	Contract research organizations (CROs) and contract development and manufacturing organizations (CDMOs) may not be feeling it yet, but the downturn in external financing for early stage bio/pharma companies is real. The impact on CDMOs and CROs will be delayed, but there is no doubt that service providers will be feeling it in coming months.
	
	Two recent articles in the financial press underscore what has been happening. An item on 

 chronicled the challenges early-stage public companies are facing as they go out for further funding beyond their initial public offerings (IPOs) (1). The article highlights the plight of two bio/pharma companies, Aldeyra Therapeutics and Ovascience, whose stock prices took big hits when they floated secondary public offerings of their stock on May 26th.  The shares of Ovascience went down 30% on the day of the offering while those of Aldeyra fell 10%.

	The Bloomberg article noted that Aldeyra and Ovascience’s experiences are reflective of what has been happening to many bio/pharma companies trying to tap public markets. According to the article, the number of secondary offerings from bio/pharma companies is down 40% in 2016, with 64 offerings vs. 106 in 2015; but the amount raised is down 70% from $9 billion to $2.6 billion. So it’s not just the decline in the number of offerings that is hurting early-stage bio/pharma; they are also raising fewer dollars per offering.

	The market has also not been friendly to young bio/pharma companies trying to tap public markets for the first time. There have been half the number of IPOs, 8 in 2016 vs. 17 in 2015, but the amount raised, just $483 million by Bloomberg’s count, is down 75%.

	Data compiled from the PharmSource Lead Sheet (

) confirms the Bloomberg analysis and also shows that venture capital (VC) investment has held up well despite the public market travails. Nevertheless, an article in the 

 described how the balance of power has shifted from entrepreneurs to investors in the venture capital world (2). According to the article, VC firms have been able to demand much tougher terms from companies they are investing in, including lower valuations and the hiring of more experienced executives. The article focuses on Internet companies, but the pinch is being felt across the start-up spectrum.


	It’s not surprising that CROs and CDMOs may not be feeling the impact of the funding downturn quite yet. After the 2008 global financial crisis, it took two years for investigational new drug (IND) filings and Phase I clinical trial starts to reflect the funding decline (

). That’s because early-stage companies focused their remaining cash on getting their lead candidates into the clinic as quickly as possible in hopes of demonstrating proof of concept (POC). POC is typically the prerequisite for licensing deals and other partnering arrangements from larger bio/pharma as well as funding from public sources. Funding from partnering arrangements is probably the most secure funding source because large bio/pharma companies now depend on in-licensed and acquired candidates for at least half of the products they ultimately take to commercial markets.

Figure 2: Investigational new drug filings 2009 to 2015.

	Early-stage companies trying to get into the clinic are an important source of business for CDMOs because most are dependent on service providers to manufacture APIs and formulated dose forms. Those companies represent the majority of customers at most CDMOs, but they typically have just a small number of new drug candidates, so CDMOs need to constantly replenish their customer portfolios to thrive. That replenishment is highly dependent on a rebound in public bio/pharma equity markets: venture capital might get candidates through discovery and into preclinical but emerging bio/pharma companies need the larger tranches of public funding to sustain a clinical development program.


	The most recent downturn in external financing is barely a year old, so CROs and CDMOs aren’t yet feeling the pinch. Emerging bio/pharma companies are using the funds they have to get their candidates into the clinic, as in past funding cycles, which is good for CDMO business in the near term. But without public markets, the industry could see a sharp drop in IND filings, and in the demand for CDMO services, as it did in 2010.

	One way for CDMOs and CROs to prepare themselves for the worst case is to focus on resurrecting their business development skills. After several years of just answering unsolicited external inquiries, they will soon have to be prospecting for new business and selling the customer on why they should use them rather than a competitor. As they were in the last downturn, new business development skills will be a key to sustained success.

“Biotech’s Vicious Secondary Cycle,”

“Start-Ups Once Showered With Cash Now Have to Work for It,”


	When referring to this article, please cite it as J. Miller, “Bio/Pharma Funding Challenges Could Hurt CDMOs in 2017," 

Articles

Perspectives on Outsourcing

CDMOs need to be aware that unfavorable public markets put emerging bio/pharma R&D spending at risk in 2017.

Bio/Pharma Funding Challenges Could Hurt CDMOs in 2017

	The International Organization for Standardization (ISO) published the long-awaited revisions to its standards for classification and monitoring of air cleanliness in cleanrooms on Dec. 15, 2015. ISO 14644-1:2015 “Cleanrooms and associated controlled environments Part 1:Classification of air cleanliness by particle concentration” (1) replaces ISO 14644-1:1999, and ISO 14644-2:2015 “Cleanrooms and associated controlled environments Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by particle concentration” (2) replaces ISO 14644-2:2000.

	The 2015 editions are the result of a systematic review and include changes made in response to requests by users and experts in the cleanroom community. In particular, the requests for reviewing Part 1 were related to “the basis for the number of sampling locations and, most importantly, the whole statistical basis of classification of cleanliness using the Student T-test for one to nine sampling locations,” notes Gordon Farquharson, convenor of the ISO TC209 working group 1, which performed the review and revisions. Because Part 2 is closely aligned with Part 1, the committee reviewed both parts together.


	The addition of “by particle concentration” to the title of the standard is a long-overdue clarification, comments Karen Ginsbury, CEO at PCI Pharmaceutical Consulting. “For years, I have heard cleanroom contractors and practitioners alike wrongly describe ISO 14644-1 and 2 as cleanroom ‘validation’ or ‘qualification’ standards.” She notes that the standards only address airborne particles, not other factors crucial to cleanroom qualification, such as smoke tests to determine airflow patterns.

	The introduction of Part 1 explains, “This part of ISO 14644 specifies classes of air cleanliness in terms of the number of particles expressed as a concentration in air volume. It also specifies the standard method of testing to determine cleanliness class, including selection of sampling locations” (1).


