Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.

Cynthia A. Challener

Originally it was thought that the only role of ribonucleic acid (RNA) was to translate genetic information encoded in DNA into protein sequences that perform critical biological functions. While messenger RNA (mRNA) is crucially important, it is just one of many types of RNA involved in a range of activities that affect the transmission of genetic information and the functioning-or dysfunctioning-of cells. Appropriately designed RNA-based therapies can thus modulate genetic information in a controlled and targeted manner, effectively acting as gene therapies. They also offer several advantages over DNA plasmid-based therapies.

	Strands of RNA by themselves, however, are unstable and degrade rapidly in the bloodstream. Delivery to specific targeted cells is one of the main challenges that developers of RNA-based gene therapies are tackling today.

	RNA technologies fall into two main categories: those that directly interfere with expressed mRNA or mRNA expression, blocking protein production, and those that cause the expression of target proteins whose deficiencies can lead to disease.

	The first type includes RNA interference (RNAi) and antisense therapies, which in effect silence the expression of specific genes that are drivers of disease mechanisms. One example is small interfering RNAs (siRNAs), which according to David Evans, chief scientific officer at Sirnaomics, are useful as therapeutics where the gene(s) to be targeted is (are) overexpressed in the diseased state and where silencing these genes may produce a therapeutic benefit.

	Indeed, Doug Fambrough, president and CEO of Dicerna Pharmaceuticals, believes that while supporting protein production is useful in a few contexts, such as treating rare genetic diseases or making vaccines, there are far more uses for silencing genes. “For example, silencing can be used to treat viral diseases, cardiovascular conditions, cancer, inflammatory diseases, fibrotic diseases, select rare diseases, and generally any condition where biological processes are inappropriately activated,” he asserts.

	Sirnaomics and Dicerna are both focusing on RNA interference therapies. Belgium-based biotech company eTheRNA immunotherapies, on the other hand, has elected to focus on mRNA-based technologies that lead to the expression of proteins the body is unable to produce on its own, according to Wim Tiest, a special advisor at the company.

	Unlike DNA-based gene therapies that must be delivered into the nucleus and create permanent changes, RNA therapies only need to reach the cytoplasm to perform their post-transcriptional activity and are not permanent, thus treatment can be modified or halted if necessary. They are also smaller, more easily manipulated and do not present the risk of unintentional genetic effects.

	“Messenger RNA and other RNA-based therapies have the advantage of being versatile (they can code for a broad range of targets), easy to produce (basically the production process is transposable between mRNA designs), and safe (due to the transient expression of the target and absence of risk for integration into the patients genome),” asserts Tiest. “Additionally,” he says, “mRNA is compatible with a range of systemic and local delivery modalities and can be designed to be nearly invisible to the host’s immune system, thus avoiding any vector-directed immune responses.”

	RNAi technologies such as siRNAs also provide high target specificity by using gene sequences that match with targeted genes and rely on naturally occurring cellular enzymes to mediate gene silencing, according to Fambrough. Due to this high specificity-and reversibility-they offer the potential of fewer side effects. “Unlike gene therapy or gene editing, which can be permanent and could have unknown, unwanted, and irreversible consequences, RNAi therapies can offer targeted, effective treatment that does not permanently edit or change DNA. An RNAi therapy’s effect is targeted, precise, and can be stopped at any time by discontinuing treatment,” he explains.

	With the rapid progress made in sequencing the human genome, there is now a wealth of information on sequences of genes, some of which have been identified as playing a direct role in the etiology of diseases, according to Evans. “Many of these therapeutic targets, however, are undruggable by traditional small molecules or antibodies but are upregulated in disease states like cancer and fibrosis,” he notes. “siRNAs are readily designed against these gene sequences and can silence expression of the proteins that are driving disease, producing therapeutic benefit to patients with disease,” Evans says.

	In addition, RNAi therapies offer a highly versatile approach that can benefit patients with an array of diseases, Fambrough points out. “Today, we can create siRNAs to match almost any messenger RNA. In effect, the RNAi approach to drug development can silence virtually any gene,” he notes.

	Another benefit of RNA therapies compared to many conventional small-molecule and biologic drugs is the possibility of reducing the treatment burden for patients, according to Fambrough. “With a longer duration of effect, RNAi therapies can be administered with infrequent subcutaneous injections, and depending on the disease modality, can be self-administered at home,” he observes.

	RNA by its nature is rapidly degraded once injected. As such, delivery to the targeted cells is the main hurdle at this time. “We need to ensure that the siRNAs are able to reach their target tissue and cell types,” Evans states. In cancer, for example, that means accumulation of nanoparticles, for instance, at the tumor site with delivery of the RNA into the tumor cells specifically. Stabilization of RNA by modification of the chemical backbone is possible, and this approach can also help to make the RNA less immunogenic, Tiest notes.

	Similarly, the inhibitory or expression effect wanes as the RNA degrades inside the cell. Self-amplifying RNA can to some extent prolong this active period, according to Tiest. The need for regular, repeated dosing and maintenance of a sufficient active dose is therefore also a major bottleneck. He does note that local administration helps to overcome some of this limitation.

	Overall, careful selection of targets that require short interventions and can benefit from the increased safety profile of RNA is required to fully profit from RNA-based therapy development, Tiest concludes.

	Like most DNA plasmid-based gene therapies, RNA therapies can also be delivered into cells using viral vectors, but the majority of these therapies under development today rely on nonviral methods, such as organic (lipid) or inorganic nanoparticle-encapsulated complexes, extracellular vesicles, and patient-derived dendritic or mesenchymal stem cells, allowing delivery even across the blood-brain barrier (1).

	Delivery technologies for intravenous, subcutaneous, and intramuscular administration are largely focused on preventing RNA-based therapies from degrading in the blood stream. For mRNAs administered in this manner, lipid nanoparticle solutions appear to be most common. Other delivery technologies are designed to enable local administration of mRNAs to the target site or organ, according to Tiest. “For cancers,” he explains, “intratumoral injection of mRNA offers advantages in safety (very local effect), dosage (no need to saturate circulation and reach the target), and easier combination with other existing therapies.”

	Lipid-based nanoparticles have also been used for siRNA-based drugs, including some that have been approved by regulatory authorities. N-Acetylgalactosamine (GalNAc)-mediated delivery of chemically modified siRNAs has been demonstrated to result in significant delivery and silencing of genes within the hepatocytes of the liver and has also resulted in an approved product.

	Dicerna’s GalXC platform is a leading example. “This approach uses a unique, proprietary, and rationally designed tetraloop configuration of double-stranded RNA molecules that interfaces effectively with the RNAi process and allows us to easily modify a compound’s chemical structure to maximize stability and potency,” Fambrough explains. He adds that it offers the potential for more convenient administration, infrequent dosing, high specificity, a high therapeutic index, and scalable, straightforward manufacturing. So far, the company has focused on treating diseases of the liver, but is now exploring the use of its GalXC platform across different organs, systems, and rare and common diseases.

	Sirnaomics uses a polypeptide nanoparticle consisting of a histidine-lysine branched-chain polypeptide (HKP). HKP mixed with siRNAs spontaneously forms nanoparticles due to the charge-charge interaction between the negative phosphates on the siRNAs and the positively charged lysines, Evans notes. An advantage of this technology over some other delivery mechanisms, he says, is its ability to deliver multiple siRNAs to a cell at the same time, allowing synergistic effects of two siRNAs to be utilized as a therapeutic.

	HKP-based nanoparticles can also accumulate near tumors, possibly mediated by the enhanced permeability and retention (EPR) effect, according to Evans. They further benefit from the presence of histidines, because these moieties have a pKa of approximately 6.3 and become protonated during acidification of the endosomes that are intermediary in the uptake of siRNAs by the nanoparticles. “This protonation helps release the siRNA payload into the cytoplasm where the siRNAs function to silence targeted genes,” he observes.

	A third challenge has been regulation of mRNA activity once it is delivered to the right cells, which is needed to ensure the right dose of the targeted therapeutic protein is expressed. A research team at the Massachusetts Institute of Technology developed one possible solution: incorporation of additional genes for RNA-binding proteins (2).

	The interaction between the mRNA and RNA-binding proteins is controlled using a small-molecule drug, and thus dosing of the small-molecule drug regulates the mRNA activity.

