parenteral drug. Small-molecule parenterals can be produced and manufactured using traditional analytical methods that are standard practice throughout the industry. However, biologics require a different approach to analytical testing and manufacturing processes, which older, less technologically advanced facilities may not be able to handle. In addition, biological parenteral manufacturing requires a state-of-the-art facility where in most cases ‘disposable/single-use’ technology is a given, and sometimes a dedicated ‘biological’ plant is needed.  

 What are the main regulatory challenges facing parenteral drug formulators? 

 Because of the difficulty in maintaining the supply for the high demand of injectable drugs while also keeping up with manufacturing quality and security at high volumes, we have seen increasing pressure by regulators on drug manufacturers to control processes more effectively and remove potential for process variation and contamination. 

	Regulators are also keeping a close eye on developments in the combination device–drug product space. Many new pharma development projects and lifecycle management opportunities are exploring the integration of medical devices with drug products and investing in developing these technologies because of their therapeutic effectiveness and potential value to patients. 

	For many combination device–drug developers, guidelines and applicable manufacturing standards have and are evolving rapidly. Such a strong growth in this sector of the pharma industry has raised significant requirements on development, formulation, clinical supply, design transfer and control, risk management, and commercial supply of these combination products, making both their engineering and compliance very challenging.

 Over the next decade, what trends do you anticipate will impact the parenteral drug formulation space?

For many pharma companies, there is a growing need to invest in facilities, equipment, and process controls associated with producing parenteral drugs and combination drug–device products. Optimal development strategies will require extensive, as well as broader expertise, often found by engaging established, competent CDMO [contract development and manufacturing organization] providers-this trend will continue to gain momentum in the coming decade.

	Industry is also witnessing the adoption of advanced technologies to ensure quality and reliability in manufacture. For example, the emergence of disposable/single-use-system injectable technologies is providing alternatives to traditional compounding-filling in stainless steel or glass-lined vessels. The capabilities for single-use compounding-filling suites will therefore be more and more needed to handle oxidation-prone compounds not compatible with traditional stainless-steel mixing vessels and transfer lines.

	1. Fact.MR, “Parenteral Drug Market Forecast, Trend Analysis and Competition Tracking-Global Market Insights 2018 to 2028,” 

, Market Report, accessed July 8, 2019.
	2. Transparency Market Research, “Sterile Injectable Drugs Market-Global Industry Analysis, Size, Share, Growth, Trends, and Forecast 2017–2027,” 


	Vol. 43, No. 8
	August 2019
	Pages: 24–25

	When referring to this article, please cite it as F. Thomas, “Formulation and Manufacturing Trends for Parenterals,” 

Formulators of parenteral drugs must be cautious of specific considerations and challenges that arise during development and manufacture.

Formulation and Manufacturing Trends for Parenterals

	Supply chain security is of crucial importance in the pharmaceutical industry. Should counterfeit or falsified drug products enter distribution channels and end up with a patient, not only is the reputation of the drug company at stake but also the health, and even in severe cases the life, of the end user.

	To combat counterfeiting within the pharma industry, regulatory authorities have been putting various measures in place. One example is the Falsified Medicines Directive (FMD), which was implemented in Europe on 9 February 2019. 

	Compliance with the new FMD regulation means that pharmaceutical companies must ensure all drug products have safety features placed on the packaging, comprising a unique identifier and an anti-tampering device. These safety features will then be used by medicines suppliers to verify and authenticate the products prior to distribution to the public.

	Radio-frequency identification (RFID) technology has been identified as a potentially useful tool to track items in the pharmaceutical supply chain. “RFID is the transmission of, at minimum, a unique identification (UID) between a transmitter and a reader using radio waves,” explains Gillian Ewers, vice-president of marketing at PragmatIC, a company that specializes in flexible electronics.

	“The transmitter could be a package that includes an RFID tag,” she says. “A tag (which is sometimes referred to as an inlay, as they are inlaid behind labels or inside packages) in this case is usually simply an integrated circuit (IC) mounted on a substrate (paper or film) that also has an antenna either etched or printed on to it. This is the simplest form of a tag, called a passive tag, as it contains no batteries and needs the reader to provide it with the energy to wake up and send the signal to the reader.”

	It has been reported that RFID is a more reliable technology than two-dimensional barcoding and is more difficult to counterfeit. However, uptake of the technology as a tracking and identification solution has been limited in the pharmaceutical industry, despite it becoming more affordable as the technology has matured over time. 

