Mark Rogers, PhD, mark.rogers@sgs.com, is the global scientific director for the SGS Health Science Drug Development group.

Mark Rogers

For several years after Crick (1) first described the genetic role of RNA, this molecular entity was perceived simply as an “inert” carrier between DNA and protein. Today, however, this view has almost entirely changed, and RNA-based molecules have been implicated in a broad range of functions including the activation/deactivation of genes, the excision of genetic material, and the transport of intercellular components. Indeed, it is expected that in the coming years, further discoveries will uncover even greater biochemical significance to this molecular type.

Given the ubiquity and variety of roles associated with RNA, it was inevitable that it would become a focus for investigators involved in the development of therapeutics. As early as 1978, Zamecnik (2) described the therapeutic use of an RNA-based oligonucleotide to inhibit replication of the Rous sarcoma virus, and, today, there are approximately 16 FDA-approved RNA therapies, 28 in clinical development, and many more expected in the near future (3).

Currently, RNA-based medicines can be segregated by their functionality and structure and include species, such as messenger RNA (mRNA), antisense oligonucleotides (ASO), small interfering RNA (siRNA), and microRNA (miRNA). Other types of RNA include aptamers which are single-stranded and form higher-order structures, and more recently described, circular RNA (circRNA or oRNA), which appears to have multiple functions prior to and following the transcription process (4). Additionally, mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) constitute components of the recently developed CRISPR technology.

 The Real Message Behind Commercial mRNA Products

The Marriage of RNA and Mass Spectrometry

Pharmaceutical Technology’s The Real Message Behind Commercial mRNA Products eBook

Mark Rogers is global scientific director at SGS.

The Real Message Behind Commercial mRNA Products

When referring to this article, please cite it as Rogers, M. The Marriage of RNA and Mass Spectrometry. 

Pharmaceutical Technology's The Real Message Behind Commercial mRNA Products

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Considerable efforts have been made over the years to resolve the key issues of stability and delivery of RNA-based therapeutics.

While not unique, the use of RNA in therapeutic applications is generally incumbered by two key issues: stability and delivery. Over many years, considerable efforts have been directed at attempts to resolve both these issues, leading to some creative solutions which, in turn, has necessitated the application of a broad range of analytics. However, one technique, namely mass spectrometry (MS), is particularly notable for: its ability to provide analysis of all RNA types, regardless of size and modification; the fact that it can be used for qualitative as well as quantitate investigations; the technology’s ability to be applied in both research and quality control (QC); and its applicability in the area of delivery systems.

Since the late 1970s, when both therapeutic biopolymers and analytical MS began a decades-long connection, there have been some considerable advancements in both therapeutic development and analytics. Most early biopolymer MS was applied to peptides and proteins, and, today, this technique has become arguably the single most important analytical tool in this field. Indeed, several laboratories within SGS, for example, were founded by one of the early innovators, H.R. Morris, whose contributions included the first combination of high-field magnet technology with soft ionization (5) and some of the first protein-mapping studies (6). These laboratories and others continue to encourage the adoption of such technological innovations, which have resulted in the application of advanced analytical services in support of the most challenging biopolymer studies.

Many of the advances made in protein MS have been adapted to make the technique appliable to the study of oligonucleotides, where attributes such as sequence, modification, and quantitation are critical.

’s report in 1992 (7), there has been increased attention given to the application of MS-based technologies in the analysis of RNA, including top-down methods that provide data from intact RNA species. In 2012, Taucher and Breuker (8) were amongst the first to report sequence coverage of full-length transfer RNA (tRNA) using Fourier transform ion cycloctron resonance (FT-ICR), combining data from electron detachment dissociation (EDD) and collision-activated dissociation (CAD) experiments. However, while such MS-based studies continue to be explored, they remain constrained by several somewhat related factors, such as the large molecular size, the inability to distinguish different species with the same mass, the high degree of purity required, and the limited availability of software to support top-down RNA analysis.

Perhaps to a lesser extent, these limitations also apply to top-down protein studies, although in this field these constraints have largely been overcome by mapping techniques. This approach involves controlled and specific digestion of the protein to yield a peptide mixture that can then be analyzed by a variety of MS-related methodologies, the most familiar being liquid chromatography–mass spectrometry (LC–MS). It was inevitable that such an approach would evolve in the RNA field, and this has now been successfully applied to species such as the mRNA coding for the SARS-Cov-2 spike protein (9). Such methods rely on digestion (partial and complete) with RNAses such as T1, RNAse A, and MazF prior to analysis of the products by LC–MS/MS. This is a very effective strategy because it can be automated with the use of immobilized enzymes (9), provides sequence confirmation as well as the ability to conduct de novo investigations, is able to identify and locate post-transcriptional and process modifications, can be used qualitatively and quantitatively, and may be applied to bioanalytical applications.