	Inhibitory RNA approaches are most advanced, while mRNA therapies are at earlier stages, with some vaccines in Phase II trials and candidates using mRNA for 

 expression of antibodies that target a specific therapeutic effect, according to Tiest.

	The two RNAi therapies that have received approval to date were both developed by Alnylam Pharmaceuticals. Onpattro (patisiran) was granted approval by FDA in August 2018 for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis (3). It has received marketing authorization from the European Medicines Agency. This drug halts the production of faulty transrethrytin.

	In November 2019, Alnylam received FDA approval for Givlaari (givosiran) for the treatment of adults with the rare genetic disease acute hepatic porphyria (4). This RNAi therapy targets aminolevulinic acid synthase 1.

	The three companies interviewed for this article alone have several RNA-based therapies in development, but they represent only a small portion of candidates moving through clinical development.

	eTheRNA is mainly active in oncology, with both vaccines against tumor targets and intratumoral injection of mRNA expressing targets involved in cell death and immune stimulation in its portfolio that leverage propriety LNP and immune stimulation (Trimix) technologies. Currently, the company has an ongoing Phase I/II program in melanoma and breast cancer. It also recently entered the field of infectious diseases and is working on a cross-protective coronavirus vaccine based on T-cell response that is administered intranasally, according to Tiest.

	Sirnaomics has developed a combination of two siRNAs targeting TGFbeta 1 and Cox2 within an HKP nanoparticle that it has formulated for intratumoral delivery (STP705 in Phase IIs in non-melanoma skin cancer) and intravenous administration (STP707 for delivery to key liver cells for fibrosis treatment; it also has demonstrated in animal models to mitigate hepatocellular carcinoma [HCC]). STP707 has further been shown to increase the activity and improve the efficacy of anti-PDL1 antibodies against HCC, according to Evans.

	The company is also working on chimeric siRNAs that combine a small-molecule therapeutic into the structure of the siRNA. “Once inside the tumor cell, the small molecule is released from the sense strand where it is designed to augment the activity of the siRNA inhibitor and produce a synergistic effect,” Evans says. The activity of these constructs has been demonstrated against a number of tumor types including pancreatic tumors, colon cancers, bladder cancer, and head and neck cancers.

	Dicerna is leveraging its GalXC technology on its own and through several collaborations. The company’s lead candidate, nedosiran, is in development to treat primary hyperoxaluria (PH), an ultra-rare and devastating condition that causes calcium oxalate crystals to form in the kidneys and often results in kidney failure. Nedosiran silences the lactate dehydrogenase enzyme, which is implicated in all three types of PH. It is currently being evaluated in the PHYOX clinical development program, which includes an ongoing registrational trial.

	Two other top candidates include an siRNA therapy to treat alpha-1 antitrypsin-associated liver disease (Phase I/II) and RG6346 for the treatment of hepatitis B virus infection (Phase I), which is being pursued in collaboration with Roche. “Between Dicerna and our collaborative partners, we currently have more than 20 active discovery, preclinical, or clinical programs focused on rare, cardiovascular, cardiometabolic, viral, chronic liver, and complement-mediated diseases, as well as neurodegeneration and pain,” Fambrough notes.

	Both mRNA and siRNA therapeutics and vaccines are advancing through the clinic, with a steady stream of readouts expected in the next few years. “Apart from clinical data, these results should validate a number of delivery and chemical modification platforms that can then open up the field into an increased development of RNA-based therapeutics,” Tiest states.

	Over the next year, Evans expects an influx of additional siRNAs targeting liver diseases and being targeted to the hepatocytes using GalNAc conjugates because this approach has already demonstrated success. “Efforts will also be focused on additional chemically modified siRNAs with different targeting ligands that enable delivery to tissues and cells outside the liver, with the biggest obstacle being avoidance of uptake by the liver for any and all systemically delivered siRNAs,” observes Evans.

	Fambrough agrees that the next frontier for RNAi therapies will be non-hepatic tissues. “There is significant interest and persistent unmet need across a range of disorders,” he says.

	Going forward, the key advantages of RNA-based therapies compared to DNA-based gene therapies will continue to drive greater interest in all types of RNA treatments. “RNA combines the advantages and versatility of genetic-based therapy modalities with the lack of (perceived) risks associated with DNA-based gene therapy. Technology has now reached a level of maturity, particularly around stabilization and delivery of RNA, and the upcoming flow of clinical data could enable the break-through of this technology onto the main stage of drug development,” comments Tiest.

	Looking specifically at RNAi therapies, Fambrough notes that while the technology is a newer approach to treating disease, it has experienced a “coming of age” in the past couple of years. “We have seen not only the first approvals in the space, but also a surge of interest among Big Pharma and big biotech companies seeking an entry into RNAi,” he says.

., 9:1208 (2019).
	2. MIT, “This RNA-Based Technique Could Make Gene Therapy More Effective,” Press Release, Oct. 16, 2018.
	3. Alnylam Pharmaceuticals, “Alnylam Announces First-Ever FDA Approval of an RNAi Therapeutic, ONPATTRO (patisiran) for the Treatment of the Polyneuropathy of Hereditary Transthyretin-Mediated Amyloidosis in Adults,” Press Release, Aug. 10, 2018.
	4. FDA, “FDA Approves Givosiran for Acute Hepatic Porphyria,” Press Release, Nov. 20, 2019.


	Vol. 33, No. 7
	July 2020
	Pages: 13–16

	When referring to this article, please cite it as C. Challener, "Can Using RNA Simplify Gene Therapy Development?” 

Articles

RNA is easier to manipulate than DNA but challenging to deliver to the right cells.

Can Using RNA Simplify Gene Therapy Development?

Residual impurity testing is essential to ensure the quality and safety of pharmaceutical products, but it can be particularly challenging with biologics because there are so many potential sources of impurities, from the media and host cells to misfolded or aggregated product to downstream purification reagents and process equipment. A risk-based approach to identifying potential residual impurities is important for understanding any risks to the product quality and potentially the patient. It also allows the development of an optimal process with a suitable control strategy and test methods in a timely fashion.

	The implementation of a risk-based strategy for the testing of residual impurities, meanwhile, offers the potential to significantly reduce the amount of testing needed while still meeting regulatory requirements and ensuring patient safety. It does require, however, a thorough understanding of the process combined with appropriate control strategies to minimize the impurities that may be present at the end of the production process (drug substance and drug product). While building a knowledge base on residual impurities requires investment, that investment pays off in the long term because it enables risk-based residual impurity control and minimizes the need for residual impurity testing.

	Residual impurities in biopharmaceutical processes can be traced back to raw materials or can form during upstream or downstream processing steps and upon storage. They can be derived from the host cell or the product itself, media components, surfactants, virus inactivation agents, inorganic or organic contaminants from permanent and single-use process equipment, the pharmaceutical formulation, and/or a breach of sterility. “A strategy for the control of residual impurities has to consider risk factors that are specific to each of these process- or product-related impurities,” asserts Michael Jahn, group head of forensic chemistry at Lonza.

	This quantity and heterogeneity of possible impurities is perhaps the single biggest factor influencing the development of risk-based strategies for the testing of residual impurities, adds Ashleigh Wake, UK chemicals and pharmaceuticals business director for Intertek.

	“Some residual impurities, such as antibiotics and stabilizers, can be relatively easily defined and a strategy put in place that may include a limit specification in a release test or validating the removal during product purification. Others are more complex to define, such as those that originate from the cellular system including residual DNA or host-cell proteins (HCPs),” Wake observes. “In the latter case, for instance, consideration must be made for total HCP levels per regulatory guidance, but also for any single HCP of particular concern, for which a more specific monitoring approach would need to be developed.”

	For some residual impurities such as HCPs, safety thresholds (at approximately 1–100 ppm) are well established, whereas quality thresholds regarding, for example, the presence of cell-derived lipases that potentially can degrade the excipient polysorbate with detrimental consequences-visual particle formation or loss of protective activity against interfacial stress on the active protein-are not established yet, according to Jahn. Leachables, meanwhile, are typically controlled in a risk-based fashion with high-risk materials assessed in laboratory studies, he notes. “A common basis for the establishment of risk factors is an in-depth understanding of the underlying processes,” Jahn summarizes.