	Ewers details some of the main applications of RFID technology in pharma. “For supply chain management, RFID technology can not only help companies to manage inventory, control stock, and, as such, manage costs effectively, but can also help in grey market prevention-where products are bought in a low-priced country and sold for a higher price in a different country,” she says.

	Through assigning an invisible unique identifier (UID) to each product, it is possible to audit complete stock at the point of sale, or anywhere along the supply chain, Ewers explains. “Key to this is it being invisible, so machine readable barcodes are no good (plus require digital printing to be truly unique), custom solutions that are not readable by commercially available RFID readers would be ideal,” she adds. “The UID can be assigned to a specific end retailer at POS.”

	As mentioned earlier, authentication is an obvious application of RFID. The technology can be used to track and trace various materials and products throughout the supply chain and, hence, reduce the likelihood of counterfeit or falsified products entering distribution channels. “Furthermore, it is possible to use RFID technology to provide consumers with information about the medicine through near-field communication enabled devices and tags,” Ewers continues. 

	The ability to connect the patient to the drug manufacturer through smart packaging has been forecasted as an area of growth in pharma (1). Through smart packaging, companies are offered additional benefits, such as improved patient adherence, which inevitably leads to higher rates of therapeutic success. 

	“Non-adherence is a significant problem across the world,” asserts Ewers. “One estimate puts the cost per year in the United States at $290 billion (2) in ‘otherwise avoidable medical spending’.” Reasons behind non-adherence are numerous, with the simplest being that patients forget to take the dose. There is an easy solution to remedy this issue, which is a reminder on the patient’s smartphone or wearable device, for example, Ewers notes. 

	“Another reason for non-adherence is failure to re-order in time, which again can be automated, either purely by time or by the packaging,” she says. “Other possibilities are regulating dosages, providing better information and reporting to remove the fear of taking a medicine and its potential side effects. There is also talk of using technology to motivate people to take medicine (3), especially in the case of ones given to reduce the probability of an event occurring.”

	“In terms of FMD compliance, RFID can be used to assign a digital UID to each medical product, which means the technology can be used in combination with an appropriate software platform for serialization,” she continues. “When the technology is integrated onto packaging, it needs to be read, and so an appropriate software package is required to ensure that the UID is actually unique and to enable reporting of any exceptions to the appropriate data controllers.”

	“RFID tags require an additional step in the production of labels and packages to insert the tags,” notes Ewers. “There are many packaging suppliers that are already able to perform this step and so pharma companies, without in-house capabilities, should be looking to partner with relevant label manufacturers and packaging converters.” 

	PragmatIC offers a technology platform that features flexible integrated circuits (FlexICs). As the flexible options are thin and low-cost compared with traditional RFID systems, they are ideally suited for pharma applications, reveals Ewers. “The first ConnectIC product family, which is aimed at RFID applications, was announced in February 2019,” she says. “Additionally, there is a range available for closed RFID systems, which when coupled with a low-cost custom reader can provide a cost-effective solution for covert tagging of products to monitor grey market division, for example.”

	The flexible tech is currently manufactured in the United Kingdom on a specific production system, FlexLogIC, which is an automated and scalable manufacturing model for FlexIC production. “As adoption of the technology is happening globally,” Ewers adds, “we envisage FlexLogIC systems being implemented around the world for the manufacture of FlexICs close to where they will be integrated.”

,” Report, March 18, 2016.
	2. HealthPrize Technologies, “

Medication Adherence: Pharma’s $637 Billion Opportunity

, [accessed July 8, 2019].
	3. Harvard Chan School, “

Increasing Cholesterol Medication Adherence with Financial Incentives May be Cost-Effective

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The potentially devastating effects of endotoxins to humans have been widely reported. At sufficient concentrations in the bloodstream it is possible for endotoxins to cause irreversible side effects and even death in severe cases.  

	Found on the outer cell membrane of Gram-negative organisms, bacterial endotoxins are one of the most potent toxins known and strongly pyrogenic (fever-causing) substances, explains Allen L. Burgenson, Testing Solutions, Lonza Pharma & Biotech. “These toxins are harmless outside of the body and in the alimentary canal. They are extremely potent, however, once introduced in the bloodstream or intrathecally,” he says.

