Chris Spivey is director of Industry Relations and Strategic Partnership, MJH Life Sciences.

Chris Spivey

Vial and injection syringe with DNA chromosome. Health care research, gene editing, molecular biology science, cell genetic analysis, medical DNA genetic engineering technology science background. 3D | Image Credit: ©Corona Borealis - stock.adobe.com

Sudden demand for large-scale manufacturing of messenger RNA (mRNA) drug substances during the COVID-19 pandemic has few parallels in the history of pharmaceuticals. Some might posit penicillin production during World War II as an analog, or perhaps early insulin availability. Pandemics and epidemics such as smallpox, typhus, cholera, influenza, HIV/AIDS, or the bubonic plague, all relied more on infection control for resolution than on manufacturing capacity. New therapeutic uses for mRNA are now ramping up good manufacturing practice (GMP) demand, especially for clinical trial supply. Moderna collaborated with CARsgen Therapeutics to combine an mRNA therapeutic cancer vaccine with a solid tumor chimeric antigen receptor (CAR)-T cell therapy, aiming to treat multiple cancers (1). Pfizer partnered with Beam therapeutics to co-develop mRNA and base editing approaches (2), while BioNTech and DualityBio have a partnership to develop differentiated antibody-drug conjugates, for solid tumors (3). In terms of upstream manufacturing for novel mRNA drug products, this technical complexity forms a tidal wave of question marks.

A prominent paper (4) examined reducing complexity by evaluating what common process could be standardized through extrapolations of clinical trial complexity, based on four published papers Phase I-II dose ranging studies. While not the amounts of product required during the COVID-19 pandemic, clinical trial doses quickly mounted up in terms of both volume and also manufacturing challenges related to techniques pursued, but also formulation delivery requirements. The authors state, “from the four studies, the average mRNA usage was 5500 μg and for the 75 trials in 2021, 412 500 μg of RNA would need to be formulated, purified, sterile filtered, and fill-finished. At concentrations of 100 to 200 μg of mRNA per mL, this would amount to 2063 to 4125 mL of drug product” (4). As an interesting foray into possible future manufacturing, the authors went on to contrast traditional mRNA with a self-amplifying method. Often, this is cited as potentially a 100 to 1 decrease in required finished product due to higher potency performances, but this study chose a picturesque perspective, saying, “assuming the entire population consists of 7.7 billion people, and each person receives two doses, a total of 15.4 billion doses or 4.5 million L of the vaccine is required. In perspective, 4.5 million L would fill two Olympic-sized swimming pools (2500 m3), whereas saRNA [self-amplifying RNA] vaccines, which we assume are 100-fold more potent, would only require 0.02 Olympic-sized swimming pools. Assuming that production costs, which account for materials, RNA, and LNP [lipid nanoparticles] production, scale linearly, vaccinating the entire population with Pfizer/BioNTech’s mRNA vaccine would cost approximately 150 billion USD. In contrast, with saRNA, it would only cost approximately 1.5 billion USD” (4).

Commercial-scale mRNA is produced through chemical, recombinant, or enzyme-based methods. For chains under 100 nucleotides, the standard chemical technique “utilizes phosphoramidite chemistry and solid-phase support promoting chain elongation from the 3′ end to the 5′ end” (2). It is amenable to automation, as is recombinant production through host cells such as 

), which has some cost advantages as it can utilize legacy protein manufacturing lines, already in GMP compliance. Enzymatic processes are newer to arrive and have tradeoffs still being worked through. A study conducted by the Duke Human Vaccine Institute explored creating standardized robust manufacturing processing steps, saying they confronted the lack of established quality standards by, in part, surveying vendor operations “through questionnaire and/or audit relating to the vendors’ quality systems and operations as they pertained to manufacturing and/or testing of the materials” (5). The study adopted the Weismann–Karikó method of production, the upstream process requiring three enzymatic steps of plasmid linearization, mRNA transcription, and DNA template digestion. In the starting out phase, fundamentals the authors emphasized were that “for optimal 

 transcription yields, it is critical to start with high quality template DNA. Some key quality attributes that are required before use include the presence of only a single band of linearized DNA, a supercoiled ratio of at least 70%, and a sequencing-verified correct poly(A) tail length” (5). They also noted that “endotoxin control was the primary raw material risk due to its variability in raw material lots and the difficulty of its removal in subsequent downstream purification steps … reaction agitation, temperature, incubation time, and reactant concentrations were also carefully examined for each reaction step in order to yield a robust upstream platform process across multiple mRNA constructs” (5).

