Extractables and leachables (E&L) must be analyzed for any product-contact material to determine the risk of leachable compounds contaminating a drug product. Container-closure systems, which contact the drug product during long-term storage, are often higher risk than process equipment, which has a short product-contact time. Processing equipment is not exempt, however. As polymer single-use systems (SUS) are increasingly being used in 
biopharmaceutical manufacturing, there is a growing need to analyze E&L of this equipment. Industry organizations and consortiums have worked
to standardize analysis protocols that can be used throughout the supply chain. Standard methods facilitate comparison between suppliers.

BioPhorum, for example, first published a standardized extractables protocol in 2014, which was updated in 2020 (1). Because the organization was previously known as BioPhorum Operations Group (BPOG), the protocol is often called the BPOG protocol. SUS suppliers can use the protocol to measure the extractables in representative components. The purpose of the extractables testing information package, says BioPhorum, is “to allow the SUS end-user to rigorously estimate the types and amounts of leachables that could be generated by the SUS component during its intended use” (1).

Another set of standard methods was recently published in the 

General Chapter <665> Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products was published in 2022 (2), but the date for it to come into effect was postponed to 2026. 

 accompanying procedural chapter <1665> became official on May 1, 2022 (3).

 documents is the use of risk assessment for end-users to determine the risk level of a component in a particular process. The analysis method depends on this risk level.

The International Council for Harmonisation (ICH) is working on a document related to E&L testing. A final concept paper for ICH Q3E was published in 2020 (4). Industry experts say that questions about how this upcoming ICH guidance will align with the USP and BioPhorum documents are yet to be answered.

The Extractables and Leachables Safety Information Exchange (ELSIE) Consortium, made up of 33 biopharma/medical device companies, is also active in this area and aims to provide expert input to efforts such as 

new chapter and the in-progress ICH Q3E, through working groups and formal commenting processes as well as white papers, workshops, and other public meetings. “ELSIE provides a collaborative environment where member companies can discuss and benchmark the approaches that companies are taking,” explains Lee Nagao, senior director of science, regulation, and policy at the ELSIE Secretariat. Harmonized guidelines or compendial chapters are crucial, because without them, “regulatory expectations can vary depending on the health authority and the experience of the specific [biologics license application] reviewer,” says Nagao.

Standardized protocols help in setting baselines and comparing differences. For end-users of SUS, standard protocols facilitate “like-for-like assessments between each supplier’s single-use technology (SUT)
products and components,” says Dan Rosen, vice-president and general manager of bioproduction at Thermo Fisher Scientific.

Extractables testing of the materials or components is used to identify which leachables might come from the material under standard conditions and in a simulation study. A challenge, however, is that different leachables could be formed during sterilization, storage, or use.

“Key challenges in characterizing E&Ls include identifying unknown molecules that arise during primary sterilization of the SUT products,” says Rosen.

The response of materials to commonly used gamma irradiation is generally well established, but due to limitations in capacity for gamma-radiation sterilization, X-ray sterilization is being considered as an alternative (5). 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).

BPSA published a guide for E&L in cell and gene therapies (CGT) in July 2020 (6) to support companies developing manufacturing lines for scale up or scale out. “Since CGT was a new modality, the drug developers were almost exclusively using single-use components for economic, safety, and technical performance. This [situation] led to a need for BPSA to help coordinate a guide to assist developers on appropriate choice of materials and components for their process development,” says Brendan Lucey, chair of BPSA’s CGT Committee. CGT processes differ from other biopharma manufacturing using SUS because in classical biopharma, process-related impurities, including process-equipment related leachables, are removed in the downstream purification process. In CGT processes, however, there may be washing steps, but not the same kind of downstream purification. Because of this process set-up and the variety of contact surfaces and contact times, E&L testing to assess and then control leachables is crucial, concluded BPSA (6). The type of polymers used in SUS for CGT manufacturing also differs from classical biopharmaceutical manufacturing. In particular, the report noted that polyvinyl chloride (PVC) polymer may be used in transfer bags and tubing sets in CGT collection, processing, and storage. Because flexible PVC typically contains plasticizers and other additives that are not present in other polymers used in SUS, leachables from these additives should be considered (6).