In contrast to polymerase chain reaction (PCR) methodologies, MS has the ability to detect and locate the more than 150 post-transcriptional RNA modifications that have been described to date. Importantly, MS is also able to identify the presence of unexpected nucleotide alterations. For many years, mass spectroscopists have taken advantage of the method’s ability to detect stable isotopic forms of elements such as hydrogen, nitrogen, carbon, oxygen, and sulfur. Nucleic acid isotope labeling combined with MS (NAIL–MS) has been used in a variety of applications to study the dynamics of RNA modification (10).

Some reports have addressed the use of MS for the investigation of dimer formation and higher-order structures, but here great care should be taken in data interpretation and extrapolation given the tendency for non-covalent molecular association within the mass spectrometer and the fundamental differences between the solution-phase environment and the environment within the mass spectrometric process. Ion-mobility MS has been used to investigate the gas-phase structures of nucleic acids such as duplexes, triplexes, and quadruplexes to try to determine if these structures are indeed similar to the structures in solution (11). Today, the use of MS for the analysis of RNA-based therapeutics has become widespread due largely to the technique’s agnostic ability to manage a wide diversity of molecular structures. From the development of Macugen (pegaptanib sodium) in the early 2000s, laboratories such as SGS and others have been highly active in analytical support for nucleic acid-based therapeutics, including today’s complex, multi-faceted structures.

Issues of stability, delivery, and binding have resulted in the development of RNA therapeutics that often involve significant modification(s) to the basic oligoribonucleic acid structure.

 provides several examples of these modified RNA structures. Changes to the phosphodiester linkage have included replacement of the free oxygen with sulfur (producing the more stable phosphorothioate linkage) and substitution with amide and boronophosphate linkages (12). Similarly, multiple, diverse structural alterations to both sugar and base moieties have been described, including methylation, methoxyethylation, fluorination, and cyclization of the 2’ sugar hydroxyl, resulting in so called “locked nucleic acid” (LNA) (10). Often, several of these modifications may be incorporated within the same therapeutic. In addition, linking of these RNA-based therapeutics with a wide range of conjugates has been exploited as a means to significantly enhance and direct their delivery and greatly improve their effectiveness. A notable example of such modification is the conjugation of siRNAs with N-acetylgalactosamine providing specific binding to asialolycoprotien receptors in hepatocytes. This structural concept has led to the successful development of the drug, givosiran, which treats hepatic porphyria by silencing the expression of aminolevulinate synthase 1 mRNA in the liver (13). Many other conjugates have been described such as lipids, peptides, antibodies (complete or fragments), vitamins, and a range of other molecular classes (14). 

Currently, the most widely used hyphenated analytical technique is LC–MS. Multiple separation methodologies have been reported including ion-pair reversed-phase chromatography, hydrophilic interaction liquid chromatography, ion-exchange chromatography, and size exclusion chromatography. A report by Santos and Brodbelt (15) provides a comprehensive review of these and other related techniques as applied to nucleic acids. Two-dimensional LC has also been described as a means to overcome incompatibility between mobile-phase composition and MS detection (16), while 3D-LC–MS—incorporating in-line digestion and hydrophilic interaction liquid chromatography (HILIC) separations—has been used for the sequencing of CRISPR-guide RNAs (17).

The majority of mass spectrometric applications involving nucleic acids have utilized either laser desorption (matrix or surface assisted) or electrospray ionization (ESI) techniques. Both are considered “soft,” thereby reducing in-source fragmentation of these large biomolecules and providing a relatively high ion current for the intact molecular species. Almost all analyzer types have been employed, including magnetic sector, time of flight, quadrupole, ion trap, and combinations of each. Today, a technology that has gained considerable acceptance both for quantitative and qualitative investigations is the so-called Orbitrap that was introduced commercially in 2005 and is shown in 

. Combining ion trap and/or quadrupole mass filters, orbitrap-based mass spectrometers provide high sensitivity with high resolution, the latter feature rising in importance as the complexity and heterogeneity of materials under examination become more acute. The implementation of multiple fragmentation functions (e.g., collision-induced dissociation, higher energy collisional dissociation, electron-transfer dissociation, ultraviolet photodissociation) on the orbitrap-based mass spectrometers also enables researchers to tackle a wide range of challenging applications. 