	The primary changes to Part 1 involve the number of samples and the selection of sampling locations. “The number of samples will increase from what was required previously,” explains Marsha Stabler Hardiman, senior consultant at ValSource. “The minimum number of samples is now determined from a lookup table (instead of an equation), and that number is set to be statistically significant.”

	According to ISO, the new method for selecting the sites and number of sampling collections uses a more consistent statistical approach based “where samples are drawn randomly without replacement from a finite population. The new approach allows each location to be treated independently with at least a 95% level of confidence that at least 90% of the cleanroom or clean zone areas will comply with the maximum particle concentration limit for the target class of air cleanliness. No assumptions are made regarding the distribution of the actual particle counts over the area of the cleanroom or clean zone; while in ISO 14644-1:1999 an underlying assumption was that the particle counts follow the same normal distribution across the room” (1). The sampling locations are to be chosen representatively, meaning that “features such as cleanroom or clean zone layout, equipment disposition, and airflow systems should be considered when selecting sampling locations” (1).

	This representative selection of sample locations is a big change from the previous random selection, says Hardiman. “A company now has to have a rationale and justification for sample site location to ensure that the sample locations selected are representative of the characteristics of that section.  Companies will have to look at the new number of sample locations and then determine where the representative sample locations will be collected.  If contracting out the classification activities, you should make sure that your contractor is now using your new, representative sample locations.” 

Classification of macro and nano-particles


	The revision makes a change regarding large (≥ 5 µm) particles, which are required to be measured for some classifications in the EU Annex 1 GMP guidelines (3) and others. “The experts working on the revision of ISO 14644-1 were of the opinion that particles ≥ 5 µm diameter should not be used to classify ISO class 5 and cleaner environments because of the uncertainty associated with particle collection efficiency and accuracy of counting low concentrations,” says Farquharson. “In order that the European Union (EU), the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme, World Health Organization, and Chinese GMPs are not left without a classification tool for their Grades A  (at rest and operational) and B (at rest), ISO 14644-1:2015 provides a mechanism of extrapolating the macro-particle descriptor for class limits of 20 and 29 particles ≥ 5 µm.”

	The new document does not address nano-scale particles, which were formerly defined as ultrafine particles  in ISO 14644-1:1999, but will address these under a new Part 12, notes Farquharson, who explains, “These particles are measured using a different particle counter, and industries such as semi-conductor monitor for concentration of these very small airborne particles at critical control points. These particles are not generally of interest to the pharmaceutical and life sciences industries.”


	ISO 14644-2:2015 now requires monitoring to provide evidence of cleanroom performance, explains Farquharson. The standard addresses airborne particle concentration, airflow, and device pressure difference. New topics include monitoring of critical parameters and setting action and alert alarms.

	The revised standard now allows companies to use risk management to set their periodic classification testing schedules, notes Hardiman. “In the past, the retesting was prescribed in a table and the timing was based on the ISO class of the cleanroom or clean zone. Now, companies can put more emphasis on the day-to-day data that they generate in their own facilities to help determine the appropriate testing and frequencies needed for continued cleanroom compliance. If a company is generating great data and the risk to continued cleanroom compliance is low, then they can set a longer periodic classification frequency,” explains Hardiman. She notes that risk-based sample site selection is crucial for environmental monitoring. “The key is understanding your unique products and processes and selecting sites that best address the risks that your products and processes present. It is important to be able to identify all of the potential contamination sources in each cleanroom and to select environmental monitoring sample locations in close proximity to these sources. It is also very important to understand the people, material, and waste flows,” concludes Hardiman.

	Pharmaceutical cleanrooms typically already have monitoring plans, which are required by Annex 1 of the EU GMPs, says Ginsbury (3). She notes that users should, however, check with their cleanroom contractors to determine whether a contractor is qualified and familiar with the revisions, because the regulators reference the ISO standards and they must be followed, says Ginsbury. “I would recommend having the discussion now so that, by 2017, your contractors and in-house staff are fully up to speed and following the new standard,” she notes. “Even if you use contractors, the responsibility for review and approval of their results and compliance with regulatory standards lies with you. ISO may not require 5 µm particles and may let you use risk assessment to determine frequency of classification. However, EU Annex 1 still requires measuring 5 µm particles as part of classification, and current industry practice (EU [3] and FDA [4]) is to perform cleanroom classification twice a year for aseptic core. We haven’t heard the regulators on this one but I wouldn’t rush to reduce that frequency based on risk assessment,” cautions Ginsbury.

ISO 14644-1:2015, Cleanrooms and associated controlled environments – Part 1: Classification of air cleanliness by particle concentration

		ISO, ISO 14644-2:2015, Cleanrooms and associated controlled environments – Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by particle concentration (Geneva, 2015).

Volume 4: Good manufacturing practice Guidelines

, “Annex 1, Manufacture of Sterile Medicinal Products,” (Brussels, 2008).

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing-Current Good Manufacturing Practice 

:
	When referring to this article, please cite it as J. Markarian, "Revised ISO Cleanroom Standards Improve Air Cleanliness Classification," 

Features

Revised versions of ISO 14644 Parts 1 and 2 introduce changes to sampling procedures and monitoring plans for cleanrooms and clean zones.

Revised ISO Cleanroom Standards Improve Air Cleanliness Classification

	Article submitted: Nov. 9, 2015. 
	Article accepted: April 5, 2016.
	

In 2014, the monoclonal antibodies market had the highest growth rate (19%) for the number of new molecules in the pipeline. DNA and RNA therapeutics were not far behind, achieving 12% year-over-year growth. Industry analytics data suggest that the RNA-based therapeutics market will reach $1.2 billion by 2020.

	This article reviews the current dynamics in the RNA therapeutics/vaccines market as well as differences between small-interfering RNA (siRNA), RNA interference (RNAi), microRNA (miRNA), and messenger RNA (mRNA). In addition, the authors outline the general production processes for these platforms, the challenges encountered during process development and production, and the strategies to overcome them.