The rapid pace of change in guidelines has required a quick response by contract labs to create experiment designs that are compliant, says Dujuan Lu, manager and global lead of extractable and leachable testing at SGS Health Science. SGS labs are following the 

 <1665> procedures, because these have been official since May 2022, but the postponement of 

 <665>’s official date is helpful for the industry, Lu says. “The transition period gives pharma/biopharma manufacturers time to requalify as needed. We are seeing lots of activity now because clients want to be ready for 2026,” she explains.

 and BioPhorum protocols, says Lu, who explains that the two differ slightly in recommended solvents for acid and base conditions. Lu points out that 

 considers the different solvents for acid conditions to be equivalent; for the base conditions, 

 recommends choosing the alkaline solvent depending on the specific process pH value.

Design of extraction studies for single-use components is complicated by multi-component assemblies and complex geometries. “The recommended ratio of surface area to volume is 6 cm2/mL. Calculating the surface area is straightforward for a tube or a bag, but can be challenging for a connector or valve, for example,” explains Lu. In designing an extraction study for single-use filters, both the housing and the membrane must be evaluated. “The procedure depends on the shape. For a capsule filter, for example, we can fill the filter with solvent and cap the ends. For a smaller filter, we might need to remove the membrane and submerge it in solvent,” explains Lu.

Because many different polymeric materials are used to construct single-use components, the extractable profile is complex and multiple analytical techniques are needed to identify and quantify the compounds, says Lu. She explains that volatile organic compounds can be measured with headspace gas chromatography (HS-GC) coupled with high-resolution mass spectrometry (HRMS) or flame ionization detection (FID). Semi-volatile organic compounds can be measured with direct sample injection in GC-HRMS/FID. Non-volatile organic compounds can be measured with ultra high-performance liquid chromatography (UHPLC) coupled with HRMS and a photodiode-array detector. “Unknown identifications are crucial for the extractables testing, so high-resolution mass spectrometers are recommended for both GC and LC testing,” says Lu. She adds that elemental impurities can be measured with inductively coupled plasma-mass spectrometry.

Calculating the analytical evaluation threshold (AET) for processing equipment is another challenge. “The AET is set up based on the toxicology concerns; above [the AET], analytes need to be identified and quantified for health risk assessment,” explains Lu. “The instrument detection limits need to be at, or lower than, the AET to make sure all compounds above the AET can be detected. The process equipment AET is typically calculated based on the final product AET by applying the dilution factor during the process. The difference between the batch size and the extraction stoichiometry during the extractable study will be considered during the calculation.”

To perform risk assessment, it is crucial to have a good understanding of the manufacturing process. “[Understanding] any clearance, concentration, or dilution steps [is] critical, particularly to help narrow down the number of materials requiring in-depth characterization,” says Nagao.

Questions remain, says Nagao, about what point in the process a risk assessment should begin, how to define component risk, and what the minimum data would be to qualify low-risk components. “For example, gaskets, seals, or connectors used upstream of a [filtration] step will be inherently low risk and unlikely to contribute significantly to the leachables profile. Is it necessary, then, to fully characterize the extractables profile? This [type of question] is where a harmonized guidance that is implemented across the globe, perhaps based on risk-management principles, would be helpful,” Nagao suggests.