 SGS uses liquid chromatography tandem mass spectrometry (LCMSMS) Orbitrap technology to solve complex analytical problems.

The use of MS in the study of oligonucleotides has become almost as significant as applications in the peptide/protein area despite the historical impact of techniques such as PCR. It is highly likely that novel RNA structures are yet to be discovered, leading to greater understanding of the function and role of this molecular type and potentially the development of more effective

therapeutic agents. As with protein chemistry, higher-order structure governs the function of many RNA species. It is likely that MS will play an important role in defining these large RNA structures that currently remain unresolved (18). Mass spectrometric techniques are just beginning to provide insights in some critical therapeutic areas where, for example, the quantitative identification of RNA modifications (genetic and epigenic) appears to provide a source of potential biomarkers and treatment monitoring in areas such as oncology (19). MS has also become important in the development and understanding of therapeutic products themselves as illustrated by the work of Packer 

 (20), who used LC–MS/MS to study mRNA modifications as a result of reaction with certain lipids used in lipid nanoparticle-vector vaccines.

There is little doubt that the past decade has seen significant advances in the understanding of ribonucleic acid functionality leading to the development of some novel and highly effective therapeutics. The growing need for sophisticated analytics to support both the investigative and routine analytical requirements of these complex therapeutics is, in large part, being met by the progression of mass spectrometric technologies. It is certain that MS will continue to maintain this close “marriage” with RNA becuase many aspects of this nucleic acid’s biochemistry are yet to be uncovered.

, 12, 138–163.
2. Zamecnik, P.C.; Stephenson, M.L. Inhibition of Rous Sarcoma Virus Replication and Cell Transformation by a Specific Oligodeoxynucleotide. 

, 75, 280–284.
3. Zogg, H.; Singh, R.; Ro, S. Current Advances in RNA Therapeutics for Human Diseases. 

, 23 (5), 2736. DOI: 10.3390/ijms23052736
4. Liu, K.S.; Pan, F.; Mao, X.D.; Liu, C.; Chen, Y.J. Biological Functions of Circular RNAs and Their Roles in Occurrence of Reproduction and Gynecological Diseases. 

, 11 (1),1–15.
5. Morris, H.R.; Dell, A.; McDowell, R.A. Extended Performance Using a High-Field Magnet Mass-Spectrometer. 

, 8, 463–473.
6. Morris, H.R.; Panico, M.; Taylor, G.W. Mapping of Recombinant DNA Protein Products. Biochem. 

, 117, 299–305.
7. McLuckey, S.A.; Van Berkel, G.J.; Glish, G.L. Tandem Mass Spectrometry of Small, Multiply Charged Oligonucleotides. 

, 3 (1), 60–70.
8. Taucher M.; Breuker, K. Characterization of Modified RNA by Top-Down Mass Spectrometry. 

, 51 (45), 11289–11292.
9. Vanhinsbergh C.J.; Criscuolo, A.; Sutton, J.N.; et al. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. 

, 94 (20),7339–7349. DOI: 10.1021/acs.analchem.2c00765
10. Heiss, M.; Hagelskamp, F.; Marchand, V.; Motorin, Y.; Kellner, S. Cell Culture NAIL-MS Allows Insight into Human tRNA and rRNA Modification Dynamics 

, 12 (1), 389. DOI: 10.1038/s41467-020-20576-4
11. Khristenko, N.; Amato, J.; Livet, S.; et al. Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process. 

, 30, 1069–1081.
12. Halloy, F.; Biscans, A.; Bujold, K.E.; et al. Innovative Developments and Emerging Technologies in RNA Therapeutics. 

, 19 (1), 313–332. DOI: 10.1080/15476286.2022.2027150
13, Honor, A.; Rudnick, S.R.; Bonkovsky, H.L. Givosiran to Treat Acute Porphyria. 

, 57 (1), 47–59. DOI: 10.1358/dot.2021.57.1.3230207
14. Hawner, M.; Ducho, C. Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules. 

