BioProcess International Peptide SPECIAL REPORT Therapeutics

Designing a Science-Led Strategic Quality Control Program

INTERTEK PHARMACEUTICAL SERVICES Your partner for regulatory-driven, phase appropriate analytical programs tailored to your molecule. Our experts help you to navigate the challenges of development, regulatory submission, and manufacturing. Peptide Therapeutics Designing a Science-Led Strategic Quality Control Program

Shashank Sharma and Hannah Lee

ince the emergence of peptide therapeutics in the 1920s with the advent of insulin therapy, the market for this product class has continued to expand with global revenues anticipatedS to surpass US$50 billion by 2024 (1). The growth of peptide therapeutics is attributed not only to improvements in manufacturing, but also to a rise in demand because of an increasingly aging population that is driving an increase in the occurrence of long-term diseases. The need for efficient and low-cost drugs and rising investments in research and development of novel drugs continues to boost market growth and fuel the emergence of generic versions that offer patients access to vital medicines at low costs. North America has been the dominant market for peptide therapeutics, with the Asia–Pacific region Insulin molecular model; the first therapeutic expected to grow at a faster rate. The global peptides use of this peptide hormone was in the market has attracted the attention of key players 1920s to treat diabetic patients. within the pharmaceutical industry, including Teva Pharmaceuticals, Eli Lilly, Novo Nordisk, Pfizer, amino acids to be peptides. Within that set, those Takeda, and Amgen. Those companies have made containing 10 or more are classed as polypeptides. available several highly successful blockbuster There is some ambiguity here. For example, insulin, peptide drugs, including glatiramer acetate, one of the most common peptide therapeutics, is liraglutide, and leuprorelin. Currently, more than 100 classed as a peptide hormone, but it contains 51 approved peptide therapeutics are commercially amino acids. Peptides are a unique category of available for multiple clinical indications such as biopharmaceutical compound. Playing on the cancer (projected to be the fastest growing segment), strengths of both small molecules and protein cardiovascular disease, central nervous system and therapeutics, they combine the size of small metabolic disorders, infectious disease, acromegaly, molecules and their ability to penetrate cell and hematological, gastrointestinal, and respiratory membranes with a protein’s high specificity for its disorders. The development pipeline is robust with target. This enables peptides to interfere with more than 100 peptides in late-stage clinical protein–protein interactions (PPIs) and target or development and more than 200 in preclinical stages inhibit intracellular molecules. Small molecules (1). Within that pipeline, 21 peptides are being struggle with larger binding sites, whereas proteins assessed to address the recent global outbreak of and antibodies cannot cross over the cell membrane COVID-19 (2) (see “Peptides” box). to reach such target sites. Although peptides occur naturally in the body Therapeutic Advantages of Peptides and throughout nature, they are unique in how they The US Food and Drug Administration (FDA) are manufactured for therapeutic benefit. They can considers all biopolymers composed of 40 or fewer be produced synthetically in a laboratory (e.g.,

2 BioProcess International 18(9)si September 2020 SPONSORED Table 1: Analytical methods to determine critical quality attributes (CQAs) for peptide therapeutics Attribute Characteristics Analytical Methods1 Comparative Primary sequence confirmation Peptide mapping approach (LC-MS/MS) covering full sequence physiochemical and confirmation, posttranslational modifications information (if applicable) structural and confirmation of disulfide (if peptide contacts cysteine) characterization N-terminal sequence Edman degradation Amino acid composition Amino acid analyzer Intact mass analysis MALDI ToF/LCMS/ESI MS Secondary structure profiling CD (near, far, and thermal denaturation), FTIR, DSC, XRD High-order structure NMR 1D (hydrogen-1 and carbon-13), 2D (TOCSY and NOESY) Optical purity GCMS Chiral confirmation GCMS/LCMS Impurity profiling Oligomer/aggregation UV-SEC-MALS, SV-AUC, DLS, SDS-PAGE, CE SDS Charge variant profiling iCIEF/IEX Peptide-related impurity profiling UPLC/HPLC-HR-MS/MS, RP-HPLC Biological comparability Cell-based assay/immunoassay Using specific cell line or kits Bioidentity test As per monograph, if available