	Other factors relate to the testing process itself, according to Lisa Sapp, biopharma market manager with Agilent Technologies. The selected isolation method-precipitation, filtration, centrifugation, extraction, etc.-should not affect the impurities with respect to concentration or composition. If derivatization is necessary to improve the sensitivity and detectability of the analyte in question, any loss in recovery must be accounted for. Filtration of samples must not cause any interference either. The chosen separation and detection technologies must also be suitable to the analysis without degradation of the impurities occurring.

	Overall, potential process-related and other residual impurities need to be evaluated with regard to their criticality and their potential to pose any risk to patient safety, asserts Stefan Braun, regulatory affairs manager with Rentschler Biopharma. “The risk assessment should aim at identifying and assessing those molecules where an element of risk may remain even at very low concentration, with potential risk evaluated in a multi-stage approach,” he notes.

	The best solution to managing risk factors is to use quality by design (QbD) to classify them, applying tools such as Ishikawa fishbone diagrams or failure mode and effects analysis (FMEA), agrees Luc-Alain Savoy, global head of biologics at SGS.

	“Critical quality attributes will be identified during this risk-assessment step and their criticality addressed from a safety perspective. Efficient purification processes will be designed and validated to remove the residual impurities. Monitoring the efficiency of the residual impurities will be guaranteed through the development of robust analytical methods,” he explains. Savoy also recommends that methods be developed following an analytical QbD approach.

	Specific factors to assess for possible impurities include requirements established in regulatory guidelines, acute and chronic toxicity, risk of co-purification, and natural occurrence in humans, according to Braun. If one or more of these factors is deemed critical, the potential impurity should be further evaluated in the next step of the risk assessment.

	For process-related impurities, Braun notes that as long as the theoretical amount of the substance in the drug substance is below the toxicologically relevant limit (acceptable level), the substance is rated as uncritical and analytical testing in the drug substance is not recommended. If the theoretical amount in drug substance exceeds the acceptable level, the molecule is recommended for testing on drug substance level. Depletion of such molecules in the final process design should, however, be confirmed during process characterization and process validation.

	There are several relevant regulations that address the identification and evaluation of residual impurities. The International Council for Harmonization (ICH) Q6B guideline (1) provides general information on setting and justifying release and in-process specifications for both process and product-related impurities in drug substances and how the suitability of any method should be addressed within the guidances outlined in ICH Q2 (R1) (2). European Pharmacopoeia monograph <0784> (3) and United States Pharmacopeia General Chapter <1132> (4) provide specific guidance on the control of host-cell-derived process-related impurities using appropriate risk-management strategies. The ICH Q9 guideline (5) discusses a risk-based approach for determining the testing requirements for monitoring the removal of reagents from processes; ICH Q3C (R6) (6) addresses residual solvents; and ICH Q3D (7) covers elemental impurities.

	At this time, one area that is not currently regulated in detail by the health authorities is extractables and leachables. “Biopharmaceutical manufacturers have to establish ‘in-house’ risk-based strategies, with the disadvantage of insecurity with regard to health authority expectations, but with the advantage of the possibility to apply process knowledge and extractables/leachables subject-matter expertise beyond a generic regulatory framework,” Jahn explains.

	Risked-based strategies work well when the process is well-defined, that is, specified impurities are analyzed and meet the specifications set forth. Issues arise, says Tilak Chandrasekaran, biopharma marketing manager for Agilent Technologies, when an unspecified impurity appears and is present at a low level. “This situation often happens if the starting material or reagents are from a different supplier and not fully characterized, and tracking the root cause for these impurities can become a time-consuming and costly process,” he explains.

	Such situations can also arise if documented knowledge from early development stages is not maintained as projects progress through the development cycle, according to Chandrasekaran. “Although full CMC [chemistry, manufacturing, and controls] information is not required until the market application dossier is submitted, a useful strategy is to prepare a comprehensive report that details the origin, genetic construct, cloning, and cell substrate preparation. Such a document should be reviewed regularly and serve as a repository for information as the project moves from early- to late-stage development,” he states.

	Finding suitable evaluation criteria that take into account the large number of different types of process-related impurities and defining a mathematically and scientifically sound way for determining toxicologically relevant limits for critical impurities can also be a challenge, according to Aline Denzel, quality control manager at Rentschler Biopharma. Furthermore, from a contract development and manufacturing organization’s (CDMO’s) perspective, she notes that the developed concepts should be applicable to many different customer projects.

	Therefore, Denzel says all raw materials used at a manufacturing site should be evaluated initially to define potential critical process-related impurities. A toxicological assessment with a relevant permitted daily exposure (PDE) value should be compiled by a registered toxicologist if required. She also notes that most PDE values are calculated based on the body weight of an adult, so application of the concept for pediatric products must be considered separately, as must different routes of administration.

	One difficulty in evaluating media and feeds, however, is the tendency of suppliers of these materials to not fully share the compositions of their proprietary products with their customers. “Other than getting suppliers to be more transparent, the best approach is to have the supplier provide a positive list of all potential raw materials and media components for the customer to use in worst-case models. The manufacturer must then agree that the toxicological limits established by the customer will not be exceeded and the media/feed does not contain any other critical substances,” Braun says.

	From a physical perspective, one of the greatest challenges to residual impurity testing relates to the difficulties in accurately measuring compounds of interest within sample matrices, particularly at the low levels observed for most residual impurities. Chandrasekaran notes that most conventional detectors lack the sensitivity to accurately measure residual impurities in process fluids. “Developing a non-denaturing method is critical to avoid inaccurate quantification.” Often the methodologies required to achieve the specification limits are more challenging to develop and validate and require the application of a wider range of technologies that are not traditionally employed for batch release, Wake adds.

	Accurately detecting impurities when performing cleaning verification is also difficult because the levels are very low, says Chandrasekaran. “The method must be sufficiently sensitive or cross-contamination can occur, which can be a safety issue, particularly with HCPs, which are known to have an immunogenic response at low levels,” he comments.

	On a company level, there may be reluctance within regulatory and quality departments to change from well-established testing strategies to a more-risk-based approach, according to Jahn. The availability of and reference to industry white papers might be convincing, he says. As an example, he points to the BioPhorum Operations Group (BPOG) and its guide for the assessment of leachables from process materials, which presents an outline for a material risk evaluation. “The numerical scoring and weighing can be used as a template for the risk categorization of the many materials that are applied in a modern single-use biopharmaceutical production suite, which cannot be assessed by individual laboratory testing,” he says.

	The most important element for the establishment of a successful risk-based strategy for residual impurity testing is the knowledge space, Jahn states. For example, he notes that if a platform drug substance downstream process has shown for different platform molecules that a supplement added during fermentation can be depleted to a satisfactory level, it might be decided that the depletion is not explicitly tested for the next platform molecule. “In the absence of the knowledge space, however, neither risk factors, nor regulatory requirements and challenges, can be appropriately addressed without extensive testing,” observes Jahn.

	Indeed, the fundamental risk with any impurity is determining if it is present and if it is, what the specification for that impurity should be, Wake says. “With process-related impurities, such as surfactants and antibodies, this is a relatively definable process and regulatory guidance is hugely relevant. The lack of safety profiles for product-related species makes the process more difficult, and hence there is a greater risk to define them and effectively specifications become more risk loaded,” she observes.

	This risk must be evaluated and defined for these impurities on a case-by-case basis. “The real challenge becomes, therefore, the ability to define ‘safe limits’ and then develop and validate a suite of methods that covers all of these species,” Wake concludes.

	Knowing all of the production and raw materials that can contribute to residual impurities becomes very important, therefore, and a key element of effective risk-based strategies, according to Braun. Sapp adds that understanding the impact of raw or starting materials is key, and selecting only those materials or batches that are fit for purpose will minimize the impact of impurities formed during production.

	“In addition, potential process-related impurities can be quickly identified and evaluated and, if testing becomes necessary, an appropriate analytical method can be developed,” Braun observes. In this context, he also notes that close contact with a registered toxicologist is an advantage for quickly determining PDE values for critical process-related impurities.

	This knowledge also allows for weighting of the risk by the probability of occurrence for those residual impurities qualified as critical, according to Savoy. As an example, he points to an antibiotic that if present as a residual impurity at certain levels would constitute a potential risk for the patient’s safety, but that if added during upstream processing and prior to all downstream purification steps would be expected to have a negligible residual quantity that would likely be below the detection limits of applicable analytical methods.