Chris Spivey is director, Industry Relations and Strategic Partnerships

Articles

A tidal wave of questions floats the need for more upstream automation.

The Making of mRNA

®, Susan Schniepp, distinguished fellow, Regulatory Compliance Associates, and co-chair of board of directors, Parenteral Drug Association, expands on the importance of maintaining a robust quality management system (QMS) in bio/pharmaceutical manufacturing. Schniepp hosts an interactive session on the impact of QMS on quality maturity at INTERPHEX, held in New York City on April 16–18, 2024.

For advanced therapy medicinal products (ATMPs) in particular, Schniepp emphasizes how fast-moving this sector is. “The regulations don't keep up with the ATMPs. That technology, and their way of thinking, is turning over quicker than the regulations,” she says.

“The changes in the regulations that are going to come are going to be around quality culture and maintaining a robust quality management system,” she adds. Ensuring documentation and keeping equipment calibrated are important practices to apply to these new fast-moving ATMPs, she states. Schniepp does not necessarily expect to see many changes in the regulations around ATMP development and manufacturing but thinks that there will likely be more guidance documents issued in the future, with one of FDA’s focuses being its quality management maturity model.

“There are some regulations out there that call out quality culture. In particular, the World Health Organization has one on data integrity. It has a definition and standard[s] on what quality culture is,” Schniepp says. She points out that a new aspect of her presentation at INTERPHEX this year is its interactive component, in which she sets up a scenario involving an audit where an incident occurs. She gives the audience three potential responses to discuss, but rather than simply asking them which response do they pick or which response is correct, she instead asks what does the chosen response say about that person or that company’s QMS and the maturity of that system?

Susan Schniepp, distinguished fellow, Regulatory Compliance Associates, demonstrates QMS impact on quality maturity at INTERPHEX.

Susan Schniepp of Regulatory Compliance Associates Explores the Impact of QMS on Quality Maturity (INTERPHEX 2024)

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Susan Schniepp, distinguished fellow, Regulatory Compliance Associates, and co-chair of board of directors, Parenteral Drug Association, demonstrates QMS impact on quality maturity at INTERPHEX.

® speaks to Bill Whitford, Strategic Solutions leader at Arcadis, on the role and impact of 3D bioprinting for biologics. Whitford is a presenter on the topic of 3D bioprinting at INTERPHEX, held in New York City on April 16–18.

Whitford notes that an important aspect of 3D bioprinting is that it is one approach to 3D cell culture, which he observes is taking over cell culture in many categories. “3D culture provides many unique capabilities it's more biomimetic. Actually, there's nothing natural about growing cells in monolayer on a styrene plate, so there are many 3D culture technologies that employ many different production methods that support many categories of application and individual products,” Whitford states.

On the workflow in a 3D bioprinting facility, Whitford remarks that many complementary and supportive synergistic technologies exist that support each other. “For example,” he explains, “machine vision now is really simple. Ordering 3D printing and the ability to both direct and inspect a nascent print job using artificial intelligence (AI) … they feed on each other. The availability of bioprinters and what we call bio ‘inks’ is, in a commercial sense, what's really driving the final applications.”

“Best practices are being developed for bioprinting,” Whitford also notes, pointing out that there are many different ways of affecting bioprinting, not just with respect to the instrumentation, but with respect to the actual printing. “There is laser-effected printing; there's extrusion-effected printing. There are many different ways of getting the bio ink into the three-dimensional structure, and they all have their advantages. They all provide either a tremendous accuracy and precision or volume.”