, 25 (24), 5963. DOI: 10.3390/molecules25245963
15. Santos, I.C.; Brodbelt, J.S. Recent Developments in the Characterization of Nucleic Acids by Liquid Chromatography, Capillary Electrophoresis, Ion Mobility, and Mass Spectrometry (2010-2020). 

, 44 (1), 340–372. DOI: 10.1002/jssc.202000833
16. Koshel, B.; Birdsall, R.; Chen W. Two-Dimensional Liquid Chromatography Coupled to Mass Spectrometry for Impurity Analysis of Dye-Conjugated Oligonucleotides. 

J Chromatogr B Analyt Technol Biomed Life Sci.

 Jan 15, 1137:121906. DOI: 10.1016/j.jchromb.2019.121906
17. Goyon, A.; Scott, B.; Kurita, K.; et al. On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC-MS System. 

, 94 (2),1169–1177. DOI: 10.1021/acs.analchem.1c04350
18. Amalric, A.; Bastide, A.; Attina, A.;et al. Quantifying RNA Modifications by Mass Spectrometry: A Novel Source of Biomarkers in Oncology. 

, 59 (1), 1–18. DOI: 10.1080/10408363.2021.1958743
19. Christy, T.W.; Giannetti, C.A.; Houlihan, G.; et al. Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP. 

, 60 (25),1971–1982. DOI: 10.1021/acs.biochem.1c00270
20. Packer, M.; Gyawali, D.; Yerabolu, R.; et al. A Novel Mechanism for the Loss of mRNA Activity in Lipid Nanoparticle Delivery Systems. 

Mass spectrometric techniques are providing insights in critical RNA-based therapeutic areas.

Marrying RNA and Mass Spectrometry 

Extractable and leachable (E&L) studies have become an integral part of any drug development program and serve to ensure compatibility between the product and its packaging. While such studies are now generally well defined in terms of experimental design and data evaluation, these have had to adapt to the ever-increasing diversity of therapeutic products. There are, for example, some unique challenges in the analysis of biopharmaceutical products as a result of certain formulation components, as well as the complexity of the therapeutic itself. This article describes a number of challenges and solutions in the E&L evaluation of biopharmaceuticals and highlights a selection of those encountered with emerging cell and gene therapy (CGT) products.

The risks posed by leachables on product quality and patient safety are an ongoing challenge for the pharmaceutical and biopharmaceutical industries. Continued investigational development in this area is primarily driven by a number of factors, including: the increased adoption of single-use technologies; progress in the medical device field, particularly combinational devices; expanded directives from industry regulators; and advances in analytical technologies.

Extractables (1) are defined as compounds that migrate from any product-contact material (including elastomeric, plastic, glass, stainless steel, or coating components) when exposed to an appropriate solvent, under exaggerated conditions of time and temperature. To study materials at risk of potential leachable components, extractions of the contact material are performed, generally using aggressive conditions including acidic, basic, organic, and aqueous solvents. These conditions are not intended to distort the material under investigation but do attempt to mimic the drug formulation. The resulting extracts are usually analyzed using a combination of analytical techniques such as liquid chromatography–mass spectrometry (LC–MS), gas chromatography–mass spectrometry (GC–MS), and inductively coupled plasma–mass spectrometry (ICP–MS) that can qualitatively and quantitatively evaluate both the organic and inorganic components, or “extractables”. Such studies are typically performed to represent a worst-case scenario and may provide an indication of possible leachable components as well as facilitate material selection during early risk assessment.

Leachables (1) are compounds, often identified during extraction studies, that migrate into a drug formulation from any product-contact material (elastomeric, plastic, glass, stainless steel, or coating components). This leaching happens as a result of direct contact under normal process or accelerated storage conditions, such as elevated temperature. However, if the leachable interacts with the drug product (as may be the case particularly with biotherapeutics) or packaging materials, new components can be generated that are known as “secondary leachables”. The complexity of many formulations, often containing a variety of buffers, surfactants, fillers, and other excipients, can lead to the formation of secondary leachables only identified after long-term stability studies.

E&L evaluation of traditional pharmaceutical medicines is now well established and may be undertaken with a certain predictability in experimental design and even outcome. In contrast, however, certain excipients used in the formulation of some biotherapeutics present unique analytical challenges for leachable determination. Among these excipients are the commonly used nonionic surfactants, such as polysorbate 80 (Tween 80), which constitute a mixture of fatty acid esters of polyoxyethylene sorbitan. These have an extremely low ultraviolet (UV) response but are highly responsive to ionization methods commonly used in LC–MS (2). This often leads to significant interference, either by co-elution and/or suppression during analysis, raising the real possibility of contaminants remaining undetected. Extraction of these surfactants prior to analysis is a possible means of mitigation but is often not highly effective due to the surfactant’s inherent heterogeneity and limited extraction efficiency. Care must also be taken in the application of such an approach since the leachable may be co-extracted with the surfactant.