	RNA-based therapeutics target the treatment of diseases such as diabetes, cancer, tuberculosis, and some cardiovascular conditions. There is currently a great deal of money being put into this relatively new class of therapeutics and vaccines, which is projected to grow 12% in 2016 and reach $1.2 billion by 2020 (1). The 2015 research and development (R&D) biotech pipeline is shown in 

. There are more than 700 nucleic acid-based therapeutics (DNA and RNA) in the pipeline and more than 60% of the nucleic acid-based therapeutic pipeline is in preclinical development. It is interesting to note that 35% of such pipeline is focused on oncology (2, 3).

	Several companies (approximately 160) and many academic institutes (approximately 65) are developing RNA-based therapeutics. 

 provides a non-comprehensive list of a few (4). Two companies have marketed RNA-based therapies: NeXstar and Ionis Pharmaceuticals. There are 12 mRNA vaccines in development, seven of which are being developed by Curevac (Germany). Based on current outlook, the RNA therapeutics market seems more promising than the market for DNA therapeutics.

	From a partnership perspective, Ionis Pharmaceuticals has entered into a global collaboration with Janssen Biotech, Inc. to discover and develop antisense drugs to treat autoimmune disorders of the gastrointestinal tract (5), and Merck & Co. (MSD) has bet $100 million on Moderna’s mRNA technology (6). Moderna also has previously announced collaborations with Alexion, AstraZeneca, and the Defense Advanced Research Projects Agency (DARPA) totalling $450 million. Moderna has raised $625 million in equity funding (7, 8).

	RNA interference (RNAi) and RNA antisense technologies appear to be dominating the market. RNAi is a gene-silencing technology in which RNA molecules inhibit gene expression by targeting and destroying specific mRNA molecules. RNA antisense technology involves synthesizing an RNA strand that binds to a specific mRNA or to a splicing site on a pre-mRNA molecule to prevent translation. The major challenges associated with the commercialization of these RNA-based therapies are toxicity and drug delivery.

With the advent of RNA-based therapeutics and their potential in treating a variety of chronic diseases, it is important to note the number of enabled technologies used to exploit the RNA mechanism/pathway, some of which are discussed in the following.


	RNAi technologies work by “silencing” or turning off a gene through the use of its own DNA sequence (

). The process is initiated by double-stranded RNA (dsRNA) that expresses either as a small or short hairpin RNA (shRNA) or as a microRNA (miRNA) transcript. Using this silencing mechanism, RNAi is commonly used to gain a better understanding of gene function, which can then be used to generate additional targeted therapeutics (9). Small interfering RNA (siRNA) and miRNA are the core elements of RNAi technology based therapeutics.


	RNAi utilizes a “dicer” enzyme to cut dsRNA into 21 oligonucleotide segments, called siRNA. These siRNAs can then bind to a specific family of proteins called Argonaute proteins, of which there are two classes: Ago and Piwi. Ago proteins bind to siRNAs or miRNAs, while Piwi proteins bind to Piwi-interacting RNA (piRNA) and are used to silence mobile genetic elements. The siRNA, miRNA, or piRNA complex bound to the Argonaute protein is called the RNA-induced silencing complex (RISC). Once bound to the Argonaute protein, one strand of the dsRNA is removed and the remaining strand binds to and directs the degradation of the complementary RNA target sequence, which then leads to the loss of protein expression (10).

It has been reported that synthetic siRNA is able to knock down targets in various diseases 

, including hepatitis B, human papilloma virus, ovarian cancer, bone cancer, hypercholesterolemia, and liver cirrhosis. Only a few molecules of siRNA per cell are required to produce effective gene silencing (11). siRNAs are most commonly delivered into cells using microinjection or a transfection agent. Many companies now offer siRNA-delivering reagents to simplify this process (12).


	miRNA do not code for proteins, as they belong to specific class of non-coding RNAs. miRNA are 19-25 nucleotides in length and are encoded within introns (i.e., the portions of the gene sequence that are not expressed in the protein) (13). miRNA acts as a guide strand for the RISC complex to its mRNA target in vertebrates. Approximately 30% of genes in the human genome are regulated by miRNA (14).

	Though siRNA silencing requires exact match between target and small interfering RNA, miRNA are non-specific and can exert action through imperfect base pairing. In addition, miRNA triggers translation inhibition (i.e., prevents the RNA from synthesizing protein from amino acids), while siRNA triggers mRNA degradation. 


	mRNA, which codes for protein, is an essential component of the central dogma of life (DNA-mRNA-protein). mRNA is transcribed from a DNA template. mRNA takes the genetic code from DNA to the ribosome where the mRNA is translated to protein. There has been a significant increase in mRNA-based therapies in large part due to the many advantages that mRNA has over DNA in relation to gene expression and transfer. While RNAi and antisense RNA technologies are used primarily for gene silencing, mRNA technologies are often used in vaccines or gene therapy (15). In both cases, after injection into the human body, mRNA is translated to protein, which can ultimately replace a missing protein (therapeutic) or induce an immune response (preventive approach). The production of synthetic mRNA for therapeutic use is relatively straightforward, and the challenges associated with its stability and delivery have been tackled through scientific advances in recent years (16).


	Conceptually, mRNA-based vaccines are a simple approach to inducing an immunological response by delivering the coding genetic element as a translation-ready molecule. Upon direct vaccination with mRNA molecules, dendritic cells (antigen-presenting cells) take-up, process, and encode the target antigen, which in turn induces an immune response. Typically, mRNA vaccines are produced by 

 synthesis through an enzymatic process. Such a synthetic process can be tightly controlled, resulting in a quality and predictable product profile. mRNA can be easily tailored to offer a specific immunogenic profile and pharmacokinetics (17). mRNA’s stability and antigenic properties can be easily manipulated by changing codon or modifying base pairs.

	Ongoing clinical trials show that mRNA can be delivered as naked mRNA; immobilized on particles or in liposome nanoparticle; or transfected in dendritic cells 

 resulting in a discernible immune response and protective efficacy. mRNA can also act as an adjuvant and mRNA also has been explored to stimulate the innate immune system through toll-like receptors (18). RNA-based vaccines are comparatively simple to produce and can be developed, manufactured, and administered in a short time period, therefore, they are suitable for pandemic situations. Thermostability of mRNA vaccines can also significantly contribute to their low cost, as they do not require cold-chain distribution.