	Residual impurity testing for biopharmaceuticals is complicated and time-consuming, even when risk-based strategies are employed. Short cuts should be avoided and will typically only lead to complications and delays, according to Jahn. As an example, he points to the importance of pro-actively justifying why certain testing can be omitted in a risk-based extractables and leachables program without impacting patient safety. “Realizing only at or after a biologics license application submission that the data package is not sufficient might endanger commercialization with a much higher financial impact compared to the costs of a testing regimen,” he says.

	Savoy adds that while previous experience and process understanding should be leveraged when developing risk-based testing strategies, it is equally important to not rely too heavily on previous experience to the point where control or verification steps are skipped.

	It is also essential to avoid taking a “tick-box” approach, according to Wake. “Any program should be bespoke and based on a true understanding of the molecule and what parameters are pertinent to truly assessing the quality, and inherently, the safety of the product. Science should be the base of everything!” she asserts.

	A rigid and inflexible approach to developing a risk-based strategy for residual impurity testing is also to be avoided, says Denzel. “The risk-based strategy should be developed in such a way that it allows the requirements of different clinical phases and projects to be addressed flexibly. The requirements for Phase I and Phase III products are different, and thus the risk assessment is a living document that should be adapted based on the status of a project, even if the process stays the same,” she observes.

	“A lifecycle management system for the risk assessment of process-related impurities should be in place that includes and evaluates relevant changes in regulatory requirements (e.g., changes in guideline PDE values), in the manufacturing process (e.g., changes to raw materials), and in clinical requirements (e.g., changes in the maximum daily dose). The impact of the changes on the analytical approaches must constantly be evaluated,” Denzel continues.

	One final consideration of note, according to Wake, is when to use a limit test or full quantitation. “Limit tests are best suited when there is a really clear specification and do work well in that situation. Full quantitation methods are often more challenging, but there are specific situations in which they are needed,” she says. Analytical limit tests, according to Denzel, are a cost-effective tool to show the absence of process-related impurities in drug substances, while quantitative tests can provide useful information during process characterization and may be employed to develop data-based models reflecting purification factors for well-known process-related impurities.

Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products

, Step 4 version (1999).
	2. ICH, Q2(R1), 

Validation of Analytical Procedures: Text and Methodology

, Products of Recombinant DNA Technology (0784) (Strasborg, France).
	4. 

, USP General Chapter <1132>, “Residual Host Cell Protein Measurement in Biopharmaceuticals,” 

, Step 4 version, (2005).
	6. ICH, Q3C (R6), 

Maintenance of the Guideline for Residual Solvents

, Final version (2016).
	7. ICH, Q3D (R1), 


	Vol. 33, No. 7
	July 2020
	Pages: 22–25

	When referring to this article, please cite it as C. Challener, "Eliminating Residual Impurities Starts with a Strategic Plan,” 

Identifying the source, assessing the risk, and removing residual impurities requires a strategic approach.

Eliminating Residual Impurities Starts with a Strategic Plan

Antibody therapies-such as those derived directly from plasma-or versions based on native antibodies but produced using recombinant technology-aim to provide individuals with a passive immunity to fight a particular disease. These therapies can be used in four ways: to prevent people at high risk, such as healthcare workers and at-risk patients, from ever becoming infected (pre-exposure prophylaxis); to prevent those infected from developing severe symptoms (early treatment); to treat people who are already experiencing severe symptoms (late treatment); and for post-exposure prophylaxis.

	“Whereas vaccines can take many weeks to produce protective antibodies, plasma-derived antibody therapies, like convalescent plasma (CP) or concentrated antibody preparations manufactured from CP (i.e., hyperimmune globulins), provide immediate passive humoral antibody protection. This feature allows plasma-derived antibody therapies to be used prophylactically in cases of exposure, which is vitally important to first responders and healthcare workers on the frontlines, or to treat infected persons who are at a high risk of a poor outcome,” explains David Bell, chief innovation officer for Grifols. “In addition, plasma-derived therapies are especially important to immunocompromised patients who cannot mount a strong immune response to a vaccine.”

	Convalescent plasma, or plasma from recovered patients containing antibodies, can be used as an emergency measure when other treatments are not available. Antibodies isolated from pooled donated plasma have been produced by patient immune systems as a defense to a foreign antigen and therefore possess specificity against that antigen. On the other hand, production of potent antibodies identified in recovered patient blood samples using recombinant technology enables manufacturing scalability and continued supply.

	The present COVID-19 pandemic, as well as the Ebola epidemics in East and West Africa, highlight the desperate need for readily-accessible, safe antiviral therapies that can be produced in a short period of time amidst rapid viral spread, according to Bell. “During an outbreak of a novel coronavirus or other agent, CP is the only antibody source available for near-immediate use,” he explains.

	Although not referred to as CP at the time, this approach-with antibodies from animals-was used by Emil von Behring to cure sick patients with diphtheria, work for which he won the first Nobel Prize in Physiology and Medicine in 1901 (1). CP was later used to dramatically reduce the fatality rate during the influenza pandemic (Spanish flu) of 1918 and during the Korean War to treat United Nations troops in the field that were sick with Korean hemorrhagic fever, also known as hantavirus. More recently, CP has been used to treat severe acute respiratory syndrome (SARS), the 2009 influenza A (H1N1), avian influenza A (H5N1), several hemorrhagic fevers such as Ebola, and other viral infections (2).

	Compared to other methods to produce antibodies (i.e., recombinant monoclonals), CP also has the advantage of possessing a breadth of antibodies to a viral antigen that vary with respect to their epitopes. “This polyclonal nature of CP antibodies allows them, without further manipulation or purification, to exhibit high overall affinity and sensitivity to an antigen,” notes Bell.

	In addition, CP harbors antibodies to a plethora of microbial antagonists to which the CP donor has mounted a prior humoral immune response that can protect against concurrent infections, Bell adds. This property is especially important to CP recipients whose immune systems may be compromised.

	A silver lining of the COVID-19 pandemic, Bell points out, is that many folks have mounted successful humoral responses to the disease, whether severe or asymptomatic, and developed antibodies against SARS-CoV-2, the virus that causes COVID-19. Presently, there are multiple clinical trials underway across the globe to evaluate the potential of CP to prevent or mitigate the severity of illness in recipient COVID-19 patients, and results are beginning to be published. In a timely report from investigators in China, CP therapy was found to be well-tolerated and to potentially improve the clinical outcomes in severe cases by neutralizing viremia (3).

	In the United States, FDA is facilitating access to COVID-19 convalescent plasma for use in patients with serious or immediately life-threatening COVID-19 infections through emergency Investigational New Drug Applications, working in collaboration with the National Institutes of Health and the Centers for Disease Control and Prevention to develop master protocols for the collections and use of COVID-19 CP, and encouraging people who have recovered from COVID-19 infections to donate plasma (4,5). The first CP treatment approved by FDA took place at Houston Methodist Hospital in late March (6). Grifols is currently conducting a clinical trial in Spain with convalescent human plasma inactivated with methylene blue.

	Ultimately, any efficacy that CP exhibits in initial clinical trials will provide an impetus to use CP to manufacture highly-purified preparations of antibodies with high neutralizing activity and longer-term potential therapy (7), according to Bell. “So-called hyperimmune globulin (H-IG) preparations, manufactured from convalescent or immunized donors’ plasma, comprise a widely tested, safe, and proven prophylactic and therapeutic intervention for certain infectious diseases [e.g., measles, diphtheria, polio, hepatitis A and B, tetanus, rabies, and disease brought on by respiratory syncytial virus (8)],” Bell states.

	CP donated from many individuals is transported to manufacturing facilities where it is pooled together. During the manufacturing process, the virus-specific antibodies from multiple donors are concentrated. The plasma then undergoes virus inactivation and removal processes and is finally purified into the H-IG therapy with a consistent amount of neutralizing antibodies in each dose.

	“This approach ensures the quality and safety of the final product, as well as provides a more realistic shelf-life for global use. H-IG therapy means a higher concentration of antibodies can be administered with lower volumes. The high concentration of antibodies, which has a known neutralization titer, will have an established benefit-risk profile in multiple populations of patients,” says Chris Morabito, head of R&D, plasma-derived therapies at Takeda.