In the growing area of CGT, there are currently no specific guidelines for E&L studies. However, such products have some unique challenges, such as the use of “ancillary materials”. These materials are used during manufacturing but are not intended to be present in the final product. They are often themselves complex biological materials, such as recombinant insulin and human serum albumin, used as media supplements, and collagen, used as a scaffold in implantable devices (3). Since these materials can potentially interact with and be affected by leachables, in the same manner as the biotherapeutic itself, the choice of materials and controls must be carefully considered to avoid adverse consequences for the manufacturing process.

The evaluation of biotherapeutics for leachables presents many challenges, some of which are somewhat unique to this type of product. For example, the biopharmaceutical itself may interfere in the testing regimen due to its high concentration, heterogeneity, and/or high detector response. A potential solution is to remove the biotherapeutic prior to analysis, but the procedure must be very carefully considered because associated leachables may be unknowingly removed and/or additional extraneous components may be introduced.

The major concern of leachables in pharmaceutical products is their specific toxicity and/or genotoxicity. The additional issue with biotherapeutics is the potential for interaction between the structurally complex biopharmaceutical itself and any leachable components. Compared to traditional pharmaceuticals, proteins and other biotherapeutics are often significantly less stable, due to their strong dependence on physico-chemical and conformational structural properties. These larger molecules often have both hydrophilic and hydrophobic regions, with many reactive sites for possible leachable binding, potentially resulting in the loss of activity via mechanisms such as unfolding, truncation, aggregation, and precipitation. Consequently, the adverse effects of extractables and leachables can be more significant and more difficult to assess on biopharmaceutical products than small molecules. Acrylic acid, for example, a leachable from syringes, has been shown to react at three different sites within a protein structure, resulting in potential changes in charge and hydrophobicity with consequential effects on the therapeutic properties of the protein-based drug (4).

As the manufacture of many biotherapeutics involves cellular-based processes, the presence of certain leachable components can have unfavorable consequences not only on the final drug product but also on the production process itself. Productivity can certainly be adversely affected by leachables that result in cellular toxicity, leading to significant loss in manufacturing yield.

During the past decade, many biotherapeutic manufacturers have migrated to the use of disposable, single-use systems (SUS) for production, and this has now become commonplace in the emerging CGT field. Every operational phase of a biomanufacturing process that uses SUS technology is at risk of leachable contamination. This contamination can be introduced as a result of contact and interaction with different materials, such as polymers, metals, and additives used to construct holding tanks, films, bags, tubing, and filters. This concern extends to containers for buffers, media, water for injection, and other liquids required for manufacturing. The possibility of leachable accumulation may also arise since concentration stages are often used in the manufacturing process.

The majority of biopharmaceuticals are injectable products and, therefore, contaminants enter the bloodstream directly, without the potential for depletion in the gut as with oral medications. In addition, many biotherapeutics, particularly monoclonal antibodies, are dosed in high concentrations (multiple mg/mL) and are produced in liquid, with complex formulations. These drugs can be stored for long periods of time, providing higher risk of leachable adulteration of the product than the solid dose forms common with traditional pharmaceuticals.

Certain products developed in the CGT area can present further challenges for E&L evaluation. Since the quantities of product can be relatively low, the ratio of contact material surface to the drug volume is very high, increasing the potential for leachable contamination. This may be further exacerbated in those treatments that are essentially customized, with each patient receiving the entire product dose.

Risk assessment in terms of E&L is now an integral part of any product development strategy. This is of particular importance for CGTs, which should not only evaluate the likely compatibility of contact materials used in manufacturing, but also include an experimental study designed to provide an accurate assessment of the extractable and leachable profiles.