Manufacturing RNA-based biopharmaceuticals


	As more experimental RNA drugs move through the clinic and into large-scale trials, the demand for efficient and cost-effective manufacturing strategies will grow (19). RNA-based biopharmaceuticals are inherently susceptible to endonucleases, so special handling is required for production and purification. Degradation of product during manufacturing adds heterogeneity and chemical instability to the product. Therefore, the manufacturing and purification methods used in RNA-based therapeutics differ from that of DNA and other proteins (20).

	mRNA purification (post-chemical synthesis) includes concentration precipitation, extraction, and chromatographic methods (including high-performance liquid chromatography) (19). The purpose of the upstream concentration and diafiltration step is to concentrate (if lower titer) and change the buffer to the necessary pH and conductivity for the first chromatography step. The objective of the final concentration and diafiltration step is to de-salt and achieve the necessary final concentration prior to sterile filtration. A 5-kD membrane cut-off is generally used for concentration and diafiltration in mRNA processes. Because siRNA are smaller than mRNA, a 1-kD membrane cut-off is used for adequate retention of the siRNA product (21).


	Since the breakthrough discoveries of catalytic RNAs in the early 1980s and RNA interference in the late 1990s, more than 50 RNA or RNA-derived therapeutics have reached clinical testing. In RNA purification, despite the different techniques such as arginine-affinity, ion-pairing reversed-phase, or pellicular anion exchange, the traditional ion-exchange (IEX) media-especially anion exchange (AEX)-remains the most popular technique  used in both pure RNA and RNA packaged for delivery (22, 23, 24, 25).

	Sm and Sm-like proteins, which can form heteromeric complexes or bind to various RNAs, were proven to contain ancient RNA-binding motifs (Sm domain) with oligo(U) specificity (26). Fractogel TMAE (MilliporeSigma), a strong anion-exchange resin, was used for the purification of small nuclear ribonucleoproteins (snRNP). The snRNP molecule was eluted with Tris/HCl and 300 mM NaCl. Ribonucleoprotein and uncoupled RNA were separated from free protein, and the sample was immediately used for negatively stained electron microscopy.

	Both AEX and reversed-phase (RP) technologies are widely used in the RNA purification process. Quarternary amine (Q) and Dimethylaminoethyl (DMAE) chemistry are among the choices for AEX (27, 28, 29, 30). One study proved that a few AEX resins can be used for RNA purification with optimized experimental conditions to achieve high dynamic binding capacity. In this study, among the 18 AEX medias that were screened, only four resins-Q Sepharose FF (GE Healthcare), POROS 50HQ (Applied Biosystems), Q Ceramic HyperD F (Pall), and Fractogel DEAE (MilliporeSigma)-showed baseline separations of RNA and plasmid DNA (31). After optimized loading and eluting conditions, Fractogel DEAE had a wider range of operation, higher dynamic binding capacity, and complete separation of RNA in the breakthrough from plasmid in the elute. The high recovery, robustness, and reproducibility also met the requirement for large-scale manufacturing. These binding and elution conditions can be utilized as a starting point for optimal experimental conditions in RNA purification.

	Overall, many biochromatography resins are suitable for RNA purification similar to use in other biomolecule separations. In many cases, the Fractogel resins showcased superior capacity and efficiency, largely due to the “tentacular” structure whereby functional groups are located at the end of long arms grafted to the bead surface, which circumvent the steric hindrance caused by large biomolecules (32).


	The most challenging aspect of RNA-based therapeutics is its delivery to target cells. Several methods have been explored and tested in clinical trials. Some of the most promising approaches are explained in the following passages.

Polymer conjugation/chemical modification


	Native RNA and RNA-based therapies are vulnerable to degradation from the many ribonucleases found within the cell. Chemical modification is one method for hardening the RNA against such enzymatic attacks. Modifications to the molecule can also increase its target affinity, decrease its undesired immunogenicity, and improve its overall efficacy. Hardening strategies include modifications to the backbone, sugar, or base of the RNA molecule.

	Conjugation of the RNA therapeutic is a strategy that is increasingly being used for improved delivery and uptake. Alnylam Pharmaceuticals has adopted a method of conjugating an amino sugar derivative of galactose, 

-Acetylgalactosamine (GalNac) to improve the delivery of siRNA therapies to the liver. The GalNac-conjugated siRNA is taken up by asialoglycoprotein receptors in the liver resulting in a fivefold increase in efficacy versus the parent molecule (33).

	Arrowhead Research is developing a competing conjugation strategy. Arrowhead’s delivery technology, termed Dynamic Polyconjugates (DPCs), is a siRNA bound to an endosomolytic polymer backbone via a disulfide bond. The endosomolytic polymer enables the quick and efficient release of the siRNA from the endosome. Arrowhead’s most recent strategy includes attaching cholesterol to the siRNA and GalNac to the endosomolytic polymer, ensuring they are both delivered to the hepatocytes. The co-injection therapy was shown to increase the efficacy of siRNA-cholesterol 500-fold with a 90% knockdown (34).


	The dominant and most-studied strategy for the delivery of RNA-based therapeutics is lipid-based delivery systems. One successful platform is the use of stable nucleic acid lipid particles (SNALPs), which are lipid particles formed from a fusogenic lipid, cationic lipid, and PEG-lipid mixture. The SNALP delivery system has been developed and championed by Tekmira Pharma; the company now refers to it as LNP technology. According to Tekmira, the LNP “encapsulates siRNAs (also mRNA) with high efficiency in uniform lipid nanoparticles that are effective in delivering RNAi therapeutics to disease sites in numerous preclinical models” (35).