BioPhorum Best Practices Guide for Extractables Testing of Polymeric Single-Use Components Used in Biopharmaceutical Manufacturing 

2. USP, General Chapter <665>, “Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products,” 

3. USP, General Chapter <1665>, “Characterization and Qualification of Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products,” 

Final Concept Paper ICH Q3E: Guideline for Extractables and Leachables

X-Ray Sterilization of Single-Use Bioprocess Equipment, Part 1: Industry Need, Requirements, and Risk Evaluation

Extractables/Leachables Considerations for Cell & Gene Therapy Drug Production Development

Jennifer Markarian is manufacturing reporter for 

Articles

Features

Single-use systems can benefit from standardized risk assessment and analysis protocols, which can facilitate the way in which processing equipment components are compared.

Considerations for E&L Analysis in Single-Use Systems

Most strategies for liquid-biopsy-based early cancer screening and detection suffer from modest sensitivity as well as insufficient specificity, and thus miss many early-stage cancers, while resulting in unnecessary, invasive, and expensive follow-up procedures. Despite the large sample volumes analyzed, the false negative rates of these technologies severely limit their utility. This is a result of the tests’ reliance for accuracy on tumors shedding sufficient amounts of material into patients’ circulation.

Glycoproteomics provides an alternative by measuring subtle shifts in the glycosylation profiles of relatively abundant circulating proteins, such as immunoglobulins and acute phase reactants. Recently, such changes have been demonstrated to show highly sensitive and specific correlations with a range of malignant diseases as well as other conditions, opening the door for the glycopro-teome to serve as a new source of highly accurate biomarkers that offer clinically relevant, profound new insights into disease processes via simple blood samples. This opportunity should not be lost on pharmaceutical companies, given its potential to produce powerful new diagnostic technologies and therapies.

Up to 70% of all proteins are glycosylated, making glycosylation by far the most common post-translational modification. More importantly, the extreme diversity of glycans—in which every compositional combination of monosaccharides may take on a wide variety of structural moieties—generates a vast repertoire of molecules, increasing the potential analyte space of the proteome by several orders of magnitude.

A large body of research has shown that protein glycosylation is highly dynamic, changing in real time with physiological and pathological states and thus—at its location far downstream the biological cascade from DNA and proximal to phenotype—predestined to be a highly differentiated and informative source of biomarkers.

Conceptually different from conventional proteomic analysis, glycoproteomic analysis does not depend on quantitative changes in abundance/expression of a particular protein. Instead, it records subtle shifts in the proportion of individual glycan moieties attached to a given glycosylation site of a protein, a domain completely missed by standard laboratory analytical approaches, such as immunoassays, which indiscriminately recognize the peptide backbone without further submolecular resolution.

Likewise, glycomic analysis, which relies on the aggregate measurement of different glycans after enzymatic separation from their cognate proteins, while occasionally yielding interesting results, discards the much more granular information provided by an analytical approach that preserves the integrity of the glycosylated protein. Glycoproteomics, therefore, allows characterization of individual glycoproteins at the resolution of both the glycan moieties and the amino-acid residues to which they are covalently attached (i.e., the glycosylation sites).

Like everywhere else, there is no free lunch in life science either. The power of analysis at the resolution of individual protein glycoforms comes at the expense of very challenging analytics. Discovery of novel protein glycoforms relies entirely on the submolecular resolution provided by mass spectrometry (MS). Likewise, measuring known, previously characterized, glycosylation isoforms of a protein depends, by and large, on MS because of the limited specificity and/or difficult generation of glycan- and glycoprotein-specific affinity reagents. MS technology has, over the past decade or so, become increasingly robust and dependable, but the instrumentation is still expensive and complex, requiring highly trained, specialized operators.

The true bottleneck,—paradoxically, a consequence of the improvements in MS technology and its throughput—however, is now represented by the volume and complexity of the data generated by MS. Glycoproteomic chromatogram processing relies on experts laboriously and manually calling and integrating the area under the ragged peaks characteristic of the primary data output generated by MS. Until recently, even with the best available software, this allowed only a frustratingly slow “one-off” approach to glycoproteomic research and analysis, which was unsuitable for translational studies, and applications in which large numbers of clinical samples need to be analyzed to arrive at valid interpretations and, ultimately, clinical actionability. As an example, while running 100 patient samples on an MS can now be carried out in the space of one to two days, interpreting/processing the output, assuming 1000 peaks per sample, will keep a PhD busy for eight months.