In the interview, Whitford also discusses the future of 3D bioprinting in the cell and gene therapy sector. “The ability now—through site-directed CRISPR [clustered regularly interspaced short palindromic repeats]-type engineering—to drive the evolution of a cell towards exactly what phenotype is required is supporting the ability to … develop the bio ink … required to get the initial print job accomplished.”

At INTERPHEX, Bill Whitford, Strategic Solutions leader at Arcadis, discusses the progress made in 3D bioprinting toward commercial biologics production.

Bill Whitford of Arcadis Discusses 3D Printing for Biologics (INTERPHEX 2024)

Bottles for injection with white powder on a color background | Mr.Ilkin

There are two main threats posed by extractables and leachables (E&L), contamination from unwanted impurities such as nitrosamine or chromium causing toxicity issues and diminution of drug efficacy or potency by destabilizing or perturbing the API. From a system-wide perspective, the E&L threat posed by manufacturing equipment is small, versus lifetime container closure and material packaging of drug products. The effects of the former are comparatively sudden and obvious (and hence easier to spot), versus the slow chemical transformation that might occur in the latter. In this sense, leachables are a subset of their parent extractables.

Recalls of nitrosamine-contaminated drugs “underscore the need for robust analytical methods and risk-based approaches for E&L and nitrosamines testing. Additionally, drug product manufacturers must ensure that toxic elemental impurities are under a state of control in their products” (1). There are many courses of instruction one can take to learn how to implement good monitoring of E&L. In line with wider industry trends, risk-based approaches to E&L testing for small molecules, especially, are becoming more commonly implemented, and the salient issue of nitrosamine contamination has certainly accelerated that trend.

Plasticizers, stabilizers, lubricants, or colorants are well-known components of medication packaging systems. The effects they have on product and patient safety partly depend on which patients, when, and through what delivery system. Aerosols, sprays, and transdermal packaging are high risk compared to tablets, capsules, or topical powders. Andrew Feilden, European E&L strategic director, Element Materials Technology, when looking back on how some regulations were formed, said, “Inhalation aerosols and sprays are deemed to have the highest likelihood of packaging component-dosage form interaction and the highest degree of concern based on the route of administration. As such, it is not too surprising that most of the early work on regulatory requirements focused on orally inhaled and nasal drug products. It was groups such as the International Pharmaceutical Aerosol Consortium on Regulation & Science that initiated and built on supply chain interaction and understanding” (2).

There is an interplay between the permitted concentration limit (PCL), the maximum daily dosage (MDD), and product material design. It is interesting to consider predictive modeling versus exposure for E&L and how far the industry has come with artificial intelligence (AI) in advancing predictive modeling and where the limits lie.

Over the past few years, there has been an increased prevalence for products to be classified as drug-device combination products. This classification leads to several additional considerations and challenges, not least with E&L study design and regulatory review of these studies. According to Nicholas Morley, principal scientist at Element Materials Technology, “Regional differences can exacerbate this and make it even more challenging for products seeking registration in multiple markets. Extractable and leachable studies performed on medicinal products typically follow standards and guidelines set out by the Product Quality Research Institute (PQRI) and the US Pharmacopeia (USP), whereas studies performed on medical devices follow standards set out by the International Organization for Standardization (ISO). Depending on the device type, there may be requirements to meet multiple different standards and guidelines which have similar aims (i.e., determining patient risk due to the product) but use different approaches to achieve them.”

No pun intended, this compounds an already complex process when trying to set minimums and maximums for E&L testing, particularly for high-potency drugs. Morley continues, saying, “When designing and performing studies on drug-device combination products, it is crucially important to have a deep understanding of the similarities and differences between the various extractable and leachable standards and guidelines, as well as the different regional expectations, and review authorities when designing E&L studies to minimize potential deficiencies and delays in approval.”