While many biotherapeutics are produced as liquid formulations, lyophilized forms of drug product are also commonplace, as part of efforts to improve stability during storage. Interestingly, the FDA Center for Drug Evaluation & Research, the FDA Center for Biologics Evaluation & Research, and the United States Pharmacopeial Convention (in 

 <1664> [5]), describe sterile powders for injection as being “low risk” for leaching, despite some evidence to the contrary. It has been demonstrated, for example, that several semi-volatile organic compounds originating from butyl rubber stoppers—part of the primary packaging system—may leach into a lyophilized product but not a liquid formulation stored in the same packaging (6). The explanation for this finding lies in the hydrophobicity of the leachables, rendering them very poorly soluble in aqueous media but able to diffuse from the stopper. This is perhaps facilitated by their volatility and the nature of the drug product.

There are multiple types of material used in the manufacture and storage of biopharmaceuticals, and most may be classified as either glass, metal, or synthetic polymer/elastomer. The latter are recognized as the most significant sources of leachables, often resulting from monomers/oligomers of the polymer itself, as well as catalysts, initiators, additives (e.g., lubricants, antioxidants, antitack, and antistatic agents), adhesives, anchoring agents, adhesive resins, colors, fillers, and degradation products of almost all these components. The diversity and nature of these potential contaminants and their often-unpredictable interactions with formulated biopharmaceuticals pose significant challenges to analytical assessment of the drug products and process materials. There are now significant guidance documents available from regulatory authorities, industry organizations, and researchers providing at least some support in the design and rationale for suitable experimental approaches.

For biopharmaceuticals, inorganic leachables (generally associated with glass and metal) are of particular concern because of their potential impact on the stability and efficacy of the drug itself. A metal commonly used is stainless steel, which has found applications in manufacturing, shipping, and storage, and is known to leach iron, chromium, and nickel, particularly if the steel is unpassivated (7). Trace levels of metal ions can potentially cause protein degradation by mechanisms such as protein oxidation and fragmentation. The latter occurs because of the metal-binding propensity of aromatic amino acids such as tryptophan, tyrosine, and histidine. Certain metal–protein binding is also known to induce secondary and tertiary structure changes resulting in the formation of insoluble protein aggregates (8).

Glass has also been demonstrated to be a source of certain metal leachables. For example, manganese and iron oxide—used as coloring agents—are present in Type I amber vials as are zinc and

barium oxides. Higher levels of manganese and iron have been shown to leach from amber glass compared to clear glass. Meanwhile, sterilization techniques, such as steam autoclaving and gamma irradiation, may also lead to the presence of metal ion contamination (9).

Some biotherapeutic excipients, and the drug itself, may also play a significant role in facilitating the extraction of metals from contact surfaces. Certain proteins and chelating agents, such as ethylenediaminetetraacetic acid, have been shown to increase the quantity of metals such as aluminum, calcium, and iron from plastic container systems. Similarly, buffers such as phosphate and lactate can also increase the metal leaching from both metal and glass surfaces.

The area of E&L investigation has developed rapidly over the past decade, resulting in technological and regulatory advancement. Ultimately, this has led to safer medications. However, developments in biopharmaceuticals and specifically in CGT are likely to present further demands on expertise in areas of manufacturing and analytics.

It is clear that a single, common strategy, for monitoring and/or eliminating leachable contaminants will never be sufficient to address all situations. Instead, engineers and investigators must be always prepared for the unexpected.

1. D.B. Lewis, “Current FDA Perspective on Leachable Impurities in Parenteral and Ophthalmic Drug Products,” Presentation at AAPS Workshop on Pharmaceutical Stability—Scientific and Regulatory Considerations for Global Drug Development and Commercialization (Washington, DC, Oct. 22–23, 2011).
2. J. Fukuda, et al., 

 B 953–954, pp. 68–72 (2014).
3. J. Solomon, et al., 

 18 (1) 1–12 (2016).
4. D. Liu, et al., 

 <1644>, “Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems,” 

, first supplement (Rockville, MD, Aug. 1, 2015).
6. S.A. Zdravkovic, 

 107 (11) 2837–2846 (2018).
7. L. Allain and Q. Wang, 

 10 (3) (2007).
8. J.L. Cleland, M.F. Powell, and S.J. Shire, 

 10 (4) 307–77 (1993).
9. D. Bohrer, et al., 

Establishing extractable and leachable studies for complex biotherapeutic molecules poses new challenges for analytical assessment.