Enter artificial intelligence (AI). Ushering in a foundational change in complex data processing, AI-driven glycoproteomic analysis can at long last be performed at scale, five orders of magnitude faster, and at superior fidelity and reproducibility than was heretofore possible.

The biotechnology company InterVenn Biosciences, for example, has developed a neural-network-based algorithm, which was trained by manually curating hundreds of thousands of features (i.e., glycoproteome chromatographic peaks), and which performs well on unseen test samples, reaching near-perfect agreement (Pearson’s r 0.99) with expert human annotation—as compared to ≈75% correlation using the most advanced currently available software packages. As a result, it can now process the 1000 features on each of 100 samples from the previous example to yield numerical, normalized data directly usable for biostatistical analysis in 6 minutes as opposed to full-time work by a spectrometrist for eight months (1).

The transformation of the liquid biopsy concept

The insights gleaned from rapidly scaling glycoproteomic interrogation of peripheral blood samples, admittedly still at an early stage and based on a limited number of pilot study examples, have been intriguing and powerful (2–4). By focusing on readily accessible high-abundance plasma glycoproteins, which undergo significant changes in their glycosylation status as part of a systemic response to localized, remote disease processes, liquid biopsy-based glycoproteomics is opening an entirely new domain of R&D interrogation, resulting in identification of new biomarkers, which promise to meet the exacting needs of the clinical markets. These biomarkers have the potential to accelerate the shift to a multi-omic approach to precision medicine, with benefits to individual patients and the healthcare system overall.

Much more research is needed –and being conducted—to understand the biology behind these glycosylation changes. Presumably, they are driven by signal cascades activated by the tumor mi-croenvironment of very early-stage tumors, or even precancerous lesions, which are not detectable by ct- or cf-DNA based methods. However, highly informative glycoproteomic biomarkers can be measured that do not depend on the presence of material shed by the tumor, representing a sea change for the role of liquid biopsy in disease management and preventive care.

is chief scientific officer and distinguished scientist at InterVenn BioSciences.

With the evolution of scalability of peripheral blood protein glycosylation, research has expanded for new biomarkers.

Using Novel Biomarkers to Facilitate AI-Driven Glycoproteomics

CSafe, a US-based provider of active and passive temperature-controlled shipping solutions, and BioLife Solutions, a US-based supplier of bioproduction tools and services for the cell and gene therapy (CGT) and broader biopharma markets, have formed a new partnership to provide a combined global service network to support CGT products. The partnership will focus on enhanced reliability, security, and quality, according to an Oct. 11, 2022 company press release.

BioLife expects to support 10,000–12,000 shipments of CGT starting materials and manufactured doses over the next 12 months using its evo cold chain management platform. The new partnership with CSafe will provide BioLife with expanded supply chain options for any CGT product at any stage of development, from early clinical-phase trials to global commercial scale. CSafe operates more than 50 service centers worldwide.

“CSafe is honored to partner with the team at BioLife Solutions, whose range of CGT bioproduction tools and services is peerless in the market. We are excited to merge these exceptional tools and services with our expertise in global, reliable scaled delivery,” said CSafe CEO Patrick Schafer in the press release. “CGT is hitting its stride and needs global support. We know how important these therapies are to patients everywhere, and it’s our mission at CSafe to protect every shipment.”

“We’re excited to work with a global partner with a strong history of reliability and performance and a deep dedication to innovative therapies,” said BioLife Solutions CEO Mike Rice in the release. “We have the best LN2 [liquid nitrogen] technology and [C]GMP [current good manufacturing practice] storage facilities in the market, in addition to our other world-class CGT solutions, and we are confident our collaboration with CSafe will extend our reach and result in even more reliability and real-time service for CGT partners.”