	“Bacterial endotoxins (also referred to as pyrogens) have the capacity to induce a high fever,” confirms John Dubczak, general manager, Microbial Solutions, Charles River Laboratories. “A large enough dose of endotoxin can lead to lung and kidney failure, intravascular coagulation, and a systemic inflammatory response that can lead to septic shock and death.” 

	As a result of the dangers of bacterial endotoxins, the importance of testing for them is plainly apparent, in the view of Victoria Watson, laboratory manager, Microbiology at Wickham Labs. “The importance of endotoxin testing is clear when looking at how susceptible and sensitive humans are to even minute amounts of endotoxin,” she says.

	“Bacterial endotoxins are ubiquitous in nature and are a menace for the manufacturers of all parenteral drugs and medical devices,” asserts Dubczak. “Not only are they everywhere environmentally, they are very difficult to remove once they are introduced into a finished parenteral product.”

	Introduction of these molecules can come from simple contact with contaminated parenteral devices or medications, notes Watson. “Ensuring that equipment and medication are free from endotoxins is particularly important when caring for vulnerable patients, including the elderly, patients in intensive care, and infants,” she says. “For example, sudden infant death syndrome has been linked to varying levels of bacterial endotoxin present in the bloodstream.”

	“In the early days of injectable pharmaceutical products, there was no method for testing for pyrogenic contaminants,” reveals Burgenson. “If a patient received an injection, there was a high likelihood that they would spike a fever, which at the time was termed ‘injection fever’.”

	It was the work of Florence Seibert and her team in the 1920s that brought forth the introduction of the Rabbit Pyrogen Test (RPT) (1), which was eventually incorporated into the 

) in 1942 as an official test, Burgenson explains. “The clotting of Limulus blood was noted for many years in injured horseshoe crabs,” he says. “Frederick Bang and Jack Levin discovered the enzymatic pathway causing the clotting of Limulus blood (2,3), and developed the Limulus Amebocyte Lysate, or LAL test.”  

	During the 1970s, pharmaceutical companies experimented with the LAL test and compared it with the RPT, finding that the LAL test was a more reliable and sensitive assay (4), and offered cost-efficacy for companies as there was no need to maintain rabbit colonies for product testing. “FDA took notice, and decided that, since LAL was derived from blood, it was a biologic subject to regulation under Section 351(h) of the Public Health Service Act, and subject to regulation and licensure by FDA,” confirms Burgenson.  

	“Since its introduction, the LAL test has become one of the most important tools used in the pharmaceutical industry,” adds Dubczak. “It has allowed for a level of bacteria and pyrogen monitoring that simply eluded manufacturers when the RPT was the only pyrogen test available. The LAL test has ensured the absence of pyrogens in raw materials, water for injection systems, in-process samples, and in the final products.”

	The LAL assay works via the reaction of the LAL reagent-an aqueous extract of blood cells from the horseshoe crab-with bacterial endotoxins and lipopolysaccharides. “There are currently three compendial bacterial endotoxin tests (BET) found in the 

 (gel-clot, kinetic chromogenic, and kinetic turbidimetric) that may be used to test injectable pharmaceutical products and implantable medical devices for release into commerce,” notes Burgenson. “There is also the endpoint chromogenic assay, which is a timed endpoint version of the kinetic chromogenic assay.” (See 

 for a list of the advantages and disadvantages of endotoxin testing methods.)

This method is the oldest of the three and the simplest, Dubczak explains. “The major advantage of the gel-clot LAL test is its simplicity,” he says. “It is easily learned and can be performed without expensive equipment.”

	The gel-clot assay works through production of a clot in the presence of endotoxins by cleavage of a coagulogen to coagulin, notes Burgenson. “This method can be performed as either a limits test or a semi-quantitative assay, giving a positive or negative result at the sensitivity of the lysate used,” adds Watson. “Dilutions are tested with lysate, and the endpoint is used to calculate approximate endotoxin level.”

	Despite the method’s advantages, such as interacting with fewer materials, thereby giving rise to a lower likelihood of results being affected by sample inhibition or enhancement, certain disadvantages have hindered its popularity. “The amount of time that is required to prepare samples using this process has made it less popular for use in raw material testing in some laboratories,” Watson says. “Additionally, the lysate sensitivities used in the gel-clot method are less sensitive than the quantitative methods.” 

	“Furthermore, the test does not allow for automation and data assimilation by computers, making it difficult to track and trend results,” adds Dubczak.