Preparing for Unexpected Issues in E&L Studies for Biopharma

Extractable and leachable (E&L) studies have become an integral part of any drug development programme and serve to ensure compatibility between the product and its packaging. While such studies are now generally well defined in terms of experimental design and data evaluation, these have had to adapt to the ever-increasing diversity of therapeutic products. There are, for example, some unique challenges in the analysis of biopharmaceutical products as a result of certain formulation components, as well as the complexity of the therapeutic itself. This article describes a number of challenges and solutions in the E&L evaluation of biopharmaceuticals and highlights a selection of those encountered with emerging cell and gene therapy (CGT) products. 

Pharmaceutical Technology Europe’s July 2021 print issue

Extractables (1) are defined as compounds that migrate from any product-contact material (including elastomeric, plastic, glass, stainless steel, or coating components) when exposed to an appropriate solvent, under exaggerated conditions of time and temperature. To study materials at risk of potential leachable components, extractions of the contact material are performed, generally using aggressive conditions including acidic, basic, organic, and aqueous solvents. These conditions are not intended to distort the material under investigation but do attempt to mimic the drug formulation. The resulting extracts are usually analyzed using a combination of analytical techniques such as liquid chromatography–mass spectrometry (LC–MS), gas chromatography–mass spectrometry (GC–MS), and inductively coupled plasma–mass spectrometry (ICP–MS) that can qualitatively and quantitatively evaluate both the organic and inorganic components, or “extractables”. Such studies are typically performed to represent a worst-case scenario and may provide an indication of possible leachable components as well as facilitate material selection during early risk assessment. 

As the manufacture of many biotherapeutics involves cellular-based processes, the presence of certain leachable components can have unfavourable consequences not only on the final drug product but also on the production process itself. Productivity can certainly be adversely affected by leachables that result in cellular toxicity, leading to significant loss in manufacturing yield.

While many biotherapeutics are produced as liquid formulations, lyophilized forms of drug product are also commonplace, as part of efforts to improve stability during storage. Interestingly, the US Food and Drug Administration (USFDA) Center for Drug Evaluation and Research, the USFDA Center for Biologics Evaluation andResearch, and the United States Pharmacopeial Convention (in 

There are multiple types of material used in the manufacture and storage of biopharmaceuticals, and most may be classified as either glass, metal, or synthetic polymer/elastomer. The latter are recognized as the most significant sources of leachables, often resulting from monomers/oligomers of the polymer itself, as well as catalysts, initiators, additives (e.g., lubricants, antioxidants, antitack, and antistatic agents), adhesives, anchoring agents, adhesive resins, colours, fillers, and degradation products of almost all these components. The diversity and nature of these potential contaminants and their often-unpredictable interactions with formulated biopharmaceuticals pose significant challenges to analytical assessment of the drug products and process materials. There are now significant guidance documents available from regulatory authorities, industry organizations, and researchers providing at least some support in the design and rationale for suitable experimental approaches.

For biopharmaceuticals, inorganic leachables (generally associated with glass and metal) are of particular concern because of their potential impact on the stability and efficacy of the drug itself. A metal commonly used is stainless steel, which has found applications in manufacturing, shipping, and storage. Stainless steel is also known to leach iron, chromium, and nickel, particularly if the steel is unpassivated (7). Trace levels of metal ions can potentially cause protein degradation by mechanisms such as protein oxidation and fragmentation. The latter occurs because of the metal-binding propensity of aromatic amino acids such as tryptophan, tyrosine, and histidine. Certain metal–protein binding is also known to induce secondary and tertiary structure changes resulting in the formation of insoluble protein aggregates (8).

Glass has also been demonstrated to be a source of certain metal leachables. For example, manganese and iron oxide—used as colouring agents—are present in Type I amber vials as are zinc and barium oxides. Higher levels of manganese and iron have been shown to leach from amber glass compared to clear glass. Meanwhile, sterilization techniques, such as steam autoclaving and gamma irradiation, may also lead to the presence of metal ion contamination (9).

1. D.B. Lewis, “Current FDA Perspective on Leachable Impurities in Parenteral and Ophthalmic
Drug Products,” Presentation at AAPS Workshop on Pharmaceutical Stability—Scientific and Regulatory Considerations for Global Drug Development and Commercialization (Washington, DC, 22–23 Oct. 2011).
2. J. Fukuda, et al., 

 Chapter <1644>, “Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems,” 

, first supplement (Rockville, MD, 1 Aug. 2015).
6. S.A. Zdravkovic, 

When referring to this article, please cite it as M. Rogers, “Preparing for the Unexpected: E&L Studies in Biopharma” 

New challenges arise in extractable and leachable studies for cell and gene therapy products.