Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

Functional characterization of protein therapeutics is an expectation of regulatory agencies. The critical quality attributes (CQAs) that impact function and efficacy of the protein therapeutic should therefore be identified and characterized for a regulatory submission. Throughout the development process, notes Khanh Ngo Courtney, director of biologics for Avomeen, bioanalytical testing must be dynamic and tailored to both the protein and the manufacturing approach. “Given the speed of enzyme and protein therapy R&D, rapidly assessing biomolecule efficacy (or potency) and determining an accurate list of CQAs are key,” she states.

Before any biologic drug enters into the clinic, it is essential to have a clear understanding of what exactly the biomolecule entails, according to Vu Truong, CEO of Aridis Pharmaceuticals. That includes both structural and physicochemical characteristics.

General structural features that must be described for a protein therapeutic include the primary amino acid sequence, amino acid composition, and any available secondary and tertiary structures, Courtney notes. Peptide mapping can be used to determine the amino acid composition, protein sequence—including N- and C-termini status—post-translational modifications (PTMs), disulfide bridges, and sulfhydryl status, for instance, although additional methods are often needed to fully characterize all these structural elements, according to Andrew Hanneman, scientific advisor for biologics testing at Charles River Laboratories.

The molecular weight profile, isoform distribution patterns, and molar absorptivity are other physicochemical properties that must be determined to support clinical studies and product release.

The specific part of the biomolecule that mediates activity should be identified as well, adds Truong. Stability profile assays must also be conducted to demonstrate under what conditions that activity is preserved and lost due to fragmentation/cleavage, unfolding, and 

 immune responses, he says. Information on impurities and the acceptable levels at which they do not adversely impact activity and safety must also be developed.

It must be kept in mind, though, Courtney stresses, that structural and physicochemical properties vary depending on the CQAs for efficacy of each drug substance. For instance, if the drug requires higher-ordered structures, such as oligomerization or other protein binding partners, for activity, showing evidence of quaternary structure might be expected. If certain PTMs are required for a protein to be internalized, such as glycosylation, then elucidation of the glycan structure important for such activity might be helpful in a filing.

There may also be varied requirements for different regulatory authorities, observes Hanneman. “Although drug development processes and submissions are increasingly harmonized among the major markets, there are some differences that need to be understood early in drug development. The characterization methods needed are developed and conducted or validated with knowledge of market-specific guidance and the expected level of oversight and data review,” he says.

A wide variety of analytical methods are needed to fully characterize therapeutic proteins. “Physiochemical properties are investigated via structural characterization and confirmation studies described in the International Council for Harmonization (ICH) Q6B Guidance (1) initiated in collaboration between FDA, the European Medicines Agency (EMA), and the Japan Pharmaceutical Manufacturers Association (JPMA),” states Hanneman.

In addition to peptide mapping, electrophoresis, high-performance liquid chromatography (HPLC), size-exclusion chromatography, and various forms of spectroscopy (e.g., Fourier transform infrared) are widely used. “The more methods that are employed, especially if they are truly orthogonal, the greater confidence the drug developer and regulators have in the nature and quality of the protein therapeutic,” Truong says. He also notes that by performing a very comprehensive set of analyses, companies set a standard that developers of biosimilars will have to meet, which raises the hurdles.

More recently, nucleic acid sequencing technologies, X-ray crystallography, cryomicroscopy, and mass spectrometry (MS) have attracted increasing attention. MS, says Hanneman, has proven highly flexible in biopharma applications because the soft ionization methods used to ionize large molecules provide a significant opportunity to manipulate large biomolecules like proteins, leading to direct and unambiguous readout of primary and, increasingly, higher order structural elements.

Protein MS, either intact or bottom-up, is commonly used in the characterization of protein therapeutics because it is a single method capable of characterizing many attributes at a time, according to Courtney. “Using high sensitivity instrumentation such as the Orbitrap, one can elucidate batch-to-batch primary sequence consistency, post-translational modifications, such as phosphorylation or glycosylation, and also damages to the protein upon stressed treatment, such as deamidation or oxidation,” she explains. A quadrupole time-of-flight (QTOF) MS system would be useful for verifying accurate intact mass of the biomolecule for use as an identity test, Courtney adds.

Quantitative peptide mapping conducted as a multi-attribute method (MAM), Hanneman agrees, holds significant promise to displace a variety of redundant HPLC and/or electrophoretic methodologies to speed up protein drug development.

Bottom up mass spectrometry is not new, but different ways to use this powerful tool are more frequently employed, Courtney says.

“MS continues to see increased applications, with newer and streamlined approaches used to manipulate large gas-phase ions. MS offers the significant advantage of precise mass measurement (ppm mass accuracy) as its output,” Hanneman agrees. He points to ion mobility and charge detection MS as two innovative methods.

Some aspects of therapeutic protein characterization still create challenges today. Truong points to demonstration of glycoform consistency and how changes in glycoform profiles can impact safety, efficacy, and immunogenicity as one area where improvements in speed and accuracy are needed.

Potency assays, such as an enzymatic activity assays and cell-based potency assays, also present difficulties, according to Courtney. She notes that both are necessary for characterization of potency and 

 cellular efficacy, but cells are intrinsically tricky “reagents” and can be difficult to control and troubleshoot, while, enzymatically, assays can be difficult to develop if the mechanism of the protein is not fully understood.

“These issues lead to higher variability for enzymatic and cellular potency assays than chromatographic methodologies,” Courtney says. The challenge, she adds, is to design such methods so they are fit-for-purpose. “Often these assays are over-designed, resulting in added complexity that leads to increased variability or being under-designed, and, thus, lack sufficient specificity. Achieving the right balance is challenging and the most difficult part of the process,” she explains.

The time it takes to complete cell-based assays is also an issue, according to Truong, but advances in nucleic-acid based methods (next-generation sequencing technologies) should ultimately overcome this difficulty. Similarly, the need to demonstrate genetic (cell-line) stability through numerous passages takes significant time.

Advances in analytical technologies are helping to increase the understanding of protein structure and function at the molecular level, providing greater insight into how proteins actually bind to targets and their detailed modes of action. “Elucidating structural and physicochemical properties of proteins and relating them to biologic activity is increasingly possible and at an ever-increasing pace,” Truong asserts.