	Another promising lipid delivery technology is the proprietary Smarticles delivery platform developed by Novosom and now owned by Marina Biotech. Similar to SNALPs, the Smarticles technology can change their surface charge to facilitate both stability and endosomal release. Smarticles are capable of encapsulating both single- and double-stranded nucleic acid therapies. Smarticles are comprised of cationic, anionic, and neutral lipids. The negatively charged Smarticles avoid the often seen toxic effects of positively charged lipids at physiological pH but convert to a positive charge in the acidic environment of the endosome, facilitating its release. Other interesting encapsulation techniques involve PLGA nanoparticles (36, 37).


	RNA-based therapeutics are a relatively new class of therapies that has bright prospects in the treatment and prevention of difficult-to-treat chronic and rare diseases. RNAi work by interfering with the transcription process, and thereby inhibit protein translation. Though such a therapeutic approach is highly selective and targeted, special care is required during the production of these therapies and vaccines because of their susceptibility to ubiquitous RNAse-induced degradation. Large-scale manufacturing of new class of therapeutics would require bioprocessing components, chemicals, and tools free from RNAse. Technology and tool providers need to consider making such products available to enable large-scale production of RNA-based therapeutics. The surmounting challenges related to potential toxicity and drug delivery need to be addressed before such products can be commercialized. However, new technologies are emerging to overcome some of these challenges, and the future of RNA-based therapeutics is very promising.
	
	ALL FIGURES ARE COURTESY OF THE AUTHORS.

“RNA Therapeutics Market is Expected to Reach $1.2 Billion, Globally, by 2020,”

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“Merck Bets $100M on Moderna and its Pioneering RNA Tech,”

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“Our Core ‘Expression’ Platform: Messenger RNA Therapeutics,”

“With Massive Venture Round, Moderna Has $450M Reasons to Stay Private,”

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 9 (2):210. doi:10.1186/gb-2008-9-2-210. Feb. 26, 2008).
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Gene Therapy of Cancer: Translational Approaches from Preclinical Studies to Clinical Implementation

, E.C. Lattime and S.L. Gerson, Eds. (Academic Press, 3rd ed., 2013), pp. 47–65.
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13 (10), pp. 759–780 (October 2014).
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98 (7), pp. 3685–3689 (March 27, 2001). 
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9 (4), pp. 596–606 (April 2004).
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804 (2), pp. 327–335  (2004).
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	Elina Gousseinov, Mikhail Kozlov, Claire Scanlan, Aaron Hammons, Ling Bei, Youssef Benchek, Karim Pirani, and Ruta Waghmare are all from MilliporeSigma. Priyabrata Pattnaik works in the Biomanufacturing Sciences and Training Centre at Merck Pte Ltd., Singapore.

 When referring to this article, please cite it as E. Gousseinov et al., "Bioprocessing Technology Trends of RNA-Based Therapeutics and Vaccines," 

Peer-Reviewed Research

The authors present an overview of the types of RNA-based therapeutics in existence and their optimal methods of manufacture, purification, formulation, and delivery.

Bioprocessing Technology Trends of RNA-Based Therapeutics and Vaccines

	While a strong focus on quality, safety, and drug efficacy is absolutely essential, biopharmaceutical manufacturers are also committed to finding ways to reduce costs and improve manufacturing efficiencies. One production step with significant costs (and potential risk to quality) is the buffer and cell-culture materials preparation process: it is labor-intensive, requires investment in storage and environmental resources, and involves repeated quality assurance (QA) testing as bulk materials are subdivided for individual process runs.

	New innovations in raw materials packaging technologies can directly impact this process--streamlining operations, mitigating risks, and contributing to operational excellence (OpEx). In this article, the author examines new methods of raw materials packaging and how they may lead to manufacturing efficiencies, elimination of raw material yield losses, reduction in QA testing time and costs, and lower costs in weigh and dispense production.

Traditional raw material delivery methods


	Upstream biopharmaceutical processes consume various raw materials, including cell-culture media, carbohydrates, amino acids, and buffers, which are typically supplied in powder form. The bioreactors and medium preparation tanks that use these materials often operate around the clock. This operation includes both large-scale reactors with 10,000 L capacity that run continuously for anywhere from 15-35 days, to newer generation single-use technologies, with multiple 2000-L bioreactors operating in overlapping sequences to achieve similar or greater productivity.

	In general, two kinds of packaging systems are in use today: Traditional steel 100 kg drums with one or two plastic liners, and smaller cardboard boxes with plastic liners holding 50 kg. Both bulk-packaging systems are part of standard practices that most raw materials suppliers have established for their supply chain systems. The end user (i.e., the biopharmaceutical producer) typically orders, receives, and stores enough salts, buffers, and other cell-culture powdered materials to last several weeks or months. The material consumption rates can be substantial: a typical buffer media preparation/usage cycle may consume between 150 and 400 kg of dry products per run, and uses multiple raw materials per cycle. These materials are then subdivided by the biopharmaceutical producer and used in smaller amounts depending on the processes they are running. In essence, this bulk material packaging and delivery methodology satisfies the raw material supplier’s operational requirements, without full consideration of how that material is used by the biopharmaceutical producer.


	Following the more traditional method described above, the biopharmaceutical manufacturer must follow multiple processing steps to properly manage and use these bulk raw materials.

		Bulk materials are received and inventoried in storage areas that must have the appropriate temperature and humidity controls to maintain the material’s integrity.

		The container’s outer packaging is cleaned and sanitized. A sample is taken to independently confirm via lab analysis that the product’s quality, purity, and characterization match what was ordered. Once this is confirmed (and analysis can take multiple days), the bulk material is cleared for use in the producer’s dispensing operation.

		Once cleared, the contents of the drum are subdivided by hand according to manufacturing requirements. For example, if 45 kg are initially required, that quantity is removed and the remainder is put back into storage. For a 2000-L bioreactor, a manufacturer may need to subdivide a 100 kg drum of material between two and five times.

		This subdivision step is time-consuming and risks cross-contaminating the remaining bulk material. Some biopharmaceutical producers conduct multiple lab analyses of these drums, each time a new quantity of material is removed, to confirm that no issues have occurred.