News

The newly formed partnership between CSafe and BioLife will expand cold chain supply chain solutions for the cell and gene therapy market.

CSafe and BioLife Solutions Partnership Will Expand Supply Chain for Cell and Gene Therapies

GSK and Tempus, a US-based precision medicine company, announced on Oct. 18, 2022 that they have entered into a new three-year collaboration agreement under which GSK will have access to Tempus’ artificial intelligence (AI)-enabled platform. GSK’s access will include Tempus’ library of de-identified patient data, which is drawn from Tempus’ work with over 40% of oncologists in US-based academic medical centers and community hospitals.

Under the new collaboration, which will utilize AI and machine learning capability, GSK and Tempus will work together to improve clinical trial design, speed up patient enrollment, and identify drug targets. These advantages are expected to contribute to GSK’s R&D success rate and provide patients with more personalized treatment faster. The agreement has a minimum financial commitment over the span of three years. GSK has made a $70 million initial payment toward that goal and has an option to extend the agreement for an additional two years.

The new agreement builds on the existing relationship between the companies, which began in 2020 and was based on the clinical-trial enrollment of patients with certain types of cancer. GSK and Tempus are currently collaborating on an open label Phase II study, under which the companies are applying an innovative, data-driven approach that is designed to accelerate and streamline study timelines. Their approach includes expediting the protocol development and intelligent site selection in under 60 days and enrolling the study’s initial patients within three months of the study launch.

“Th[e] [new] collaboration will provide GSK with unique insights to discover better medicines and transform drug discovery. Tempus complements the work our team is already doing at the intersection of genomics and machine learning across both early discovery and clinical trials,” said Tony Wood, chief scientific officer, GSK, in a company press release.

“GSK’s data-first approach to therapeutic research aligns with our own, and we believe that Tempus has the resources and capabilities to complement GSK’s dedication to data science in a way others can’t, given the breadth and depth of our platform. We both share a commitment to providing patients with more personalized therapeutic options to help them live longer and healthier lives,” said Eric Lefkofsky, founder and CEO, Tempus, in the release.

Under the new collaboration with Tempus, GSK will have access to de-identified patient data to accelerate drug discovery, improve clinical trial design, and speed up enrollment.

GSK Expands Precision Medicine Collaboration with Tempus to Accelerate R&D

Alexion, a group within AstraZeneca focused on rare diseases, recently announced a definitive agreement to acquire LogicBio Therapeutics, a genomic medicines company. The deal, which is expected to close in November 2022, will see Alexion purchase all outstanding shares of LogicBio for $2.07 per share; a total sum was not disclosed.

According to a press release from Oct. 3, 2022, the intent of the acquisition is to grow Alexion’s genomic medicines through LogicBio’s technology as well as that company’s experienced rare disease R&D team and expertise in preclinical development. LogicBio has developed various technology platforms for the delivery and insertion of genes to address genetic diseases. The company has also designed a platform to improve the viral vector manufacturing process.

"The proposed acquisition of LogicBio is a significant development for our growing research in genomic medicine,” said Marc Dunoyer, CEO, Alexion, in the release. “LogicBio’s people, experience, and platforms provide new scientific capabilities by adding best-in-class technology and expertise to our genomic medicine strategy. The scientific collaboration between Alexion and AstraZeneca has been a substantial area of focus since last year’s acquisition and the addition of LogicBio will expand this foundational work.”

“We are excited about the opportunity to bring our science and expertise in genetic medicine to Alexion, which shares our commitment to discovering treatments for rare conditions and improving the lives of patients,” said Fred Chereau, president and CEO, LogicBio, in the release. Through this acquisition, we strive to accelerate our research in gene editing and AAV capsid development and together move the field of genomic medicine forward.”

Alexion’s acquisition of LogicBio is designed to accelerate the company’s growth in genomic medicines.