Courtney refers again to MS as a powerful tool not only for understanding the molecular characteristics of proteins, but also as a read-out method for bioactivity and cellular potency. “If a protein therapeutic can elicit a cellular response, then perhaps a representative biomarker resulting from that response could be monitored by MS using selective ion monitoring or multiple-reaction monitoring,” she observes.

Significant effort has also been underway to use “native mass spectrometry” for direct MS measurement of proteins in their native conformation, including complexes of increasingly higher molecular weight. Improved separation modes allow for direct analysis of native proteins, including charge variant species, according to Hanneman.

There are hurdles to using MS for potency and routine testing, however, including the need for specialized equipment and personnel not usually found in a quality control (QC) laboratory. “I look forward to seeing how mass spectrometers can be simplified as bench-top instruments that aren’t so intimidating for routine use and could be run by non-specialized personnel,” Courtney comments.

Hanneman notes that instrument companies and software developers continue to work closely to provide advanced “push-button” MS data applications under 

 Part 11) (2)-compliant electronic data platforms for routine use in QC labs. For instance, MS tool kits include improved workflows and streamlined data analysis approaches for profiling and quantifying impurities, including host-cell proteins (HCP) and other process impurities, which will continue to grow in their implementation.

New formats and improved techniques for conducting 

 bioassays are also evolving, including new labeling and imaging approaches, such as innovative time-resolved fluorescence methods and reagents, Hanneman observes. “These tools for measuring target binding typically offer increased signal-to-noise ratio resulting in higher sensitivity and greater confidence in demonstrating specificity,” he says.

New and improved methodologies for aggregate and particle separation, including field flow fractionation (FFF), as well as instruments providing high-definition particle imaging and detection will see increased application, according to Hanneman. These new tools and approaches for detecting and measuring sub-visible particles and determining the size of protein aggregates (as well as viral particles) are undergoing innovation and developments to improve rate of throughput.

Regardless of what characterization methods are selected, they are targeted and tuned toward uncovering and reliably measuring drug product CQAs because they ultimately impact protein bioactivity 

 and safety, including potential immune reactions. The methods chosen, therefore, should provide overlapping and correlative information without “knowledge gaps”, Hanneman asserts.

“Comprehensive protein structure determination ranging from primary to tertiary/quaternary elements currently requires a wide variety of complementary techniques (some truly ‘orthogonal’), and each may fill a different piece of an overall large puzzle. The overlapping data sets then need to be synthesized into an overall description, which can be a complex process for large, heterogeneous, or multimeric protein drugs,” says Hanneman.

Truly comprehensive structural determination remains an elusive goal, however, according to Hanneman, due to protein higher order conformation as well as proteoform heterogeneity within a given sample. “[On a positive note], the development of computational bioinformatics tools and shared database resources go a long way toward simplifying the processing, visualization, and sharing of large protein characterization and comparability data sets among collaborating stakeholders,” he observes.

The advantages and disadvantages of various methods and approaches should, in addition, be weighed in terms of the “intended use” of each analytical method and whether each method needs to be validated, according to Hanneman. “Information-rich characterization methods need to be developed and applied as early as possible in a drug development program and used to direct best use of effort, then continually refined (or re-defined) all the way through to drug commercialization,” he says.

Furthermore, with cell and gene therapy programs gaining ground in the biologic marketplace, Hanneman believes the intended use and implementation of protein characterization methods will see changes and refinements in response to the need to detect the presence of genetic material and potentially characterize oligonucleotides as well as to analyze increasingly smaller sample aliquots produced using new and varied manufacturing processes.

Underlying the need to select the right set of methods for each given protein therapeutic is the critical importance of having the right talent working on a project. “People with the right skillsets and experience are needed to design studies that can overcome the various analytical challenges faced with respect to protein characterization,” Courtney states. “The right team,” she says, “will include someone with a fundamental understanding of protein biochemistry, enzyme kinetics, and/or cell biology to enable development of the right model system and a person who is QC-centric to help edit the method to ensure that it is fit-for-purpose.”

The team should also leverage best practices and, wherever possible, a set of analytical methods that have been successfully demonstrated in prior filings, according to Truong. “Evaluation of optimal sets of assays used in past applications can be very informative when selecting assays for a new protein candidate that will afford the best specificity and speed,” he says.

Going forward, adds Hanneman, cloud-based computing and “paperless” corporate infrastructure will greatly improve drug development timelines by supporting faster clearer data sharing. “These developments take significant time and resources to implement but once in place are highly advantageous,” he says.

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

 Title 21, 11 (Government Printing Office, Washington, DC) pp. 110–114.

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


Vol. 34, No. 1
January 2021
Pages: 34–37

When referring to this article, please cite is as C. Challener, “Using Protein Characterization to Support Regulatory Submissions,” 

Articles

Having a clear and detailed understanding of exactly what a biomolecule entails is essential for therapeutics development.

Protein Characterization Can Support Regulatory Submission

Despite the COVID-19 pandemic, FDA’s approval rate in 2020 remained high. As of Dec. 17, 2020, 50 new drugs had been approved by the agency’s Center for Drug Evaluation and Research (CDER) (1), surpassing the 48 new drugs approved in all of 2019 (2). An additional 65 first-time generics were granted marketing authorization in 2020 (3). As the year wound to a close, six small-molecule and two biologic drug applications had Prescription Drug User Fee Act (PDUFA) decision dates scheduled before the end of 2020 (4), creating the potential for 58 approvals, which would be comparable to 2018 (5).

A few interesting firsts and trends are highlighted in the following.

The first approval for a COVID-19 therapy

One of the most exciting approvals went to Gilead Sciences for its new treatment for patients with COVID-19 requiring hospitalization (6). Veklury (remdesivir) originally was granted Emergency Use Authorization (EUA) in May 2020. Unlike the EUA, the approval does not include pediatric patients weighing less than 40 kg. The drug received accelerated approval under FDA’s Coronavirus Treatment Acceleration Program. Remdesivir was also granted fast track and priority review designations and a material threat medical countermeasure priority review voucher.

COVID-19 wasn’t the only disease for which first therapies were approved. In fact, many of the drugs granted marketing authorization by FDA in 2020 are first treatments. In May, FDA approved the first drug for treatment of severe malaria (7). Amivas’ artesunate for injection was previously only available to patients through FDA’s expanded access program. Artesunate received priority review and orphan drug designation.