	Much of this activity precedes, and is not directly related to, the value-added process of protein manufacturing. It is time and effort spent on materials management and warehousing activities, particularly the sub-dividing step, to supply the bioreactors with the precise amount of material needed for production.

Complications due to material caking/clumping

Various raw materials, such as salts, buffers, amino acids, and carbohydrates, have an intrinsic propensity to form clumps or cake due to their crystal structure and surface moisture content. The presence of available surface moisture catalyzes the process of caking when free moisture migrates onto the surface of the crystals and dissolves a small portion, forming a salt bridge. This caking, or clumping (

), is a common problem with packaging, storing, and sub-dividing powdered materials in bulk containers.

	Changes in ambient temperature or humidity are the principal factors driving this process; the number of temperature-change cycles will increase the strength of the cake. Severe cases of caking can result in complete solidification of the entire package. Caked materials must be completely broken up in order to measure out the precise amounts needed for bioreactor processes. This is a time-consuming manual activity with an open container, which is at risk for cross-contamination and absorption of additional moisture, potentially extending the problem. This practice also creates a potential safety risk, as operators work to manually break up clumps while the container is open, and can lead to material loss.


	Over the past few years, pharmaceutical and biopharmaceutical manufacturers have worked with bulk material suppliers to modify packaging approaches. The goal was to enhance operational excellence and reduce wasted time and impact on process and product quality. The initial focus was on bulk material container sizes, which typically held many more materials than were needed, and forced on-site storage and subdividing steps. While continuing to offer the standard 50 kg and 
	100 kg containers, suppliers began to provide smaller, more manageable container sizes, such as 12 kg and 25 kg.

	This approach reduced the length of time containers were kept in storage and the number of times a container was subdivided. Subdividing was still required, however, because the exact amounts of sugars, buffers, salts, and other powdered bulk materials varied greatly by manufacturer, based on the specific process, protein, and bioreactor equipment being used. Subdividing also adds a measure of uncertainty to the amount of raw materials dispensed in a particular process step. Biopharmaceutical production uses extremely tight control on process parameters to protect the final drug’s safety and quality, and finding ways to strictly control the amount of raw material that goes into bioreactors can enhance operational excellence and process yield.

Figure 2: To help ensure a free-flowing dispensing system and eliminate clumping, some bags have outer and inner layers with special desiccant materials installed between the two layers. The inner layer uses a vapor permeable material; any moisture that develops within the bag passes through this material and is controlled by the desiccant, to maintain the correct moisture levels.

New packaging for pre-weighed, free-flowing materials


	Innovative chemicals suppliers have begun offering single-use, pre-weighed product bags that provide biopharmaceutical producers with an easy-to-use method for dispensing salts, buffers, and other cell-culture materials directly into their media or buffer preparation tanks, in the exact amounts they specify for a given process. The packaging options that are now used essentially complete the evolution from 50 kg and 100 kg bulk product drums, to individual direct dispense bags. The packaging is constructed of transparent polymers, the same materials that have already been used to line the traditional bulk drums; therefore, biopharmaceutical manufacturers do not have to re-validate the material as safe for use.

The packaging’s design offers biopharmaceutical manufacturers more choices with regard to packaging size. Manufacturers can order the materials in a wide range of smaller, precise quantities (e.g., from 250 g to 100 kg). The size, shape, sealing, and seams of these bags are designed so that when they are inverted, they dispense virtually all the pre-weighed material into the bioreactor. An important consideration here is that pre-weighed amounts should be within a 1% tolerance of the amount of material required. This is crucial, especially when the direct dispense bags are used in single-use bioreactors. If a biopharmaceutical manufacturer has determined that an exact amount of glucose or sodium citrate is needed to achieve maximum yield on a process, the ability of the packaging to freely deliver that amount to very tight tolerances must be assured.
	To help ensure a free-flowing dispensing system, the bags also incorporate design features to eliminate clumping, including outer and inner layers with special desiccant materials installed between the two layers (

). The inner layer uses a vapor permeable material already in use in other container-lining applications. Any moisture that develops within the bag passes through this material and is controlled by the desiccant, to maintain the correct moisture levels and reduce clumping to an absolute minimum (

Direct dispense bags simplify sampling and testing


	Traditional large-volume bulk container packaging also necessitates the time-consuming process of sampling and testing to verify the material properties of a newly delivered drum of product. Direct dispense bag systems use transparent polymers that are compatible with non-destructive identity testing tools, such as contact-free Raman spectroscopy. With Raman testing, there is no need to open the bag and take a physical sample to verify the product, the closed bag can be scanned and verified upon delivery, saving multiple testing steps. The packaging is also tamper-evident to ensure validity and supply chain security.

	In addition to near Raman testing, some suppliers also will provide a tailgate sample along with the bags. Biopharmaceutical producers that are required to conduct full analyses of all materials used in their processes don’t need to open the bag to obtain a product sample. The tailgate sample process must be validated to ensure that the material in the tailgate sample is equivalent to the materials packed in the bag.

Direct dispense systems: Time and cost savings


	Expanding the use of these direct dispense systems can help advance operational excellence initiatives and reduce costs within the biopharmaceutical industry’s supply chain. By adopting flexible manufacturing technology, such as the direct dispense systems, biopharmaceutical manufacturers may be able to reduce their operating costs in certain areas by up to 40%. There are multiple savings associated with the use of these systems:

		Labor: Eliminates the time and cost of personnel who need to weigh, subdivide, and dispense materials from bulk containers. The savings can be significant. Depending on the process, each 1000 L run of a cell-culture reactor may require approximately 1000 to 2000 kg of more than 50 raw materials for the production process. The media/buffer preparation activities could require 30-50 labor hours for dispensing and adding the materials to the reactors.

		Facilities: Use of direct dispense systems can eliminate the need for dedicated raw material preparation areas, drum storage and handling equipment, and environmental (temperature and humidity) control equipment. In some cases, these systems can reduce floor space needs by 40-70%.