Where possible, the industry is accelerating migration toward continuous manufacturing and single-use systems (SUS). With regard to E&L, setting baselines and comparing differences can be helped by standardized protocols, which can facilitate “like-for-like assessments between each supplier’s SUS products and components” for end-users of SUS, according to Dan Rosen, vice-president and general manager of bioproduction at Thermo Fisher Scientific (3).

As said earlier, leachables being a subset of extractables, extractables testing can show which leachables might emanate from the material under standard conditions, or extreme conditions, and through simulation or modeling. However, one wrinkle to pay attention to is that new or different leachables might form during sterilization, storage, or use (3).

There is talk about a migration away from the well-characterized and established response of materials to gamma irradiation versus (due to lack of gamma testing capacity) employing less understood X-ray sterilization methods. “Current theories suggest that the E&L profile should be similar after X-ray or gamma sterilization, but it is important to perform extractables testing, says the Bio-Process Systems Alliance (BPSA)” (3).

According to Lalit Saxena, senior director of MSAT at Samsung Biologics, “Regulatory requirements are becoming increasingly stringent with respect to patient safety, and thus more in-depth risk assessment to implement single-use technologies for late-stage downstream unit operations is required” (4). Saxena pointed to the European Union’s Annex 1 as an example of regulatory expectations. Challenges are also being created by compliance with 

 <85> (for endotoxin certification) for single-use materials. Customized single-use assemblies increase the complexity, especially with components from multiple vendors, according to Saxena (4).

The impact on E&L study design can be easily overlooked. To understand this, it is important to determine who is responsible for the review of the extractable and leachable studies.

“Different E&L guidelines apply to pharmaceutical container closure systems (e.g., USP or PQRI) compared to medical devices (ISO). In additional, the different regional organizations can have their own specific requirements or interpretations. Extractables studies on container closure systems and single-use manufacturing process components often employ binary solvent systems,” states Morley. “Whereas CDRH [the Center for Devices and Radiological Health] prefers extractable studies to be performed using pure solvents. When a product is classified as a drug-device combination product (e.g., a pre-filled syringe containing an active drug-product), extractable studies may need to meet the requirements of the pharmaceutical container closure guidelines as well as the medical device standards simultaneously. Within the EU, if a drug-device product is classified as integral as is the case for pre-filled syringes, a notified body opinion is required prior to receiving a MAA [marketing authorization applications] approval. When designing and performing studies on drug-device combination products, it is crucially important to have a deep understanding of the similarities and differences between the various extractable and leachable standards and guidelines,” he concludes.

One of the great new tools for pharmaceuticals is the rise and integration of AI models and programing. Opinions vary in degree of enthusiasm, with many highly supporting greater inclusion, implementation, and reliance on such systems. But there are minority reports circulating among the E&L expert community. When asked how far the industry has come with AI in advancing predictive modeling for E&L, Rick Reiley, technical director, Extractus, replies, “We have not come far at all.” He points out that, “this is about the data sets available to train AI. The first problem is the data itself: data needs to be marked up with mass assignments, and modern software suites (Masshunter, UniFi/masslynx, and xcalibur) can do this. [However,] it is important to have correct identification of compounds as chromograph systems can vary the elution profile (ideally, compound assignments should be confirmed with a standard or reference material which are not always commercially available).”

Reiley continues to draw attention to a lack of extractable data sets that directly link to leachable data sets.“The vast majority of extractable data sets,” emphasizes Reiley, “do not have a corresponding leachable data set to train the AI, and those that do exist are not available for AI teams as these are company secrets. If you make a product and you disclose data to a regulatory body, the data [are] flat (no data files, just summary data and chromatograms/spectra) as giving more information invites greater scrutiny. If an AI algorithm were to find additional peaks from raw data, it would trigger review of a product license and potentially pull a product from the market.”