Tepezza (teprotumumab-trbw) is an antibody drug conjugate (ADC) from Horizon Therapeutics Ireland DAC for the treatment of thyroid eye disease that can potentially spare patients from multiple invasive surgeries (8). It was granted priority review, fast track, and breakthrough therapy designations and supported in part by the FDA Orphan Products Grants Program.

Ultragenyx Pharmaceutical and Alnylam Pharmaceuticals both received approvals for first treatments for two metabolic disorders: Dojolvi (triheptanoin), a highly purified, synthetic, seven-carbon fatty acid triglyceride as a source of calories and fatty acids, for the treatment of pediatric and adult patients with molecularly confirmed long-chain fatty acid oxidation disorders (9) and Oxlumo (lumasiran) for the rare genetic disorder, primary hyperoxaluria type 1 (PH1) (10), respectively. Effort to achieve the latter approval was coordinated by the Oxalosis & Hyperoxaluria Foundation and the Kidney Health Initiative.

FDA also approved Zokinvy (lonafarnib, Eiger Biopharmaceuticals) as the first treatment to reduce the risk of death due to Hutchinson-Gilford progeria syndrome and for the treatment of certain processing-deficient progeroid laminopathies in patients one year of age and older (11).

Several first therapies approved in 2020 are targeted against very specific diseases, and many fall into the class of drugs known as kinase inhibitors. Examples include Ayvakit (avapritinib, Blueprint Medicines Corporation) for the treatment of adults with unresectable or metastatic gastrointestinal stromal tumor (GIST) harboring a platelet-derived growth factor receptor alpha (PDGFRA) exon 18 mutation (12); Koselugo (selumetinib, AstraZeneca Pharmaceuticals), the first treatment of neurofibromatosis type 1 (NF1), a genetic disorder of the nervous system causing tumors to grow on nerves (13); Pemazyre (pemigatinib, Incyte Corporation), the first treatment approved for adults with certain types of previously treated, advanced bile duct cancer (cholangiocarcinoma) (14); Tabrecta (capmatinib, Novartis) for the treatment of adult patients with non-small cell lung cancer (NSCLC) that has spread to other parts of the body and whose tumors have mutations that lead to MET exon 14 skipping (15); and Retevmo (selpercatinib, Loxo Oncology, Inc., a subsidiary of Eli Lilly and Company) for the treatment of three types of tumors with alterations of the “rearranged during transfection” gene (16).

Three drugs with notable mechanisms of action were approved by FDA in 2020. Rukobia (fostemsavir, ViiV Healthcare) is a new type of anti-retroviral medication for adults living with human immunodeficiency virus (HIV) who have tried multiple HIV medications and whose HIV infection cannot be successfully treated with other therapies because of resistance, intolerance, or safety considerations (17). Rukobia received fast track, priority review, and breakthrough therapy designations. Winlevi (clascoterone cream 1%, Cassiopea) for the treatment of acne in patients 12 years and older is the first acne drug with a new mechanism of action to receive approval in nearly 40 years (18). It operates via a non-antibiotic approach, targeting androgen receptors directly in the skin.

Blenrep (belantamab mafodotin-blmf) is a first-in-class anti-B-cell maturation antigen therapy for patients whose disease has progressed despite prior treatment with an immunomodulatory agent, proteasome inhibitor, and anti-CD38 antibody (19). It is one of five drugs from GlaxoSmithKline approved by FDA in 2020 and one of 10 ADCs receiving agency approval (1). Blenrep comprises a humanized B-cell maturation antigen (BCMA) monoclonal antibody (mAb) conjugated to the cytotoxic agent, auristatin F, via a non-cleavable linker. The drug linker technology is licensed from Seattle Genetics; the mAb is produced using POTELLIGENT Technology licensed from BioWa.

Tauvid (flortaucipir F18, Avid Radiopharmaceuticals) is the first drug designed to help image tau pathology (20). It is used with positron emission tomography of the brain to estimate the density and distribution of aggregated tau neurofibrillary tangles, a primary marker of Alzheimer’s disease.

In July, Kite, part of Gilead, became the first company with multiple chimeric antigen receptor (CAR) T-cell therapy approvals (21). Tecartus (brexucabtagene autoleucel) is the first approved CAR T-cell therapy for the treatment of adult patients with relapsed or refractory mantle cell lymphoma, a rare form of non-Hodgkin lymphoma. The autologous anti-CD19 CAR T-cell therapy uses the XLP manufacturing process, which includes T-cell enrichment, a necessary step in certain B-cell malignancies in which circulating lymphoblasts are a common feature.

Several new drugs approved by FDA in 2020 address both the disease in question and patient needs for ease and/or convenience. Vyepti (eptinezumab-jjmr, Lundbeck) was approved as the first intravenous preventive treatment for migraine (22). The therapy allows many patients to experience relief within days and then over a period of several months. This ADC binds to calcitonin gene-related peptide (CGRP) ligand and blocks its binding to the receptor. Biohaven Pharma’s Nurtec ODT (rimegepant) is another CGRP receptor antagonist for treatment of migraine, but the only one available in a fast-acting orally disintegrating tablet (ODT) (23). According to the company, a single oral dose of Nurtec ODT 75 mg can provide fast pain relief and return patients to normal function within one hour, with many also experiencing sustained relief for up to 48 hours

Inqovi (decitabine and cedazuridine) tablets from Astex Pharmaceuticals, a subsidiary of Otsuka Pharmaceutical, is a treatment for myelodysplastic syndromes and chronic myelomonocytic leukemia that can be taken at home (24). Previous intravenous therapies required a visit to a healthcare facility. Evrysdi (risdiplam, Genentech) for the treatment of spinal muscular atrophy is also an oral therapy and the first for this disease (25).

International collaboration was emphasized by FDA in 2020. Two drugs were approved as part of Project Orbis, an initiative of the FDA Oncology Center of Excellence (OCE) that provides a framework for concurrent submission and review of oncology products among international partners (26). Collaboration among international regulators may allow patients with cancer to receive earlier access to products in other countries where there may be significant delays in regulatory submissions, regardless of whether the product has received FDA approval, according to the agency. With a framework for concurrent submission and review of oncology drugs, Project Orbis also facilitates a collaborative review to identify any regulatory divergence across review teams.