		Testing/validating: Use of Raman testing and tailgate samples greatly simplifies the testing/validating step. Products do not have to be re-validated each time material is sub-divided from a bulk container. In addition, the primary packaging material typically used for these bags is the same as standard drum liners, so contact material does not have to be re-validated.

		Quality: Pre-weighed direct dispense systems eliminate the need to clean the weighing and dispensing area for another operation, saving time and eliminating risk of cross-contamination and employee exposure.

		Material stability and efficient use: Anti-clumping packaging design improves raw material yields by avoiding material non-conformities and inaccurate ingredient measurements from clumped materials.

		Safety: Anti-clumping packaging leads to sound environmental, health, and safety practices, as employees no longer need to engage in the potentially unsafe practice of breaking up clumps that can form in large drums.

		Raw materials savings: Pre-weighed amounts in direct dispense bags that match specific biopharmaceutical process requirements eliminate the need to buy and store material in bulk, reducing overages, out-of-date materials, and disposal costs.

Advances in bulk material packaging focus on improving overall OpEx


	By evolving raw material packaging and delivery options to align more closely with the operational requirements of biopharmaceutical producers, raw materials suppliers are helping to eliminate inefficiencies and drive down costs within the overall supply chain and production environment. The success of these bag-based, pre-weighed, free-flowing direct dispense systems is also encouraging the development of streamlined systems for other types of materials beyond bulk powders. Some raw-materials suppliers are investigating new packaging methods to deliver ready-made liquid solutions to customers, which would eliminate the biopharmaceutical manufacturing step of taking solid materials and creating solutions. While the transportation and storage considerations for liquid solutions are more complex than solids, there are opportunities to apply innovations to raw-materials packaging designs to improve the efficiency, productivity, safety, and quality of biopharmaceutical manufacturing.

:
	When referring to this article, please cite it as N. Deorkar, "Raw Materials Packaging Innovations for Biopharmaceutical Manufacturing," 

Supply Chain

Recent trends in raw materials packaging may impact manufacturing, quality, and cost of biopharmaceuticals.

Raw Materials Packaging Innovations for Biopharmaceutical Manufacturing

	Single-use technologies (SUT) have made significant inroads in biopharmaceutical and vaccine manufacturing. Greater adoption in the years to come can be anticipated, given their broad use in pre-clinical and clinical manufacturing and expanding use for approved therapeutics. With increasing regulatory oversight of SUT processes, it’s worthwhile to review basic concepts of design and qualification that apply to single-use components and systems (SUS).
	
	

Equipment Design Regulations and Guidance


	While drug and vaccine manufacturers are subject to regulatory review and inspection of how equipment is used, that is not the case for the manufacturers of process equipment, including SUT components and systems, even when sold for use under good manufacturing practice (GMP). Suitability of design and qualification for use must be determined by the therapeutic manufacturer. The 

 211.63), Section 211.63 states, “Equipment used in the manufacture, processing, packing, or holding of a drug product shall be of appropriate design, adequate size, and suitably located to facilitate operations for its intended use and for its cleaning and maintenance” (1). While this is the responsibility of the user, equipment manufacturers need to understand and design to user requirements for what may be considered appropriate, adequate, or suitable.
	
	US 21 

 does provide some information for process equipment designers: Section 211.65, paragraph (a) states, “Equipment shall be constructed so that surfaces that contact components, in-process materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements” (1).  While not specifying those requirements, this requirement highlights the need for SUT equipment designers to consider operational performance criteria, as well as potential chemical interactions and equipment-derived impurities or particulate contaminants that may be introduced into applicable processes and potentially impact intermediates or final dosages. 

	Much of SUT use occurs in therapeutic protein API production. International Council for Harmonization (ICH) Q7, European Medicines Agency (EMA) Q7, and FDA 
	Q7A 

Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients

 incorporate essentially the same design requirement statement: “Equipment should be constructed so that surfaces that contact raw materials, intermediates, or APIs do not alter the quality of the intermediates and APIs beyond the official or other established specifications” (2-4).

	Fortunately, established SUT manufacturers have significant experience working with drug and vaccine companies and have learned to adopt SUT equipment designs to meet user needs in GMP-regulated processes. Adoption of established good engineering practices for design and deployment of SUTs is crucial and requires close collaboration between users, component designers and system engineers having experience in polymer materials, device manufacturing, human interface engineering, and other related practices (5).
	
	


	Design considerations for SUT manufacturers can be divided into six categories:

		Physico/chemico properties cover selection and performance of the materials of construction and finished component under anticipated use conditions, with reasonable safety factors. These properties can include pressure requirements (e.g., operating, burst, creep, pressure drop), max/min temperature requirements (e.g., operating, thermal sterilization, melt, cryogenic, impact of temperature on pressure performance), mechanical properties (e.g., flexibility, rigidity, and tensile strength), optical properties (e.g., clarity or opacity), cleanliness (e.g., surface or embedded particles), biocompatibility, material oxidation and radiation stability, gas barrier properties, polymer additives, transmissible spongiform encephalopathy-bovine spongiform encephalopathy (TSE-BSE) status, and other raw material supplier data, etc.

		Fluid contact properties that should be considered in product design include chemical compatibility (e.g., polymer solubility, swelling, embrittlement, etc.), chemical reactivity, extractables under appropriate solvents and extraction conditions, particle shedding, and protein binding/adsorption to contact surfaces (along with any other formulation ingredients).

		Form, fit, and function relates to the “fitness for purpose” of the physical design of the component(s) and assemblies. It includes handling and other ergonomic properties, accessibility for installation and removal, microbial barrier and physical and fitment (leak) integrity properties, sterilizability, drainability, incorporation into an automated platform as the disposable fluid path, etc.

		Manufacturability ensures that the SUT production equipment and environment are suitable to effectively produce the component or system as designed and maintain quality specifications with a high degree of assurance. Manufacturability should also include operator training and in-process and final product testing (as needed) for quality factors such as integrity (i.e., retention properties of filters, leak absence of containers, connector and hose fitments, microbial barrier, etc.) bioburden, endotoxins, and particle contamination or generation.