While on this subject, Reiley goes on to discuss a third hurdle regarding extractable data sets. “Very few have good data (meaning that peaks are not identified confidently, performed with rigor [usually single extraction with single injection] or separated chromatographically). There are problems with sensitivity limit of quantification (LOQ) that is not low enough), poor chromatography, poor identification (either none or letting the library decide) as well as inappropriate separation technique so the peaks are lost in noise or solvent peaks,” he states.

This last hurdle could be overcome by original equipment manufacturers (both instrument and packaging/material vendors) “as they don’t directly market drugs/medical devices, but they have incentives to avoid the research. You will have to source the materials for an extractable and leachable study, but it would need to be a large data set. Ideally, you would want to pick something with multiple suppliers (you don’t want to alienate a potential or current client), and it would require a lot of guesswork as E&L studies start with a risk assessment (calculating analytical evaluation threshold [AET] will vary depending on a number of factors). The alternative would be an independent body or an academic research group, but it would require a lot of funding (industry would not fund this) as E&L studies are expensive due to instrumentation and time required,” concludes Reilly.

Traditional analytics for identifying extractables and leachables are liquid or gas chromatography combined with mass spectroscopy (LC–MS or GC–MS). But according to Fran Adar, principal Raman applications scientist, Horiba Scientific, “From my discussions with Mark Witkowski [at FDA], I understand that the standard LC–MS and GC–MS techniques have difficulty identifying inorganic materials. In contrast, Raman spectroscopy is equally active for inorganic and organic compounds” (5).

Adar went on to say, “What Raman can do that those standard techniques cannot is identify minute amounts of material—typically 1–2 μm2 × 1–5 μm deep. And there are no waste products from the analysis that need to be disposed of. Is the information the same? Maybe yes and maybe no. The measurement is done on intact material whereas MS tears the molecule apart. There may be cases where information is lost in tearing the molecule apart” (5).

It could be that new tools from other application areas may migrate over to help out the E&L field before too long. There is certainly interest there, and some room for improvement.

Panzade, P. Nitrosamines, Elemental Impurities, Extractables and Leachables: A New Normal for the Pharma Industry. Webinar. Catalent. 

Feilden, A. Material Management and the Impact on Extractables and Leachables,” 

Markarian, J. Analyzing Extractables and Leachables in Single-Use Systems. 

Challener, C. Benefiting from Single-Use Tech Downstream. 

Adar, F. Exploration of the Use of Raman Microscopy to the Identification of Extractables and Leachables from Polymeric Containers. Spectroscopy 2022 37 (11) 8-13. DOI: 

Chris Spivey was previously the editorial director of 

When referring to this article, please cite it as Spivey, C. Testing the Limits of Extractables and Leachables. 

What are the lessons gleaned from a product’s E&L lifetime?

Testing the Limits of Extractables and Leachables

® spoke with Carl Allenspach, director of business operations, manufacturing, science, and technology organization at Bristol Myers Squibb about expectations and significant benefits to be gained at this year’s INTERPHEX, to be held at the Javits Center in New York City on April 16–18, 2024.

Allenspach’s background includes solids formulation and processing, with more than 20 years of experience in formulation development. Much of his experience involved hands-on work with equipment, operations, and processes formulations. Allenspach has also moved into the biologics space, gaining exposure to biologics drug substance, drug product, and packaging.

About this year’s INTERPHEX, Allenspach remarks on the addition of scientific-focused sessions, stating that, “INTERPHEX is well known and established for bringing in an impressive array of pharma vendors—covering all aspects of the industry. So, attendees already go there to meet with the vendors to see the new equipment, the new materials, and processes that are available. So, adding on the scientific content really adds another aspect to the meeting. The [meeting] brings in an international audience because of the location; it's a place where scientists can collaborate and exchange ideas.”

“By adding in this—the learning lab that INTERPHEX is offering—it's going to give [attendees] this curated scientific content that the attendees will really be able to benefit from—getting the information on these technologies, processes, regulatory changes—that they can then bring back and utilize in their daily life,” he adds.