The kinase inhibitor Tukysa (tucatinib, Seattle Genetics) was approved in combination with chemotherapy (trastuzumab and capecitabine) for the treatment of adult patients with advanced forms of HER2-positive breast cancer that can’t be removed with surgery or has spread to other parts of the body (27). FDA collaborated with the Australian Therapeutic Goods Administration (TGA), Health Canada, Health Sciences Authority (HSA, Singapore), and Swissmedic (SMC, Switzerland) on the review—the first time the agency worked with HSA and Swissmedic. Tukysa was also reviewed using the real-time oncology review (RTOR) pilot program to streamline data submission and the assessment aid to facilitate discussions among the regulatory agencies and regulatory review. The drug received priority review, breakthrough therapy, fast track, and orphan drug designations.

Qinlock (ripretinib, Deciphera Pharmaceuticals), another kinase inhibitor, is the first new drug specifically approved by FDA as a fourth-line treatment for advanced gastrointestinal stromal tumor (28). FDA collaborated with TGA and Health Canada on the review of this application as part of Project Orbis. Qinlock received the same four designations as Tukysa, and the RTOR and assessment aid were used for its review.

Finally, FDA granted approval to the first treatment for Zaire ebolavirus (Ebola virus) infection in adult and pediatric patients to Inmazeb (atoltivimab, maftivimab, and odesivimab-ebgn), a combination of three mAbs from Regeneron Pharmaceuticals (29). The data used to evaluate Inmazeb were collected from a clinical trial and through an expanded access program conducted in the Democratic Republic of the Congo (DRC) during an Ebola virus outbreak in 2018–2019. FDA stated in its press release about the approval that international collaboration was key to achieving success in the fight against Ebola virus.

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FDA Approves Only Drug in U.S. to Treat Severe Malaria

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FDA Approves First Drug to Treat Rare Metabolic Disorder

,” Press Release, Nov. 23, 2020.
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FDA Approves First Treatment for Hutchinson-Gilford Progeria Syndrome and Some Progeroid Laminopathies

,” Press Release, Nov. 20, 2020.
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FDA approves the first targeted therapy to treat a rare mutation in patients with gastrointestinal stromal tumors

,” Press Release, Jan. 9, 2020.
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FDA Approves First Therapy for Children with Debilitating and Disfiguring Rare Disease

,” Press Release, April 10, 2020.
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FDA Approves First Targeted Treatment for Patients with Cholangiocarcinoma, a Cancer of Bile Ducts

,” Press Release, April 17, 2020.
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FDA Approves First Targeted Therapy to Treat Aggressive Form of Lung Cancer

,” Press Release, May 6, 2020.
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FDA Approves First Therapy for Patients with Lung and Thyroid Cancers with a Certain Genetic Mutation or Fusion

,” Press Release, May 8, 2020.
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FDA Approves New HIV Treatment for Patients with Limited Treatment Options

,” Press Release, July 2, 2020.
18. Cassiopea, “Cassiopea Receives FDA Approval for Winlevi (clascoterone cream 1%), First-in-Class Topical Acne Treatment Targeting the Androgen Receptor,” Press Release, Aug. 27, 2020.
19. GSK, “FDA Approves GSK’s BLENREP (belantamab mafodotin-blmf) for the Treatment of Patients with Relapsed or Refractory Multiple Myeloma,” Press Release, August 6, 2020.
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FDA Approves First Drug to Image Tau Pathology in Patients Being Evaluated for Alzheimer’s Disease

,” Press Release, May 28, 2020.
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22. Lundbeck, “FDA approves Lundbeck’s Vyepti (eptinezumab-jjmr)—the First and Only Intravenous Preventive Treatment for Migraine,” Press Release, February 22, 2020.
23. Biohaven Pharma, “Biohaven’s NURTEC ODT (rimegepant) Receives FDA Approval for the Acute Treatment of Migraine in Adults,” Press Release, February 27, 2020.
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FDA Approves New Therapy for Myelodysplastic Syndromes (MDS) That Can be Taken at Home

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FDA Approves First Drug for Fourth-Line Treatment of Advanced Gastrointestinal Stromal Tumors

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29. FDA, “

FDA Approves First Treatment for Ebola Virus

Cynthia Challener, PhD, is a contributing editor to 


Vol. 45, No. 1
January 2021
Pages: 26–29

When referring to this article, please cite it as C. Challener, “FDA Ends 2020 with High Drug Approval Rate,” 

FDA approval rate speeds up despite COVID-19 complications.

FDA Ends 2020 with High Drug Approval Rate

Highly potent compounds present numerous challenges with respect to process development, formulation, and delivery. “The key features at the interface of drug delivery and highly potent APIs (HPAPIs) are the ability to maintain operator safety through containment of the API during processing and to deliver a drug product in which the HPAPI is evenly dispersed, even when doses are as low as micrograms,” asserts David Lyon, a senior research fellow with Lonza.

Doing so is becoming more challenging as the molecular complexity of HPAPIs
continues to increase significantly. “These molecules are much more specific in how they interact with the body and different cellular targets,” says Brian Haney, director of technical operations for AMRI. “As a result, it makes them much more difficult to manufacture, more difficult to test, and more unstable,” he observes.

In fact, HPAPIs have largely been too fragile for traditional oral solid-dose formulation, and the majority are delivered parenterally via infusion or intravenous injection, according to Haney.

Historically, HPAPIs have been almost exclusively associated with oncology therapeutics. “Highly potent molecules for such acute indications could be delivered by intravenous infusion even if an oral drug product could not be developed,” Lyon explains.

Currently, however, HPAPIs include a growing number of classes of molecules and new chemical entities for a broader range of indications, thus covering a much wider swath of the developmental pipeline. “For many of these indications,” Lyon comments, “oral delivery is required due to the chronic or frequent dosing needed for the molecule and indication. The need for oral solutions has thus led to adaptation of existing pharmaceutical processes to enable the handling [of] HPAPIs in a safe and efficient manner,” he adds.

Examples include microdosing, liquid-filling of capsules, and wet granulation, which help balance therapeutic benefit versus risk. These processes address safety concerns for operators by properly containing the HPAPI, according to Lyon.

“Liquid-fill and wet granulation processes remove much of the concern with operator exposure because the powder risk is removed once the HPAPI is dissolved in a lipid or solvent for either filling into a capsule of spraying onto a solid support dispersible,” explains Lyon. Once the HPAPI powder is dissolved or emulsified, further processing of the liquid becomes simpler and less costly, requiring less strict containment. Lyon also notes that these processes allow for highly accurate dosing down to low API levels, helping ensure content uniformity and reproducible dosing for caregivers and patients.