	A key benefit of a plasma-derived immune globulin therapy is that the production of the H-IG is based on already-approved manufacturing systems, according to Morabito. Plasma-derived intravenous immunoglobulin (IVIG) therapy also has an established safety profile based on years of clinical evidence. “In addition,” Morabito notes, “a plasma-derived polyclonal immune globulin therapy can bind multiple antigens on the virus, including the ones that have been shown to be effective in clearing the infection from the recovered donor, whereas a monoclonal approach covers one single antigen.”

	And while a direct plasma approach may be quicker to develop than an H-IG therapy, before the former method can be put into wide use, scientists must work out safety issues, such as making sure the plasma taken from recovered patients is free of other viruses and toxins. The antibody levels also vary significantly from individual to individual, so the potential effectiveness of the convalescent plasma is dependent on the specific donor. Furthermore, larger volume dosing is likely to be required as the virus-specific antibodies in the CP would not be concentrated through a manufacturing process.

	Plasma-derived hyperimmune globulin therapies have shown preliminary signs of effectiveness in the treatment of severe acute respiratory infections of viral etiology and may present a potential treatment option for individuals with COVID-19, as well to potentially prevent infection in those at high risk of developing COVID-19 from exposure to SARS-CoV-2, such as healthcare workers, according to Morabito notes.

	Takeda is leveraging its experience in the development of plasma-based antibody therapies in the fight against COVID-19. The company successfully produced an H-IG to address the 2009 H1N1 influenza pandemic and manufactured two full-scale lots using its novel Gammagard Liquid (GGL) IG process from approximately 15,000 L of CP, resulting in approximately 50 Kg of hyperimmune globulin product. “GGL has a proven track record of safety and efficacy for 16 years and is available in 39 countries (marketed as Kiovig outside of US and Canada),” Morabito comments.

	Recently, Takeda announced that in order to accelerate development of an H-IG treatment, it is partnering with other plasma therapy developers (CSL Behring, Biotest, Bio Products Laboratory, Octapharma, and LFB) in the United Kingdom, Switzerland, Germany, and France to develop a single, nonbranded product. “We believe that by collaborating on plasma collection, clinical trials, and manufacturing, the CoVIg-19 Plasma Alliance can accelerate bringing a potential therapy to market and increase the potential supply,” states Morabito.

	CoVIg-19, a polyclonal H-IG, is an investigational treatment prepared from the pooled plasma of individuals who have fully recovered from COVID-19 and whose blood contains high titers of antibodies that can fight SARS-CoV-2, the virus that causes COVID-19.â¯ This plasma-derived therapy has the potential to treat COVID-19 patients and to possibly prevent infection in frontline healthcare workers.

	“The CoVIg-19 Plasma Alliance is collaborating closely with regulatory agencies, governments, and healthcare partners to advance this program. If successful, it is anticipated that this potential therapy could be available within nine to 18 months from the time work was initiated in January 2020,” Morabito says.

	Grifols is another company leveraging experience in the development of H-IG therapies for the fight against SARS-CoV-2. Specifically, Grifols has developed a platform to produce hyperimmune globulin from disease-state CP in an isolated, dedicated manufacturing facility, which has been used to successfully produce a purified, concentrated intravenous immune globulin preparation enriched with antibodies to Ebola virus.

	The company is currently collaborating with FDA, the US Biomedical Advanced Research Development Authority (BARDA), and other federal health agencies for collection of CP using its network of FDA-approved plasma donor centers. Grifols is manufacturing hyperimmune globulins in its purpose-built Clayton, NC facility and conducting preclinical and clinical trials. The company also is participating in discussions for the potential development of a clinical trial in IVIG, according to Bell.

	Emergent BioSolutions, which has extensive experience with the development and licensure of six FDA-approved hyperimmune products, also initiated the development of two hyperimmune treatments for COVID-19. COVID-Human Immune Globulin (COVID-HIG), manufactured from human plasma with antibodies to SARS-CoV-2, is being developed as a potential treatment for COVID-19 in severe hospitalized patients and high-risk patients; COVID-EIG, manufactured from plasma of immunized horses with antibodies to SARS-CoV-2, is a potential treatment for hospitalized patients with severe symptoms. The company’s goal is begin a clinical trial as early as the summer of 2020.

	Emergent is collaborating with the US government to expedite the development of COVID-HIG, receiving funding from BARDA and participating in a study with the National Institute of Allergy and Infectious Diseases (NAID), part of the National Institutes of Health (NIH), to assess potential treatments for COVID-19.

	“We are expanding our partnership with the US government working closely with BARDA, NIH/NIAID, FDA, and other key agencies to stay aligned on our development goals and near-term response plan,” said Laura Saward, senior vice-president and therapeutics business unit head at Emergent BioSolutions. “COVID-HIG will leverage the platform that was established in partnership with BARDA through their investment in our treatments for anthrax and smallpox vaccine complications, and it provides a sustainable capability for responding to emerging infectious diseases such as COVID-19.”

	The technology Emergent is employing for the COVID-19 therapies has been used by the company to develop other FDA-approved plasma-based treatments for different diseases. “As we develop these new drugs, we will be able to leverage many of the same elements around the safety and [pharmacokinetic] PK profiles, as well as the validated manufacturing controls and assays,” says Saward.

	Recombinant antibody therapies based on antibodies generated in recovered patients can be either polyclonal or monoclonal. Due to their recombinant nature, a single donor can be used to generate millions of doses, according to David Johnson, CEO and co-founder of GigaGen, a biotechnology company developing a recombinant polyclonal antibody therapy.

	“Administering therapeutic antibodies that target and neutralize the virus can provide protection much faster than vaccines and is a safer, more effective, less variable, and scalable treatment approach than convalescent serum,” adds Carl Hansen, cofounder and CEO of AbCellera, a biotechnology company developing a monoclonal antibody therapy.

	In addition, the specific antibodies against COVID-19 are not selected for in serum- or plasma-derived therapies. “In the collected serum, there are antibodies against all types of diseases, and those diseases might have been ones that the patients were exposed to months ago and not necessarily coronavirus,” Johnson observes. He goes on to add that there is also greater batch-to-batch consistency with recombinant therapies.

	One challenge for some recombinant therapies is the need to select clones with a cost-effective production yield. It is also necessary for all recombinant therapies to develop robust upstream and downstream processes yielding products that fulfill the required specifications for human use from the point of view of purity, identity, and potency, according to Bell.

	GigaGen is developing the recombinant polyclonal therapy rCIG (recombinant anti-coronavirus 19 hyperimmune gammaglobulin), which according to Johnson, combines the advantages of a polyclonal approach and a recombinant approach.

	“Polyclonal antibody therapies have the advantage of ‘polyclonality’; they contain many different antibodies. Unlike monoclonal antibody (mAb) therapies, which focus on a single antibody and thus only target the virus at one specific location, polyclonal antibody therapies have a broader scope,” Johnson states. He adds that because they provide patients with a diverse and comprehensive library of antibodies, the chances of efficacy are increased.

	“That comprehensive set of antibodies coats the virus at various locations, making it difficult for the virus to attach to the human cell, and ultimately stopping disease progression,” Johnson explains. The company has shown that recombinant polyclonals can have much higher potency than other therapeutic options, which may result in better clinical outcomes.

	“The primary indication of our polyclonal antibody therapy is for people actively suffering from COVID-19. We want to help infected patients survive the worst part of the disease,” Johnson comments. In many cases, he adds, if a patient gets through the worst, they will retain immunity through their own immune system, so they wouldn’t need more of GigaGen’s drug. If the patient is generally immune incompetent, however, they would need follow-on doses as a prophylactic against re-infection.

	GigaGen is using its proprietary Surge single-cell technology to capture and recreate complete libraries of antibodies from COVID-19 convalescent patients. Blood is collected from COVID-19 convalescent patients and their B-cell repertoires (antibody producing cells) are used to replicate their natural diversity of antibodies at high concentrations. Specifically, antibody-coding DNA is captured from millions of B cells and used to engineer a library of antibodies into cell lines that produce the drug.

	“Our plan is to use antibodies from the best 5–10 donors/responders. Not all patients respond the same way. Our technology enables us to select the best donors and replicate their antibody repertoires in the laboratory to generate a drug that can treat millions of patients,” Johnson asserts.