The KTA is one of the quantitative assays in which the measurement of absorbance versus endotoxin concentration is used to create a standard curve, explains Watson.

	“During the LAL-endotoxin reaction, the solution mixture becomes increasingly turbid as the gel begins to form,” adds Dubczak. “The time required for these turbidity changes to occur is inversely proportional to the amount of endotoxin present in the sample (i.e., more endotoxin requires less time). The endotoxin in a sample can be estimated by comparing the time it takes to reach a defined level of turbidity to the times obtained from a series of endotoxin standards (standard curve).” 

	Sensitivity can be higher with KTA over the gel-clot method, he asserts, and although it is possible for KTA to be performed in test tubes, it is more common to perform the assays on plastic, 96-well microtiter plates. “The spectrophotometers that are used to measure turbidity changes can be captured by the use of appropriate software, and the assimilated data are used to generate quantitative reports,” Dubczak continues. “Those same data can be uploaded to a LIMS database for tracking and trending.” 

	Watson adds that the most significant advantages of quantitative methods over the gel-clot method are the increase in sensitivity and ability to extrapolate results. “Being able to extrapolate quantitative results can be invaluable when testing raw materials because it can offer insights into potential sources of endotoxin contamination,” she states.

The third method, the chromogenic substrate test, is a further evolution of the LAL test. “Instead of cleaving coagulogen to coagulin, the same enzymes cleave a substrate-releasing para-nitroanaline (pNA), and a yellow color develops,” reveals Burgenson. “Similar to the turbidimetric assay, the time of color development to exceed a threshold is measured. The more endotoxin, the faster the substrate is cleaved, resulting in a deeper yellow color.”

	For Dubczak, these chromogenic tests have an elegant methodology and feature many of the advantages that are found with KTA, such as increased sensitivity and extrapolation of quantitative results. “Chromogenic tests are, however, more expensive,” he says. “The chromogenic substrate material requires months to synthesize and as such, is very expensive to manufacture.” 

	“The latest evolution of the LAL assay is the use of recombinant Factor C, derived from the DNA of a horseshoe crab,” says Burgenson. “The gene sequence for Factor C is inserted into cells and expressed in cell culture. This recombinant Factor C (rFC) is the same as the natural Factor C. The assay using rFC does not produce a gel, turbidity, or a yellow color; it produces fluorescence proportional to the amount of endotoxin in a sample. The more endotoxin in a sample, the more light is generated.”

	Another in-vitro alternative is the monocyte activation test (MAT), which was developed on the principle of the human immune system response, reveals Watson. “When challenged by the pyrogens entering the body or coming into contact with the bloodstream, the host’s innate immune defense mechanisms cause the monocytes/macrophages to produce prostaglandins and pro-inflammatory cytokines such as interleukin-1 (ILâ1), interleukin-6 (ILâ6) and tumor necrosis factor-α (TNFâα),” she says.

	Additionally, the RPT is also an option, although this should only be considered as a final option once the LAL in-vitro methods have failed, Watson notes. “RPT is generally used for highly complex products that interfere with the in-vitro methods, and for release of products that require full pyrogen evaluation in markets that do not accept MAT as an alternative,” she explains.

	There are some products, namely biological ones, that are exempt from the rabbit test as stated in US 21 Code of Federal Regulations (CFR) 610.13(b), clarifies Burgenson. Therefore, in his opinion it is prudent for the developer to look at every approach that may be able to help overcome any interferences with the LAL assay. Some formulations include a chelator (such as citrate), with a non-ionic surfactant (such as polysorbate) causing the endotoxin to be lost in the product formulation. “A magnesium chloride solution has been successfully used to overcome interferences by chelating agents such as ethylenediaminetetraacetic acid or citrate buffers. There are other proprietary products, such as Pyrosperse (Lonza) or a glucan blocker that have also been used.”

	If difficulties persist, however, Burgenson recommends the best course of action is to contact the LAL reagent manufacturer, so that the work can be done in partnership. “These manufacturers have seen all types of interferences many times over, and, therefore, should be able to help,” he says.

	As LAL assays are performed using an extract of blood cells from the Atlantic horseshoe crab, the prehistoric marine arthropod plays an important role in patient safety. “This animal is under legal protection,” emphasizes Watson. “Without this protection, the horseshoe crab population would be threatened.” 