Managing poor solubility/bioavailability challenges

Handling a highly potent compound across the oral drug development process becomes even more complex when the compound is also bioavailability-challenged. The simplest approach, according to Lyon, is to fill the micronized active ingredient into an empty capsule shell using equipment that can be deployed in a contained space (microdosing).

Many HPAPIs, however, have material properties (sticky, semisolid, liquid, too light, too fluffy) that preclude the use of conventional microdosing. The powder may also have poor flow properties or the size and shape of the HPAPI particles may be inconsistent. In some cases, Lyon observes, HPAPI compounds may be so potent that a safe containment level cannot be easily achieved or the dosage itself may simply be below the weighable range of microdosing.

In these cases, liquid-filled hard capsules (LFHCs) can effectively minimize operator exposure to the solid HPAPIs. “The liquid-filling process is straightforward, involving only four steps: dispense, mix, fill, and seal. LFHCs contain either room-temperature liquids or thermo-softening materials manufactured as molten liquids at temperatures of up to 65 °C.
Typically, sticky semisolid and liquid HPAPIs are miscible in liquid excipients, forming a homogeneous mixture, no matter how low the dosage is,” says Lyon.

In addition, the use of solubility-enhancing excipients and liquid lipid-based options such as self-emulsifying drug delivery systems (SEDDS) and self-micro emulsifying drug delivery systems (SMEDDS) is also possible. Milling and micronization of HPAPIs to produce nanoparticles is also safer when performed in an oily solvent.

Size reduction, in fact, through “top down” approaches such as micronization and “bottom up” techniques such as crystallization, expands the surface area-to-mass ratio, thereby increasing the dissolution rate of the HPAPI, according to Ilan Avni, vice president of business development with Wavelength Pharmaceuticals. Other techniques for increasing bioavailability of HPAPIs, he remarks, include spray drying and hot-melt extrusion to generate amorphous solid dispersions in polymeric matrices and the formation of cocrystals with appropriate conformers to improve physicochemical properties.

One of the challenges to formulating highly potent APIs is the fact that often only small quantities of the drug substance are required in each unit dose. Only formulation approaches that can ensure homogenous distribution throughout the product, even at extremely low doses, are therefore appropriate. These constraints also require accurate analytical methods; again, preparing liquid solutions or emulsions for further processing (encapsulation, wet granulation, then tableting) is effective.

The melt-spray-congeal (MSC) process is also now being successfully applied to advance potent oncology compounds in lipid multiparticulate (LMP) formulations, according to Lyon. “This technology is increasingly used to address controlled release, taste-masking, and/or low solubility challenges,” he says.

In the MSC process, the API is suspended or dissolved in a molten lipid formulation at elevated temperatures (70–90 °C) and then pumped to a spinning disk where the solution/suspension is atomized and then congealed, forming a multiparticle of 100–300 microns. Notably, Lyon says, these lipid multiparticles can contain from as little as a few percent API to 50% API, making them an effective vehicle for diluting HPAPIs.

Particularly for targeted oncology therapies, it takes time to deliver the HPAPI to the specific cancer cells of interest. “Sustained- or modified-release formulation makes it possible for the active substance to stay in the body longer as it travels to where it needs to go,” says Haney.

To achieve this goal, newer delivery approaches include conjugation of the HPAPI via a chemical linkage to another inert molecule that has an affinity toward the target cells. “Antibody-drug conjugates (ADCs) are an ever-growing class of drugs in this category. The HPAPI is conjugated to a monoclonal antibody that carries it to the target cells, where it is released for targeted action,” Haney explains.

AMRI, Haney adds, has been seeing other conjugation approaches as well, such as linkage of HPAPIs to polyethylene glycol (PEG) resins. “These PEG polymers, often 5000–20,000 Daltons in size, are slowly degraded by the body. The conjugated system consequently remains intact for much longer than the inherently unstable HPAPI would by itself, providing sustained release,” he comments.

Similarly, extended-release parenteral formulations based on bioabsorbable poly(lactide-co-glycolide) (PLG) microparticles can improve therapeutic efficacy and increase patient compliance. In some cases, HPAPIs can be delivered from PLG microparticles for weeks and months following a single administration.

Other approaches can also be used. For instance, if a simple modified release profile—such as delivery of the API to the duodenum—is required, a simple fill of either the solid or dissolved API into an enteric capsule may provide the desired release profile with minimal handling, according to Lyon.

Other release profiles, however, such as a zero-order release profile, will likely require a wet granulation step to obtain a homogeneous drug product intermediate prior to the final tableting step, Lyon notes. “Of course, each of these unit operations must be appropriately contained and validated for the required occupational exposure levels for the operators,” he adds.

Because many highly potent drug products are low-dose, small-volume drugs, it is important to understand and manage development of the formulation process, according to Iain MacGilp, director of GMP manufacturing at AMRI. During final formulation, for instance, aseptic filtration can potentially lead to loss of costly drug product via adsorption, which is not acceptable for complex HPAPIs.

With respect to delivery techniques for parenteral HPAPIs, MacGilp adds that it is important to understand and minimize interactions between the drug product and the delivery device while also keeping in mind the volume requirements during different phases of drug development.

There is also significant effort focused on further removing operators from the process to minimize risk, both for operator protection and product assurance, according to MacGilp. That has translated, for example, into the development of robotic systems for product filling. “While these systems can be costly, highly potent drugs often address unmet therapeutic needs and there is a drive to implement advanced formulation filling and delivery technologies to enhance specific safety requirements,” MacGilp observes.

Lyon adds that the biggest recent advances are, in fact, largely around containment. “As HPAPIs become increasingly potent, the need for containment during processing becomes increasingly rigorous,” he states. It is important to remember, he points out, that contained processing is required across HPAPI development and manufacturing, including any particle engineering and other processing required to generate a finished dosage form. “Each of the technologies used is able to be contained to different levels based on its footprint and the need for powder handling,” Lyon adds.

One of the ways to reduce risk during the manufacture and formulation of HPAPIs, at least for liquid HPAPI solutions, is to employ single-use containment systems. “Disposable solutions can be designed to be much more specific for a particular process and are just as safe in terms of band 5 containment, but significantly less expensive than traditional barrier isolation systems,” Haney explains. In particular, disposable solutions are ideal for early-phase products for which it is undesirable to dedicate manufacturing capacity and suites.