The collaborative spirit at INTERPHEX offers the opportunity for experts from different fields to meet and engage, which significantly helps accelerate different innovations as well, Allenspach points out. “We hope that attendees will gain some actionable strategies that they'll be able to apply. I think everybody that attends a conference goes in there with some problem that they're having, something that they want to know more about, and [most of the time] by talking to the other people there and by listening to presentations, by asking questions … I've taken something back that I've benefited from. So … we really hope most people who go there get inspired from listening to some of these talks, and they're going to leave with some excitement and enthusiasm that will be able to give them a boost in their career,” he explains.

Carl Allenspach, director at Bristol Myers Squibb discusses the interactive and collaborative important of INTERPHEX.

 Bristol Myers Squibb’s Director of Business Operations for MSAT, Carl Allenspach, Highlights Collaboration and Interactive Exchange at INTERPHEX 

Carl Allenspach, director of business operations, manufacturing, science, and technology organization at Bristol Myers Squibb discusses the interactive and collaborative important of INTERPHEX.

Bristol Myers Squibb’s Director of Business Operations for MSAT, Carl Allenspach, Highlights Collaboration and Interactive Exchange at INTERPHEX 

Fabian Gerlinghaus, co-founder and CEO, Cellares

The number one priority for every early-stage biotech is speed to clinical data. Standardizing processes and regulatory paths are an overarching priority for the commercial cell and gene therapy (CGT) field, as this not only advances timelines, but also encourages further investments. The Office of Tissues and Advanced Therapies (OTAT) at FDA currently lists 35 approved CGT products (1). Clearly this is an amazing success story for saving many lives, but some of these products have a troublesome future commercially, while others have a difficult road ahead in terms of manufacture and mechanical deployment. However, all faced regulatory uncertainty alongside a burdensome filing requirement to become “products”, and all of those that follow (currently numbering into the several thousands) are subject to the same pressures, uncertainties, and requirements.

According to Fabian Gerlinghaus, CEO of Cellares, companies today typically generate on the order of 400 pages of paper-based batch records for every individual patient. “They're writing a book for every single patient. That doesn't scale well,” he says with some exasperation. “Try and do that manually, paper-based, for hundreds of thousands of patients every single year. It's a nightmare. However, our interactions with the FDA have been incredibly positive, and we view FDA as a very collaborative partner.”

Cellares was extended an FDA Center for Biologics Evaluation and Research (CBER) Advanced Technologies Team (CATT) meeting resource. The scope of these gatherings pertaining to advanced manufacturing technologies “focus on novel technologies that can have a significant impact on product development, manufacturing process and control strategies, and may also have regulatory implications. This includes manufacturing and analytical methods for those products or classes of products for which the center has limited experience with the manufacturing or development process” (2).

https://www.pharmtech.com/view/cellares-expands-agreement-with-bristol-myers-squibb-for-second-car-t-program

“FDA understands that they need automation the standardization afforded by this automation, Gerlinghau continues. “Peter Marks has been very outspoken about what we want, what the cell therapy industry needs, is effectively a soda machine where you press the Coca-Cola button and out comes Yescarta, and you press the Pepsi button and out comes Breyanzi. And that is what we have built. And moreover, Congress at this point has actually given directions to the FDA to standardize the processes. The FDA has since implemented this direction from Congress, and they came out with the Advanced Manufacturing Technologies designation, which is really an effort to standardize a cell therapy manufacturing technologies.”

“On the topic of standardization, we talked about the benefits of standardization for the drug sponsors, for the FDA. If there is a manufacturing standard, it means less work, less effort for the drug sponsor to create their regulatory filings and less work for the FDA to review those regulatory filings. If you can just reference a standardized self-manufacturing technology.”

Watch part 4 of our interview with Gerlinghaus below:

Can Cellares deliver on the goal to meet total patient demand for cell therapies globally? CEO Fabian Gerlinghaus discusses.

Automation of Clinical Data Key in Meeting Cell and Gene Therapy Demand

Approved Cellular and Gene Therapy Products