		Packaging and shipment containers must be designed to effectively protect the SUT component or assembly during transport from the manufacturing or assembly site, as well as ease and security of unpacking and installation at the user site.

		Documentation should also be considered as part of product design, to include support data for performance claims, design and production validation data reports, operation guides, and other information that may be requested by users or from regulatory authorities. 


	Qualification is the action of proving that any equipment works correctly (as designed) and can be expected to perform as intended. For the user, qualification includes confirming that the equipment is the right tool for the job. While the term validation is sometimes applied to incorporate the concept of qualification, validation means verifying (and documenting) that the equipment consistently functions within a specified range of operations to produce an intended result. Qualification studies are, therefore, done with representative samples prior to use. Validation is conducted on process equipment (or scaled-down models) under actual use conditions.
	While the word qualification is not specifically mentioned in 21 

 211, the interpretation of these regulations by both industry and regulatory agencies has introduced terms such as design qualification (DQ) (i.e., is the design suitable for user requirements?), installation qualification (IQ) (i.e., is the equipment installed properly?), operational qualification (OQ) (i.e., does it operate according to the manufacturer’s specifications?), and performance qualification (PQ) (i.e., does it consistently perform to meet the user’s requirements?). Additional terms that combine these concepts include factory acceptance qualification (FAQ) or test (FAT) (e.g., DQ, IQ, OQ) and site acceptance qualification (SAQ) or test (SAT) (e.g., IQ, OQ, PQ).

	The first step of qualification should be DQ. For SUT, this occurs in two phases: confirmation by the equipment supplier that the equipment meets its’ design and operation criteria, and evaluation by the user that the component design and performance is suitable for use in the intended application. To the degree that the equipment supplier can anticipate the user’s requirements, some portion of the manufacturer’s qualification data can also serve as the user’s evaluation and be incorporated into the user’s documentation to be presented to regulatory authorities. This is of course provided that the manufacturer/supplier procedures and data are suitable for GMP use, the user review of the data is documented, and a user audit is conducted to confirm data validity.

	Conformance of the SUT design with the manufacturer’s claims and user’s process requirements should be demonstrated and documented.  According to European Union GMP Annex 15, Section 3, “Qualification activities should consider all stages from initial development of the user requirements specification (URS) through to the end of use of the equipment, facility, utility or system.” Once the URS is developed, “The next element in the qualification of equipment, facilities, utilities, or systems is DQ where the compliance of the design with GMP should be demonstrated and documented. The requirements of the user requirements specification should be verified during the design qualification” (6).

Supplier Quality System and Qualification Practices


	SUT and SUS are unique from traditional stainless equipment in that the user depends on the supplier’s quality system. Review of the supplier’s quality system should be part of supplier qualification. Furthermore, users cannot normally perform incoming testing prior to using SUSs intended for implementation in production, so are also more dependent on suppliers’ product qualification practices. 

	Supplier qualification of SUT products can be summarized in product qualification and process validation reports suitable for user documentation and submittal to regulatory authorities. Applicable test methods for pre-use qualification are detailed in Bio-Process Systems Alliance (BPSA’s) 

 document, along with supplemental BPSA guides with specific recommendations on qualification of extractables, sterilization, and particulates testing (7). Additional tests for SUS qualification have been described by users (8-12). The United States Pharmacopeial Convention (USP) has published a draft standard on extractables testing for plastic process equipment (13). A BPSA guide on SUS leak testing is also under development.  In addition, BPSA provides an industry consensus-generated Quality Agreement Template that can be used to document accountabilities (7), and audits should be used to review these under the supplier’s quality management system.
	Also important for SUS users is to qualify the SUT equipment supplier’s reliability and delivery history. Potential supplier performance can be assessed by the experience of integrators and users who have previously worked with them. Attendance at industry meetings, participation on scientific association SUS committees, or trade associations such as BPSA provide important access to suppliers, integrators, and users to aid in determining appropriate supplier/integrator qualification practices.

 211.63), Section 211.63, Current Good Manufacturing Practice for Finished Pharmaceuticals (FDA, April 2014).
	2. ICH,  Q7 

Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients

 (ICH, November 2000).
	3. EMA, Note for Guidance on Good Manufacturing Practice for Active Pharmaceutical Ingredients (CPMP/ICH/4106/00), November 2000.
	4. FDA, 

Guidance for Industry, Q7A Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients

, August 2001
	5. M. Botterill and B. Rawlings, 

EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use

, Annex 15: Qualification and Validation, February 2014.
	7. BPSA, Component Quality Test Matrices, Gamma Irradiation and Sterilization, Extractables and Leachables, Particulates, Quality Agreement Template, www.bpsalliance.org
	8. PDA, 

Technical Report No. 66, Application of Single-Use Systems in Pharmaceutical Manufacturing

  16 (7) (November/December 2013).
	11. W. Ding, 

. 28 (9) (September 2015) pp. 32-39.
	12. D. Riedman and J. Martin, 

 9 (S2) (2011) pp. 28-35.
	13. USP, <661.3> (draft): Plastic Systems Used for Manufacturing Pharmaceutical Products, 


	When referring to this article, please cite it as J. Martin, “Design and Qualification of Single-Use Systems," 

Troubleshooting

The author provides a review of the concepts of design and qualification that apply to single-use systems.

BioPharm International-07-01-2016

Bio/Pharma Funding Challenges Could Hurt CDMOs in 2017

Revised ISO Cleanroom Standards Improve Air Cleanliness Classification

Bioprocessing Technology Trends of RNA-Based Therapeutics and Vaccines

Raw Materials Packaging Innovations for Biopharmaceutical Manufacturing

Design and Qualification of Single-Use Systems

Managing Biomanufacturing Capacity Expectations

Microbiological Testing: Time is of the Essence

Recognizing Biopharma Industry Excellence

Modular Bioreactor Simplifies Process Development

FDA and Manufacturers Intensify Concerns about Data Integrity

Forced Degradation Studies for Biopharmaceuticals

BioPharm International, July 2016 Issue (PDF)