At its multiproduct sites, AMRI has implemented a single-use philosophy because it reduces risk of cross-contamination, which is a key issue for HPAPIs. “Anything that is used for our formulation process or the pathway for delivery into the vial is one-use and then disposed of,” MacGilp says. In fact, the company has moved the upstream formulation process to a single-use isolator that can contain any compound below 0.1 µg/m³. The system has been fully qualified by SafeBridge and is used as a single-use system on a routine basis.

“We are excited to blend the manufacturing controls of traditional barrier isolators and hard-sided containment with new opportunities that are more flexible and single use,” concludes Haney.

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


Vol. 44, No. 12
December 2020
Pages: 20–22, 46

When referring to this article, please cite it as C. Challener, “HPAPI Complexity Prompts New Delivery Mechanisms,” 

Operator, caregiver, and patient safety are at the forefront when selecting the best options and dosage forms.

HPAPI Complexity Prompts New Delivery Mechanisms

Biologic drugs are highly valuable in treating diseases, and assurance of sterility and proper final product quality is essential. Appropriate testing must be conducted to ensure the filled vials, syringes, cartridges, etc. contain the correct quantity of sterile, contaminant-free product. Aseptic process simulations are crucial for the assessment of aseptic filling capabilities.

The main goal during aseptic processing is to prevent contamination of the final product by microbes, particulates, or pyrogens. That prevention requires control of incoming materials and active supplier quality management, appropriate facility design, and advanced technologies involved in final formulation and filling processes, as well as consideration of sensitive product characteristics for biological drugs (i.e., sensitivity to light, temperature, mechanical stress, and oxygen), according to Oliver Kurz, vice president quality assurance at Vetter. Management of the ‘human’ factor also must be addressed. “A holistic approach considering all relevant aspects is key to the successful validation of aseptic filling operations. The intent is to validate the process from an aseptic perspective and any negative impact on sterility must be ruled out,” he asserts.

A number of elements must be incorporated to ensure quality filling processes. April Peters, director of quality assurance at Catalent, lists a full cleaning and sanitization program, area clearances to ensure reduced risk of product mix-ups, use of checklists for setting up equipment, and use of engineering tools to ensure that equipment is aligned and ready for operation among the essentials. She also notes that the use of an automated environmental monitoring system reduces operator intervention and risk of bioburden introduction, while automated clean-in-place and steam-in-place systems maintain sterility assurance for lines.

As importantly, Peters says, strong aseptic technique training and intervention programs provide a solid foundation to ensure operators maintain the Grade A environment. Frameworks must also be built to ensure smooth communication and proper escalation. “Catalent Biologics’ facility in Bloomington, IN, has incorporated a shop floor [quality assurance] QA team to observe operations and review documentation while fills are in progress. By partnering with manufacturing, corrections are addressed in a timely manner, reducing deviations,” she observes.

An assessment of each process step should be made to identify risks and describe the problems so that they can be evaluated and eliminated, or suitable management mechanisms can be constructed, according to Jim Donovan, vice-president of Pfizer CentreOne Operations. “A robust contamination control strategy (CCS) based on the risks identified ensures the required controls, preventions, and detection systems are in place to maintain product quality,” he states.

CCS, Donovan adds, is a holistic approach that considers but is not limited to aspects related to the environment (cleaning, sanitization, and environmental monitoring programs), the product/process (introduction of appropriate and effective bioburden-reducing and sterilization steps), the equipment and technology (fit-for-purpose, validated, and established through use of quality by design [QbD] and innovation), and personnel (trained and qualified).

The best approach, agrees Maria Lacourt, director of manufacturing operations for Alcami, is to conduct a holistic assessment of all the controls for the process and work toward a facility design that ensures proper execution of the process. “Beyond meeting requirements for the relevant room classification, the right equipment must be identified that enables optimum performance within appropriate containment systems such as isolators, which ensure reduced intervention of operators during aseptic processing,” she says.

In addition to proper training and qualification of staff executing the filling process, Lacourt also stresses the importance of managing the materials used in the process. “It is important to identify vendors, establish container closure integrity, and assess all of the properties of the materials that can impact product quality attributes,” she explains.

Sterile products can be aseptically filled into a variety of containers, such as vials, prefilled syringes, cartridges, and ampoules. While the general process steps (washing, sterilization/depyrogenation, filling) do not vary depending on the type of container processed, the complexity and details can vary significantly, according to Kurz. “With containers of different shapes and opening sizes, the aseptic exposure risk will be accordingly different,” adds Yiwei Li, senior engineer of drug product 4 (DP4) at WuXi Biologics.

The type of filling line—open Grade A, restricted access barrier system (RABS) or isolator—can also impact the number of steps involved, as can the method used for sterilization (filtration vs. thermal or chemical treatment) and whether or not lyophilization is required, according to Donovan.

Peters gives several examples. “Sterility assurance steps may vary depending on the components and filling line utilized for a product. Vials intended for one line may be ready-to-use and ready-to-sterilize for another filling line requiring washing and depyrogenation. Likewise, stoppers may be ready-to-use or need to be steam-sterilized and require additional processing steps.”

“Facilities that manufacture different product presentations or products filled in multiple container types may apply a family or matrix approach to qualification, although there should be a strong scientific and/or risk-based justification for doing so,” Donovan notes.

Aseptic process simulations (APS), also referred to as media fill studies, are critical to qualification of an aseptic facility, according to Li. They demonstrate, adds Lacourt, the ability of the filling process to operate consistently all of the time due to the design of the process, facility, equipment, and the training of the personnel. “The goal is to determine the microbiological risk associated with the performance of specific interventions and to challenge the way those interventions are executed in a simulation to confirm that they do not in any case affect the sterility of any manufactured product,” she says.

The APS is used to challenge the aseptic capability of the manufacturing process from the point of view of product, equipment, and component sterilization through container closure and subsequent processes that may impact unit integrity, adds Donovan. “Their purpose is to assess the entire process from beginning to end for weaknesses, which may result in microbial contamination of your final product,” he says.

In Vetter’s experience, according to Kurz, all aseptic operations should be assessed within a properly designed media fill. The testing should take into consideration careful inherent and corrective interventions as well as worst-case conditions like maximum permitted holding or duration times. “A comprehensive understanding of the process and its related aseptic criticalities that takes into consideration the variances during routine operations is essential to effectively conduct successful aseptic process simulations,” he remarks.