	The biggest challenge for GigaGen going forward at this point is securing sufficient funding to scale production. The tens of thousands of clones making different antibodies will need to be produced at larger scale. Toxicology studies in monkeys also need to be performed before GigaGen can move into clinical trials. “We expect to do tech transfer to a manufacturing facility sometime this summer [2020] for GMP [good manufacturing practice] production, with plans to start first clinical doses at the beginning of 2021, dependent on our conversations with FDA,” says Johnson. He notes that it would be helpful to receive clearer information from FDA regarding current policies related to expedited review and expedited paths to the clinic.

	AbCellera has elected to develop a mAb-based therapy for COVID-19. The problem, says Hansen, is that the human body makes billions of unique antibodies, each unique type secreted by a single antibody-producing immune B cell. The challenge is to find the few cells among billions that make the very best antibodies for fighting SARS-CoV-2. “We’ve adapted our platform for exactly this scenario. Through our participation in the Defense Advanced Research Projects Agency’s P3 [Pandemic Prevention Platform] program, we’ve successfully tested our platform against MERS coronavirus and pandemic flu,” he remarks.

	AbCellera’s drug discovery platform combines high-throughput microfluidics, big data, artificial intelligence, machine learning, and genomics to search and analyze the immune system to find the best antibodies against a disease or virus. “We can screen millions of immune cells (B-cells) in days-not weeks or months-to quickly find the best antibodies for fighting diseases,” Hansen states.

	A microfluidic device approximately the size of a credit card possesses arrays of more than 200,000 nanoliter-volume reaction chambers where individual B-cells are isolated, each secreting a unique antibody. The antibodies in each chamber are tested to determine their ability to bind the spike protein on SARS-CoV-2 and identified using machine vision. Celium, AbCellera’s proprietary antibody visualization software, is applied to narrow the thousands of antibodies that can bind the target down to the best antibody candidates.

	“Because we don’t need to create cell lines or clones and we can search the natural immune system so thoroughly, we can quickly and cost-effectively find thousands of diverse antibody drug candidates. The speed, depth, and integration of our technology has redefined how antibody drugs are discovered,” Hansen asserts. Indeed, the company was able to go from a recovered patient sample to a group of 24 lead drug candidates in just three weeks, a process Hansen indicates could normally take a decade.

	Being able to deeply and rapidly search the diversity of the natural immune system also affords AbCellera the best chance of finding the most potent, neutralizing antibody candidates. For very potent antibodies, the amount of antibody needed for dose is reduced, which is one of the most effective ways to alleviate some of the manufacturing challenges, according to Hansen. “Antibodies from natural immune systems also require less engineering to become a drug, which reduces safety risk and time to clinical trials,” he adds.

	To manufacture at the speed and scale needed for the COVID-19 pandemic, AbCellera has partnered with Eli Lilly, which has global capabilities for rapid development, manufacturing, and distribution of therapeutic antibodies. Lilly has already started GMP manufacturing of an antibody drug candidate that has demonstrated potent neutralization of SARS-CoV-2 and expects to begin clinical trials in July 2020.

	Vir Biotechnology is also producing potent neutralizing human mAbs against SARS-CoV-2. The company’s leading candidates VIR-7831 and VIR-7832 have demonstrated high affinity for the SARS-CoV-2 spike protein and are highly potent in neutralizing SARS-CoV-2 in live virus-cellular assays. They were isolated from SARS-CoV-1 recovered patients using its proprietary platform technology, which identifies rare antibodies from survivors that have the potential to treat and prevent rapidly evolving and/or previously untreatable pathogens via direct pathogen neutralization and immune system stimulation

	Vir has signed agreements with both GlaxoSmithKline (GSK) (9) and Generation Bio (10) to accelerate development of solutions for coronaviruses, including SARS-CoV-2. The collaboration with GSK will use Vir’s proprietary mAb platform technology to accelerate existing and identify new anti-viral antibodies that could be used as therapeutic or preventative options against COVID-19 and GSK’s expertise in functional genomics, as well as both companies’ clustered regularly interspaced short palindromic repeats (CRISPR) screening and artificial intelligence capabilities (9). The collaboration with Generation Bio is intended to extend the impact and reach of Vir’s mAbs against SARS-CoV-2 using Generation Bio’s proprietary ceDNA non-viral gene therapy platform and scalable manufacturing process (10).

	Vir has also signed a $363-million agreement with Samsung Biologics for the large-scale manufacturing of its two COVID-19 mAb candidates (11). Production is expected to begin as early as October 2020. This deal follows agreements Vir signed in February with WuXi Biologics and mid-March with Biogen to scale up cell-line development, process development, and clinical manufacturing for its mAbs. Vir is hoping to move VIR-7831 and VIR-7832 into Phase II clinical testing within the next three to five months.

	Rather than start only with antibodies isolated from plasma obtained from recovered COVID-19 patients, Regeneron is using a two-pronged approach that also includes generation of hundreds of neutralizing antibodies against SARS-CoV-2 obtained from humanized mouse models exposed to the virus (12).

	The company previously used its proprietary VelociSuite technologies, including VelocImmune mice that have been genetically-modified to have a human immune system, to rapidly develop a promising mAb triplet therapy for Ebola, moving into clinical trials just four months after the August 2018 outbreak. REGN-EB3 is currently under review by FDA (13). Regeneron is developing a two-mAb candidate therapy (REGN3048-3051) against MERS-CoV using the same technology.

	Isolation of the hundreds of virus-neutralizing, fully human antibodies from the mice took just one month (12). Regeneron has also isolated antibodies from humans who have recovered from COVID-19 to maximize the pool of potentially potent antibodies. Both sets of antibodies have been screened, and Regeneron has selected a small number to move into cell line production.

	Each of the mAbs ultimately selected for a combination treatment will elicit a robust response but target slightly different parts of the virus to provide protection against multiple viral variants. Regeneron is using its VelociMab technology to prepare manufacturing-ready cell lines as lead antibodies are selected, so that clinical-scale production can begin immediately (12). In mid-April, the company moved the most potent antibodies into pre-clinical and clinical-scale cell production lines (13).

	The cocktail could potentially be used as a prophylactic for at-risk people or as a treatment for people who are already infected, according to Regeneron (12). “Our three decades of investment in our VelociSuite antibody technologies, which accelerate and improve the traditional drug discovery process, have hopefully prepared us for this critical time and to meet this important challenge,” said George D. Yancopoulos, co-founder, president, and chief scientific officer of Regeneron, in a press release (12).

	Regeneron is on track to begin human clinical trials in June 2020 and plans to scale production to have hundreds of thousands of lower prophylactic doses or tens of thousands of treatment doses per month by the end of August 2020 (14). Regeneron is also working with BARDA and other organizations to further increase manufacturing capacity (12).

	For development of antibody-based therapies for COVID-19 disease, the main challenge that must be overcome is the current limited knowledge and understanding about the virus infectivity process itself and the disease caused by it, according to Bell. “There are still many uncertainties that complicate identifying and validating the right therapeutic target that could also be dependent on the timing of the infection process, so, there could be a target for the initial steps of the infection process and a therapeutic target for posterior phases where the main objective is to treat the clinical symptoms,” he says.

	Understanding the biology of the disease, Bell believes, will lead to identifying what proteins or processes are the optimal to be targeted with a therapy. Then, the challenge will be to develop a targeted mAb product that has the needed potency and selectivity in order to have an adequate efficacy and safety profile.

	As importantly, Johnson stresses that multiple health systems need prophylactics (i.e., active vaccines, passive vaccines) and therapeutics. “Vaccines are very cheap and often help 90% or so of the population. Passive vaccines help people with immune deficiencies, for example people who don’t respond to the active vaccine, or people at high risk (nurses in emergency rooms). Therapeutics help people who get sick despite the availability of both active and passive vaccines. It is important that a variety of treatment options be developed so that we have multiple avenues to target this virus,” he explains.

	Bell adds that there is even precedent for concomitant administration of hyperimmune globulin (H-IG) and vaccine in order to ensure protection while vaccine-generated antibodies increase in titer. Others have proposed the combined use of convalescent plasma and H-IG in a complementary fashion to treat patients infected with SARS-CoV-2, the virus that causes COVID-19, both presently and in subsequent infectious waves (15). In the absence of a vaccine, though, as is the case with the current COVID-19 pandemic, Bell stresses that plasma-derived antibody therapies can bridge the early stages of an outbreak until the time one is available.