	Dubczak concurs, highlighting the fact that reduction of animal use for pharmaceutical testing has been a well-established influencer of the industry over the years, which has seen the LAL in-vitro test replace the traditional RPT to prevent the use of hundreds of thousands of rabbits per year to test for product safety and contamination. The LAL test is an animal-derived test, meaning the animal itself is not tested upon. “The horseshoe crab’s blood is collected only to a point where the crab stops bleeding as part of a natural mechanism to protect itself,” he notes.

	Additionally, there has been much work and commitment by manufacturers of the LAL reagent to conserve the horseshoe crab. “Work has included sustainability partnerships to enhance the horseshoe crab population along the mid-Atlantic shores,” says Watson. “Such projects have focused on increasing the survival rate of the horseshoe crab in early stages of life. Eggs are collected, and the crabs are grown in predation-free, controlled environments then released into their natural environment when older and stronger.” 

	Other projects include the “Just Flip Em” program where volunteers scan the beaches to rescue horseshoe crabs stranded on the beach on their backs, adds Burgenson.  Each year many thousands of stranded crabs are rescued.

	“Without the protection of the biomedical industry, these prehistoric creatures would surely become endangered, if not extinct. For this reason, it is critical that we serve as advocates for the humane treatment of these animals and strive to achieve balance between our need for this valuable material and the life of the animal that provides it,” stresses Dubczak.

	Raw material quality: Regulation required?

	“There has been an increase in raw material manufacturers requiring their products be tested for endotoxins,” notes Sophie Bell, section head for Bacterial Endotoxin and Cell Culture at Wickham Labs. “These requests come in usually due to pharmaceutical manufacturers requiring materials that do not contain detectable levels of endotoxins. The responsibility of the manufacturer is to source adequate materials for their products, so regulation of raw materials seems unnecessary; especially as different endotoxin specifications in end-products can affect the level of endotoxin contamination allowed in raw materials.”

	In agreement, Dubczak highlights that product regulations are designed to set forth the standardized expectations for finished drugs, biologics, and medical devices, and as such the responsibility of sourcing adequate raw material is that of the manufacturer. Using an example from FDA guidance on pharmaceutical good manufacturing practices (5), he states that it is clear that regulators expect the sound application of science and associated risk-based rationales to be the basis for a product’s development and manufacturing process. 

	“For instance, manufacturers should determine the level of risk for both active and inactive raw materials, and there should be a comprehensive assessment of critical raw material formulations, ingredients of in-process buffers, and final product formulations,” he adds. “However, to place strict regulations on this process would be counter to the intent of this approach.”

	Furthermore, Bell emphasizes the point that raw materials are not the only source of endotoxin contamination in end products. “Manufacturing processes can equally be a source of contamination, so the requirement for endotoxin testing in the product is still necessary. Testing of products from the beginning, middle, and end of manufacture is still advised to ensure uniformity across the manufacturing process,” she says. “Products that can be terminally depyrogenated will not require as clean raw materials compared to those that cannot.” 

	Despite the fact that raw materials going into pharmaceutical products are not specifically regulated, drug manufacturers use the highest grade of raw materials obtainable, emphasizes Burgenson. “Many of these materials are USP grade,” he adds. “Manufacturers will test raw materials and purified waters for the presence of endotoxins, and determine if the raw material meets its specifications and is suitable for manufacturing use.”

	“What is important is that the quality of the raw materials used in production meet the standards appropriate for their intended use,” clarifies Dubczak. “Raw materials or reagents do require science-based evaluation to establish the presence or absence of deleterious endogenous or adventitious agents.”

 71 621–651 (1925).
	2. J. Levin and F.B. Bang, 

 115 337–345 (1964).
	3. J. Levin, P.A. Tomasulo, and R.S. Oser, 

 75 903–911 (1970).
	4. F. Pearson, “LAL Assay: Applications and Equivalence,” in 

Pyrogens: Endotoxins, LAL Testing, and Depyrogenation

, Marcel Dekker, pp. 148–155 (New York and Basel, 1985).
	5. FDA, “Pharmaceutical cGMPs for the 21st Century-A Risk Based Approach,” Final Report (September 2004). 


	Vol. 43, No. 7
	July 2019
	Pages: 38–42

	When referring to this article, please cite it as F. Thomas, “Putting Endotoxins to the Test,” 

This article discusses current methods of endotoxin testing, its importance to the industry, sustainability issues relating to the process, and best practices.