If done correctly, media fill studies provide information on processing operations that may affect the sterility of the final product and the performance of aseptic filling personnel under operating conditions, according to Donovan.

Media fill execution is also used to certify the personnel who will participate in the sterile manufacturing process, according to Lacourt.

Each APS must reflect the processes used on the targeted filling line and consider the specific processing elements used for manufacture on that filling line, states Donovan. “There are many elements to the manufacturing process that can impact or contribute to the sterility of an aseptically manufactured product. Balancing these considerations into the APS in a representative way will likely be a primary topic during a regulatory inspection. If the tests are performed correctly then the boundaries of the manufacturing process will be demonstrably represented by the media fill process. The negative growth of all units in the media demonstrates the line’s capability to aseptically manufacture,” he continues.

Critical components to consider for aseptic simulations include the frequency of media fills, the type of filtration used, the number of aseptic connections, the interventions, personnel qualification, environmental monitoring, allowable rejects, incubation temperatures and durations, who is completing the inspection, when samples are taken for growth promotion, accountability for filled units, and processing hold times, according to Amanda Adams, director of validation and quality engineering for Catalent. Others, according to Donovan, include the line speed, vial size, the number of operators, the length of time an operator can remain in the aseptic area, and equipment and component hold times.

Design of an APS should be scientific, risk-based, and mimic the production process, incorporating the contamination risk factors that occur on a production line, Donovan asserts. It should also be designed not to impede the recovery and growth potential of the media used in the process. In addition, the APS should reflect the overall process validation and simulate manufacturing operations including “worst case” activities and conditions as identified during risk assessment, according to Li.

“Setting up an APS program should include defining the aseptic processes, performing a risk assessment for each process, and evaluating each process for key control points and key factors that could present a risk of microbial contamination of the 
product,” Li says, noting that at WuXi Biologics, a bracketing approach is used because multiple products or dosage forms are being filled or processed in the same facility.

Lacourt agrees that the key to performing successful media fill studies is development of a validation master plan that includes the strategy that will be followed, the components that will be used, the aseptic process times to be challenged, and the validation approach. Personnel must also be trained to ensure proper execution of each step and appropriate documentation must be maintained, she adds.

There is significant complexity involved in coordinating and scheduling an APS in a dynamic manufacturing environment that manufactures multiple products with variants in batch size, on multiple filling lines using variants in primary packaging components and supported by personnel on multiple shift patterns, according to Donovan.

In addition, often not everyone understands the need and requirements of media fills, Adams observes. It is also important to ensure operators understand that units that may be rejected during a routine manufacturing batch would not be considered a reject during a media fill. “Communicating the rationale to execute specific requirements can become a challenge as a result,” she comments.

The best way to manage these challenges, observes Lacourt, is the use of a structured program within a good quality system combined with planning the execution of media fills.

Demonstrating the quality of a filling process requires performance of effective and validated analytical methods. While each product will dictate what analyses are required, there are a few critical aspects that must be incorporated for those that are manufactured in aseptic filling lines, according to Adams.

If aseptic filtration is performed, then filter integrity testing data are key to support sterility of the batch. “Pre- and post-use filter integrity testing is essential to ensure that material is sterile-filtered. Implementing a filter testing protocol specifically for a given filter design can be challenging, but these difficulties can be overcome by working with filter or sterile filtration assembly manufacturers,” Adams says.

Periodic assessment of fill weights should also be conducted during aseptic filling to ensure that the required volume per container meets specifications, according to Adams. This information provides continuous feedback that the line is functioning as intended.

During media fills (APS), microbiological testing should encompass all of the testing completed during the manufacturing process itself, according to Lacourt, including pre- and post-use filter testing and sterility testing. Sterility tests, adds Adams, are critical to assessing the successful execution of aseptic processing, and procedures on sample collection and testing performance should be documented.

Each unit in a media fill must be inspected for turbidity by qualified inspectors to confirm that the aseptic filling operation can produce sterile units, Adams continues. Furthermore, the number of units filled and incubated should be documented, as should the number of units rejected pre-incubation with the cause for rejection, according to Li. After incubation, he notes that the number of units experiencing positive growth and their relevant tray identities should be recorded, and a detailed investigation conducted to determine the root cause and appropriate corrective/preventive actions.

Growth promotion testing of filled units is also important to demonstrate that microbes of concern can in fact grow under the processing conditions, adds Kurz. “Contact of the nutrient solution with all interior surfaces in the aseptically filled units must be assured. All filled containers should be incubated at two different temperatures with each followed by a visual inspection. Additionally, training of visual inspection operators including the methodology applied to identify microbiological contamination is important to consider,” he says. The resulting microbiological analyses and visual inspection will demonstrate the process capability for product sterility.

Environmental personnel monitoring during the execution of an APS is also important, according to Donovan, including viable and non-viable air sampling, surface sampling (contact plate and swabbing), and evaluation of process air and water for injection. The results are used to assess contamination controls in the manufacturing area and may be used in the event of a failure of the APS to help identify a root cause.

To ensure an effective simulation and result, Li observes that quality unit oversight of the entire process, including observation in real time, is required. “Ensuring appropriate quality oversight during media fills and performance of interventions is essential,” agrees Adams. “Quality oversight provides verification that media fills and interventions are performed and documented appropriately, ensuring the operations' validity,” she asserts.

“Specifically, observation of an APS performed by qualified personnel whose responsibility it is to document aseptic behaviors and maintain a log of any issues that may have occurred is important,” Donovan says. This information can be used as feedback on personnel behaviors for continuous improvement and for root cause analysis.

A final report providing an evaluation of the entire media fill and including a conclusion on the acceptability of the APS is also required, Li remarks.

It is not sufficient to run a media fill once. Routine requalification is an important part of the manufacturing landscape and is performed at a minimum twice per year per filling line, according to Donovan. “Performing ad-hoc APS beyond the routine requirement should be considered in response to significant modifications to equipment or facilities, changes in personnel, facility shutdowns, or as a result of a product sterility failure during routine manufacture,” he adds.

In addition, it is important to recognize that validation of the aseptic capability of filling operations is only one stage in the validation of the overall process, says Donovan. “Other stages that contribute to the success of the aseptic filling validation include: primary packaging container qualification, extractable and leachable studies, equipment qualification, filter validation, environmental monitoring performance qualification, personnel training and qualification, robust manufacturing procedure and batch record development, utility qualification, cleaning validation, cleanroom validation, and sterilization cycle validation,” he explains. Collectively these activities contribute to demonstrating the capability of a process to consistently produce product that will meet the pre-determined product quality attributes.