Alexion, AstraZeneca Acquire Genomic Medicines Company

Schrödinger, a pharmaceutical company that utilizes a specialized computational platform for drug discovery, announced a collaboration with Eli Lilly and Company (Lilly) on Oct. 6, 2022. The collaboration is focused on discovering and optimizing small molecule compounds for a designated target.

According to a company press release, Schrödinger will be responsible for the discovery and optimization process, while Lilly will handle completion of preclinical development, clinical development, and commercialization. Schrödinger will receive an upfront payment and be eligible to receive up to $425 million, contingent on various discovery, development, and commercial milestone payments. Schrödinger will also be eligible to receive low single - to low double-digit royalties on net sales of any products emerging from the collaboration.

Source: 

Eli Lilly and Company will partner with Schrödinger to develop and optimize small molecule compounds.

Lilly and Schrödinger Ink $425 Million Small Molecule Partnership

Cytiva has acquired CEVEC Pharmaceuticals, a Germany-based provider of high-performance cell line development and viral vector manufacturing technologies, in a move that strengthens Cytiva's capabilities to offer biomanufacturing solutions.

With this acquisition, Cytiva gains CEVEC’s scalable producer cell lines for vectors based on adeno-associated virus and adenovirus, two widely used vectors for gene therapy delivery, according to an Oct. 6, 2022 company press release. Cytiva also gains producer cell lines (PCLs) enabled by CEVEC's ELEVECTA technology, which promotes yield, scalability, and robustness in the manufacturing process. The ELEVECTA technology provides all the components necessary for vector production integrated into the genome of the PCL, thus offering an alternative to technologies based on transfection or the use of a helper virus.

Under the agreement, CEVEC and its 46 scientific experts become part of Cytiva, effective immediately. CEVEC's products, services, and technologies will continue to carry the ELEVECTA and CAP brands in the immediate future and will be available to customers worldwide.

"[CEVEC's] innovative technology complements Cytiva's offering and brings the promise of improved viral vector manufacturing. With Cytiva's global reach and ability to scale, more customers will have access to [CEVEC's] technology and expertise to help accelerate and advance therapeutics for the benefit of patients," said Emmanuel Abate, president, Genomic Medicine at Cytiva, in the release.

"We are excited to become part of Cytiva. Combined with Cytiva's global presence and leading brands, this enables the whole gene therapy industry to embark on new and powerful solutions for scalable vector manufacturing and start a new era for therapies made available to patients in need," said Nicole Faust, CEO of CEVEC Pharmaceuticals, in the release.

Cytiva’s acquisition of CEVEC Pharmaceuticals strengthens the company’s cell line development and biomanufacturing capabilities.

Cytiva Acquires CEVEC, Boosts Cell Line Development Capabilities

Freudenberg Medical, a medical and pharmaceutical devices, components, and tubing manufacturer, announced the launch of the HeliFlex Thermoplastic Elastomer (TPE) tubing on Sept. 29, 2022. The Heliflex is a high purity TPE designed for biopharmaceutical fluid transfer applications.

According to a company press release, the HeliFlex is ideal for bioprocessing applications because it can be heat-sealed to allow for easy, fast, and safe fluid transport and transfer. Additionally, the product can be welded to existing tubing lines, is sealable, translucent, and can be used in peristaltic pumping applications.

The tubing is offered with various sterilization options, including autoclave, gamma irradiation, x-ray, and e-beam. According to the release, it is a more environmentally friendly option than silicone, as it can be recycled.

“Silicone has always been the gold standard for biopharma applications,” said David Schwass, director, Sales & Marketing Biopharm, Freudenberg Medical, in the release. “But with silicone material in short supply over the past few years, Freudenberg wanted to offer global customers another single-use tubing alternative.”

New Technology

Freudenberg Medical’s Heflix TPE tubing is a high purity TPE intended for bioprocessing applications.

Pharmaceutical Technology's In the Lab eNewsletter, November 2022