	1. D. Roos, “Before Vaccines, Doctors ‘Borrowed’ Antibodies from Recovered Patients to Save Lives,” 

, March 31, 2020.
	2. A. Casadevall and L.Pirofski, 

. Online, 10.1172/JCI138003 (March 13, 2020).
	3. K. Duan, et al. 

 117 (17) 9490-9496 (April 28, 2020).
	4. FDA, “

Coronavirus (COVID-19) Update: FDA Coordinates National Effort to Develop Blood-Related Therapies for COVID-19

,” Press Release, April 3, 2020.
	5. FDA, “

Coronavirus (COVID-19) Update: FDA Encourages Recovered Patients to Donate Plasma for Development of Blood-Related Therapies

,” Press Release, April 16, 2020.
	6. Houston Methodist Hospital, “FDA Approves First Plasma Therapy for Houston Methodist COVID-19 Patient,” Press Release, March 30, 2020.
	7. S. Jolles, W.A. Sewell, S.A. Misbah, 

 142:1–11 (2005).
	8. J.L. HsU, N. Safdar, 

North Am 25:773–88 (2011).
	9. Vir Biotechnology, “GSK and Vir Biotechnology Enter Collaboration to Find Coronavirus Solutions,” Press Release, April 6, 2020.
	10. Generation Bio, “Generation Bio and Vir Biotechnology to Collaborate on Research to Leverage Scalable Non-Viral Gene Therapy Platform for Durable Production of Monoclonal Antibodies Against Coronavirus That Causes COVID-19,” Press Release, March 30, 2020.
	11. Vir Biotechnology, “Samsung Biologics and Vir Biotechnology Enter into Agreement for Large Scale Manufacture of SARS-COV-2 Antibodies for Potential COVID-19 Treatment,” Press Release, April 9, 2020.
	12. Regeneron, “Regeneron Announces Important Advances in Novel COVID-19 Antibody Program,” Press Release, March 17, 2020.
	13. Regeneron, “FDA Accepts for Priority Review Biologics License Application for REGN-EB3 to Treat Ebola,” Press Release, April 16, 2020.
	14. Regeneron, “Regeneron’s Covid-19 Response Efforts: Developing A New Antibody Medicine,” 

, accessed May 1, 2020.  
	15. J.D. Roback and J. Guarner, 

Vol. 33, No. 6
	June 2020
	Pages: 16–21, 35

	When referring to this article, please cite it as C. Challener, “Plasma-Based Antibody Therapies Battle Against COVID-19," 

Therapies for early and late treatment and passive immunization of COVID-19 are needed and can be developed using antibodies from recovered patients.

Plasma-Based Antibody Therapies Battle Against COVID-19

Bioassays for use in biologic drug development and final product release must be robust and meet an array of regulatory requirements. As the complexity of next-generation biopharmaceuticals increases, so does the complexity of the bioassays necessary for effective evaluation of their performance across a spectrum of activities. Collaboration is essential between drug developers, testing laboratories, and regulatory authorities to ensure that new bioassays consistently serve their intended purpose.

	MOA, stability, validatability, and more

	Regulators expect bioassays for any new biologic to reflect the mechanism of action (MOA) of the drug in question, have stability-indicating properties, and be validatable, as outlined in International Council for Harmonization (ICH) Q2(R1) (1) and United States Pharmacopeia (USP) General Chapter <1033> (2). “Assays need to be adequately robust, accurate, and precise to ensure consistency in batch manufacturing and to provide meaningful data to support stability claims,” asserts Sharon Young, scientific manager, analytical and formulation sciences with Thermo Fisher Scientific.

	MOA is important because it uncovers important clues-sometimes the most important clue-as to what is happening in the patient in vivo. “While the MOA rarely mimics exactly what is happening in the patient, in the best-case scenario the bioassay will mimic what is relevant for the patient (i.e., what makes the drug efficient),” explains Ulrike Herbrand, scientific director, global in-vitro bioassays at Charles River Laboratories.

	Developing bioassays intended to measure the potency of biologics that truly reflect the relevant MOAs involves selecting appropriate indicator cell lines and relevant readouts that measure expected cell responses, adds Weihong Wang, technology development manager, cell and molecular biology services at Eurofins Lancaster Laboratories. “A master and/or working cell bank should be created for indicator cell lines and be well-characterized in order to assure meaningful interpretation of assay results and avoid artifacts unrelated to drug effects,” she says.

	In addition, a quantitative method is generally expected for potency measurement and results are usually expressed as percent relative potency when compared to a reference standard that is fully characterized, according to Wang. Sponsors should avoid using a surrogate assay unless they can provide sufficient justifications that address scientific rationale (MOA) and comparability to traditional assays that might be practically challenging/less robust.

	A bioassay should also be able to determine the stability of a drug and for how long it will be stable. Stress tests are conducted over a prolonged time at moderate conditions, including the recommended storage conditions, and the results help determine how a drug should be stored (at what temperature and for how long), according to Herbrand. She also notes that accelerated stress testing at various stress conditions (e.g., temperature, oxidation, deamidation, shear-stress, repeated freeze-thaw cycles) is required to determine which types of alterations the chosen bioassay is capable of detecting by showing altered activity.

	“Another key expectation for a bioassay is fulfillment of validation requirements by meeting the preset validation acceptance criteria, which is required for lot release and stability testing of the biologic at the end of clinical Phase II at the latest,” Herbrand observes.

	Young adds that bioassays should employ statistical monitoring to detect any shifts in performance over time and to detect any changes in the reference standard potency in real time. The use of more advanced statistical tools such as computational simulation can raise questions from regulators because they may seem unnecessarily complex, but such approaches can provide a much larger data set to determine assay acceptance criteria (3), she cites. The use of animal models is discouraged, Young comments, but is sometimes required and is acceptable if reasonably justified.

	Ensuring the bioassay is suitable for transfer to other labs is not exactly a key requirement from a regulatory perspective, but nevertheless, Herbrand says it is an important point to consider. “A bioassay that is ridiculously difficult for other labs to use can delay programs since it takes too long to establish the method in a GMP-compliant [quality control] QC lab,” she explains.

	Reporter-gene assays are a category of surrogate assays, notes Wang, that have been used more often in the past several years, especially in place of assays that require primary cells where assay performance can be quite variable and lack robustness desired as a QC testing method. They are particularly becoming common in early development and are increasingly being accepted by regulatory authorities, adds Young.

	As an example, Wang points to antibody-dependent cell-mediated cytotoxicity (ADCC) reporter-gene assays, which have been used to measure Fc receptor mediated response for monoclonal antibodies (mAbs) and in place of traditional peripheral blood mononuclear cell (PBMC)/primary natural killer (NK) cell-based cytotoxicity assays. “Instead of looking at direct cell killing (by primary NK cells), the assay measures activation of signal transduction associated with the cellular event,” she explains. The resulting reporter gene assay is easy to perform with much shorter assay duration and in general delivers excellent assay performance.

	The key to successful use of reporter-gene assays, adds Young, is to have additional bioassays that really reflect the biological mechanism(s) of action and then show that results using the reporter-gene assay are comparable, or better, to those bioassays that measure the cellular response(s) directly. “Bridging results need to include a large number of lot release samples as well as stability and force-degraded samples. Bridging studies should also include isolated impurities in these comparisons to directly demonstrate that all potential changes in the molecule can be detected in the reporter-gene assay comparably, or better, than in the direct assay(s),” she observes.

	Wang highlights that the increasing use of reporter-gene assays is a good example that mechanism-of-action is the first and foremost important characteristic, and when this feature is proved, solid method performance including robustness is another key element of a successful bioassay.

	The top trends in bioassay development observed at Thermo Fisher Scientific include the implementation of ready-to-use cell banks and automation. “Thaw-and-use cell banks can be thawed and plated directly in the bioassay, eliminating the need to spend a large amount of resources on maintaining actively growing cultures. It also simplifies monitoring of assay performance because impact from the age (passage number) of the culture need not be tracked and can reduce method variability attributable to variations in culture conditions between testing occasions,” Young explains.