Aseptic filling involves complex interactions and therefore requires close coordination between the personnel, sterilized product, filling system/line, cleanroom, support facilities, and sterilized filling components to ensure the quality of filling operations for biopharmaceuticals. In recent years, aseptic filling technology has evolved from restricted access barrier systems (RABS) to isolator to even robotic aseptic solutions, drastically improving how drugs are produced in an aseptic manner, according to Li.

WuXi Biologics has several traditional fill lines, but recently added an aseptic filling facility leveraging Vanrx SA25 robotic aseptic filling technology (Vanrx), which greatly simplifies the aseptic fill/finish process via the use of a gloveless robotic isolator and ready-to-use (RTU) and single-use materials. “The system eliminates all human intervention into the filling area and provides an advanced aseptic assurance level,” says Li.

With RTU materials, Li notes that vial washing/depyrogenation, stoppers, and tooling sterilization are no longer required. The use of disposable technology reduces the risk for cross contamination, and thus also the sterility and particle risk. Aseptic media fill studies are also simplified due to the minimized need for intervention simulation. Combined with the smaller footprint, these factors help to reduce the cost of drug product operations, according to Li.

In addition, the system can handle a wide variety and sizes of contamination control strategies (CSS), such as vials, cartridges, and pre-filled syringes. “By design, the CCS formats reduce rejection rates caused by particles and other part defects that are more common in traditional rubber stopper and aluminum crimp seal configurations,” observes Li.

Key in-process and product release analyses with this system include the bioburden and concentration for drug products, the weight check for filling accuracy, and sterility test for drug products, Li notes. The weight check for filling accuracy is performed inline using an automatic sampling and weighing system included with the machine. Sterility and bioburden tests are performed offline in the quality-control lab.

Since the first good manufacturing practice run using the robotic aseptic filling line was completed in WuXi Biologics’ DP4 facility in July 2019, no environmental monitoring excursions have occurred, all five media fills passed with a 100% success rate, and 16 client quality audits revealed no critical findings. Through the end of Q2 2020, the company has successfully filled more than 50 batches of drug product with an acceptance rate of up to 99.8%.

“Since constructing and implementing the Vanrx SA25, we have been able to demonstrate its value for the industry with much improved aseptic assurance, flexibility, capacity, and reliability to support our clients’ complex drug filling needs, particularly in terms of reduced time to market,” Li asserts.

Because aseptic filling is a complex process, a holistic approach to validation and assurance of the quality of this critical operation is essential. “The establishment of a thoroughly designed systematic approach is key to the successful validation of aseptic processes, including fill/finish,” states Kurz.

As a result, building and validating a quality program for aseptic filling operations requires the support of manufacturing, engineering, and technical teams, as well as quality control, according to Peters. “All functions have vested interests in the proper execution of media fills and aseptic fill processes to ensure patients receive life-enhancing and life-saving medicines,” she observes.

Donovan comments that a high level of process understanding is fundamental to successful validation of aseptic filling operations and manufacturing a product that meets quality standards. Using tools like TrackWise (Sparta Systems), an automated tracking system to manage change control and manufacturing investigations; good manufacturing practices; and monitoring process performance through continued process verification are key, he adds.

“The most common failure modes include contamination resulting from human intervention and exposure of critical surfaces before and during filling,” Donovan continues. “Therefore, continued investment in personnel training and development and facility maintenance through preventative maintenance and calibration programs is important. Adopting a QbD approach also results in fewer defects, failures, and investigations, higher yields, and lower costs, all indicators of a well-controlled, managed, reliable quality process,” he states.

For Lacourt, it is important that everyone involved in aseptic filling operations understands that aseptic processes rely heavily on process protocols, personnel practices, equipment utilization, and facility design and controls. “It is the combination of these elements that ensures the exclusion of microorganisms from sterile processes, components, and products. It isn’t sufficient to have the best techniques for intervention if the equipment is not properly designed or the facility is not qualified to ensure contamination control,” she concludes.

A holistic approach to validation and quality assurance is essential.

Prepping Fill/Finish Systems to Ensure Quality Output 

The pharmaceutical industry is no stranger to fermentation. Synthetic biology has been used for the production of both biologic and chemical APIs for decades. While it has not been the predominant method in either sector, the greater sustainability and ability to access novel structures provided by fermentation processes are driving greater interest, particularly for small molecules.

Synthetic biology “will propel companies that understand the right strategy for the distinct value propositions it offers at each step in the production life cycle,” said Gihan Hewage, previously a Lux Research analyst, in a press release (1). “Companies with products in the ideation and early development stages will differentiate most from flexible production and the ability to create novel products, whereas products in later stages of development can leverage the environmental and marketing benefits of the technology,” he added.

Cell culture, while often more suited for the production of biologics, such as proteins and monoclonal antibodies, is more expensive and often more complex. Microbial fermentation tends to be, according to Jose Luis Barredo, biotechnology business director with AMRI, more straightforward and scalable. One key reason is that the organisms have faster and more stable growth patterns and lower intrinsic metabolic burden, notes Filippo Giancarlo Martinelli, head of global industrial partnerships development at Abolis.

In addition, fermentation often involves the conversion of cheap and readily available raw materials (sugar or other cheap substrates) into high-value products with high yields/titers, ensuring the commercially viable manufacture of high-quality products, observes Nikolay Krumov, senior project leader, upstream development microbial at Lonza.

Processes typically proceed at room temperature and ambient pressure in water, avoiding the use of harmful or toxic solvents or chemical raw materials and production of dangerous waste, according to Martinelli. Operators benefit from safer work environments, and drug makers avoid issues associated with the use and production of hazardous materials. Fermentation facilities and equipment may also be less costly to build and install.

Synthetic biology can in some cases also provide access to molecules that cannot be produced via traditional synthetic organic chemistry. “While this concept is not novel per se, over the last several decades, the development of improved tools and understanding have also enabled greatly increased yields and decreased development costs and risks,” Martinelli states. He points to steroids, analgesics, opioids, and some anti-cancer drugs. Similarly, he observes that some phytochemicals that cannot be practically extracted from plant sources may also be produced via fermentation.

Biological and analytical advances enable modern fermentation processes to deliver safe and effective next-generation medicines.