	Laboratory/assay automation, from simple pipetting robots to end-to-end automation, is helping to increase throughput and reduce the risk of laboratory error, according to Herbrand. It also allows for miniaturization, which she says enables performance of six to seven tests simultaneously, affording higher throughput, the need for fewer reagents and lower cost.

	For instance, the use of automated liquid handlers is becoming increasingly common in bioassays because it significantly reduces assay variability attributable to pipetting, Young says. “Automation also mitigates variability attributable to positional bias by enabling randomization of plate layouts within and between plates without concern for analyst error, which significantly mitigates plate position and sequence (order of addition of reagents) sources of variability,” she remarks.

	Cell-therapy and gene-therapy challenges

	With the fast growth of the gene- and cell-therapy pipeline, more potency assays for this category of product are being submitted to the regulatory agencies, according to Wang. Potency assays for cell- and gene-therapy products are generally more complicated than recombinant therapeutic proteins, as they need to demonstrate several characteristics of the product.

	These products, according to Herbrand, require bioactivity testing, often in a multistep approach. For the first step, molecular methods such as a quantitative polymerase chain reaction (PCR) or droplet digital PCR are often the methods of choice to confirm the desired re-expression or suppression effect in vitro at the RNA level. In addition, she says the re-expression or suppression of a gene of interest can be shown on the protein level using enzyme-linked immunosorbent assay (ELISA) testing, or in the case of receptors, via flow cytometry. In some cases, a reporter-based method might be applicable as an MOA-reflecting, cell-based bioactivity assay to confirm the effect on the functional level.

	“An assay for a gene therapy usually involves transfecting/infecting a cell line followed by measurement of the expression of the intended target gene at the nucleic acid or protein level. In addition, whenever possible, the function of the expressed protein should be confirmed, for example, by enzymatic assays, or receptor binding, etc., depending on the MOA. Therefore, often times several assays are needed to fully address all aspects of the product characteristics,” explains Wang.

	Potency assays for cell therapy products also have their unique challenges. “First of all,” states Wang, “it may be more difficult to correlate in-vitro potency measurements to clinical efficacy compared to typical recombinant protein therapeutics.” Second, depending on the product, she notes that it is sometimes difficult to truly establish a “reference standard” to which subsequent lots may be compared. Third, performance of functional assays, in general, tends to be less robust.

	Because of these issues, setting specifications can also be challenging, according to Wang. “Last but not least,” she observes, “some cell-therapy products require fast delivery to patients; therefore, the balance between thorough characterization and short assay turn-around time may need to be carefully considered.”

	Because multiple assays may need to be deployed, from phenotypic (e.g., flow cytometry), to functional confirmation (e.g., cytokine release and/or cytotoxicity for chimeric antigen receptor T-cell therapies), these assays need to be developed as part of a ‘matrix approach’ in order to fully characterize the product and assure efficacy in order to gain regulatory acceptance,” Wang says.

	A recent approach noted by Young that has gained approval is flow cytometry detection of biomarkers that correlate with function, such as cell survival or differentiation. “Analysis of the selected biomarkers must be able to detect unacceptable behavior of the cells, and not simply rely on cell identity markers that are unlikely to do so under conditions that affect cell function,” she notes. The rationale for selection of the biomarkers must be clear, particularly when a complex assay matrix is proposed, and the assay must be shown to be capable of rejecting a sub-par batch, she adds.

	One of the main challenges, asserts Herbrand, is that there currently are no clear guidelines on how to evaluate bioactivity for cell-therapy and gene-therapy products. “For now,” she says, “these therapies are being considered on a case-by-case basis by regulators. Many of the gene therapy companies are currently running tests that more or less reflect what would be required for the development phase, but they haven’t nailed down what they need for QC release of a marketed drug.”

	What isn’t in question is the complexity of cell and gene therapies. “What is understood is that the safest course to take is to make sure we reflect that the drug is doing its job on all relative levels. Twenty years ago, we pondered the same questions with mAbs and what was required for lot release. Today, at least we have imprecise guidelines,” Herbrand comments.

	Many molecules have more than one mode of action, particularly bispecific antibodies, and regulators increasingly want a single assay that can reliably detect changes in potency through those multiple mechanisms, according to Young. “Combined approaches measuring the activities of both epitopes within the same assay rather than reporting two independent results for the activities of the individual arms of the bispecific have an excellent chance of being accepted by regulators,” Herbrand agrees. The reason: it is important that bioassays for potency of bispecific molecules reflect any additive or synergistic effects of the bispecific mechanisms of action.

	“This need,” Young notes, “can create challenges in developing the method, particularly in cell-line selection, as well as in interpreting the results. Results for a bispecific with a synergistic effect will show changes in EC50 as well as in asymptotes, for which tests of parallelism are not appropriate. Differences in asymptotes is traditionally unacceptable per USP guidance, as sample curves must be similar to reference curves for calculation of relative potency.”

	Currently, although the ideal assays from a regulatory and a sponsor’s standpoint would be dual reporter systems, it is still more common to independently test the bioactivity using two different assays to measure each arm of the bispecific antibody, Herbrand remarks. “While easier to develop, they do not allow one to fully see the synergistic effects and combined activity of the antibodies,” she notes.

	Young observes that at the CASSS Bioassays 2016 conference, Bhavin Parekh presented an approach for calculating relative potency/efficacy for a bispecific product with synergistic activity that he developed based on the field of pharmacology (4). She thinks this simple approach to reach a discrete value to compare concomitant shifts in EC50 and asymptotes may be able to establish a precedent for regulatory acceptance.

	3D cell culture is garnering some interest

	Common in drug discovery, three-dimensional (3D) cell culture models have recently been attracting interest among biologics manufacturers for use in bioassay development as well. “The use of 3D cell cultures and 3D bioprinting has the potential to enable assays in more complex cellular environments, more like tissue structures,” Young explains. “Such models,” she adds, “are ideal for mimicking the complex cancer microenvironment as well as for culturing cell lines that rely extensively on the extracellular matrix environment for growth, differentiation, and function, such as skeletal muscle cells.”

	“These 3D systems are often a better reflection of the in-vivo situation than what is observed for traditional two-dimensional cell-culture-based assays and provide better information regarding what is occurring in the patient,” agrees Herbrand. She does note, though, that it can be tedious to validate such tests, and they are often still time-consuming. Young also says that it can be challenging to control variability, find suitable methods of quantitation for potency results, and implement such complex techniques into a QC environment.

	Potential for protein tagging and human primary cell assays

	Protein tagging is based on peptide sequences that are attached to proteins to facilitate easy detection and purification of expressed proteins, according to Herbrand. They can also be used to identify potential binding partners for the protein of interest. The tags, which Herbrand notes are typically applied using CRISPR/Cas9 gene editing technology, are of particular interest because they are highly sensitive down to the endogenous level of the protein, often with over seven logs of dynamic range.

	Because human primary cell assays are closer to the situation in patients, Herbrand suggests they may be more translatable than cell lines, which are immortalized/modified in their key characteristics and thus could exhibit modified cell behaviors. “However,” she cautions, “human primary cell-based assays are not without risks.” Human primary cells are subject to lot-to-lot variability and availability of suitable sourcing material. In addition, even if it was possible to identify the perfect lot, it wouldn’t last forever. “There are no guarantees you won’t have to adjust the assay. And even if you manage to get the assay running again, you have to revalidate it, at least partially, which requires a change in the regulatory filing and months of delays,” Herbrand explains.

	Increasing bioassay complexity requires consideration and caution

	Caution is warranted in general when it comes to newer bioassays, according to Young. “As cell culturing techniques advance and increasingly complex multifunctional molecules are developed in the biopharmaceutical industry, bioassay development will become increasingly complex as well,” she says.
	One way the industry has responded, according to Wang, is to develop bioassays to support therapeutic biologics as early as possible, particularly when more complex products are involved. “While science continues to be the driving force for bioassay development, sponsors are encouraged to engage regulatory agencies early in order to obtain guidance on their assay development approach and avoid unnecessary surprises and delays in their product development programs,” she says.

Next-generation therapeutics and regulatory requirements create demand for complex, fit-for-purpose tests. 

Author | Cynthia A. Challener

Articles

Can Using RNA Simplify Gene Therapy Development?

Eliminating Residual Impurities Starts with a Strategic Plan

Plasma-Based Antibody Therapies Battle Against COVID-19

Building Better Bioassays