1 LC = liquid chromatography, MS = mass spectroscopy, MALDI = matrix-assisted laser desorption/ionization, ToF = time of flight, ESI = electrospray ionization, CD = circular dichroism, FTIR = Fourier-transform infrared, DSC = differential scanning calorimeter, XRD = x-ray diffraction, NMR = nuclear magnetic resonance, 1D = one dimensional, 2D = two dimensional, TOCSY = total correlation spectroscopy, NOESY = nuclear Overhauser effect spectroscopy, GC = gas chromatography, UV = ultraviolet, SEC = size-exclusion chromatography, MALS = multiangle light scattering, SV-AUC = sedimentation velocity analytical ultracentrifugation, DLS = dynamic light scattering, SDS = sodium dodecyl sulfate, PAGE = polyacrylamide gel electrophoresis, CE = capillary electrophoresis, iCIEF = imaged capillary isoelectric focusing, IEX = ion-exchange chromatography, UPLC = ultraperformance liquid chromatography, HPLC = high-performance liquid chromatography, HR = high resolution, RP = reversed phase oxytocin) but also can be produced using bacteria guidelines for peptide therapeutics. Depending on and other living organisms as well as recombinant their length, intended clinical indication, and DNA technology (e.g., insulin). That gives peptides manufacturing technology, peptides can be regarded another dynamic advantage over their protein and as conventional chemical molecules or as biological small-molecule counterparts. In recent years, entities or biosimilars. Regulatory guidance is peptides of smaller size with optimum balance of outlined by the International Council for conformational rigidity and flexibility have become Harmonisation of Technical Requirements for promising candidates for targeting challenging Pharmaceuticals for Human Use (ICH) for small- binding interfaces with satisfactory binding affinity molecule drugs and their impurities (3). Regulatory and specificity. guidance for biologicals also is available, and guidance for biosimilars is being developed. Regulatory Expectations and Challenges Currently, however, the industry lacks guidelines for The FDA regulates peptides under the Food, Drug, the regulation of peptide drugs, resulting in a and Cosmetic (FD&C) Act, whereas a protein is regulatory vacuum. That presents a challenge to regulated under the Public Health Service Act. With peptide manufacturers. many peptide drugs already on the market, the main As per the FDA mandate, peptide products that regulatory challenge is to establish quality control have fewer than 10 amino acids and are made parameters of active pharmaceutical ingredients through chemical synthesis fall into the jurisdiction (APIs) and/or drug substances before manufacturing of new drug approval (NDA) pathways; those made finished drug products. When developing a quality through recombinant DNA (rDNA) technologies must control (QC) strategy, several tests — including those go through the biologics license application (BLA) to evaluate impurities and high–molecular-weight process. A product approved as an abbreviated NDA impurities as well as amino-acid analysis (AAA), (ANDA) is considered to be a generic and moisture determination, and microbiological testing interchangeable with its reference product (4). — should be considered when setting specifications However, a product approved through biosimilar to evaluate the final quality of a product before its pathways will need to demonstrate interchangeability release. because of the differences in manufacturing An additional challenge within the regulatory technologies between biosimilar peptides and those of landscape is the limited availability of specific reference products. During drug development,

SPONSORED September 2020 18(9)si BioProcess International 3 Peptides: A Potential Therapeutic Against COVID-19 The unprecedented impact of COVID-19 is accelerating the for cell entry offer a potential treatment option by blocking development of next-generation vaccines and therapies key regions of the spike protein, disabling the virus, and around the world to help prevent infection and to aid patient preventing infection in human cells. As intrinsic signaling recovery. The range of COVID-19 therapy and vaccine molecules for many physiological functions, peptides technology platforms is broad, with products based on present an opportunity for therapeutic intervention to disrupt nucleic acid (DNA and RNA), antibodies, small molecules protein–protein interactions (PPIs) and target or inhibit and peptides, virus-like particles, and live–attenuated or intracellular molecules such as receptor tyrosine kinases. inactivated viruses. According to GlobalData’s Pharma Because peptides generally offer greater efficacy, safety, Intelligence Center Pipeline Drugs Database, 21 peptide and tolerability in humans than do small molecules, and they drugs are in development to target COVID-19, including 15 can penetrate cell membranes because of their smaller size synthetic peptides in development for treatment of compared with proteins, peptides have emerged as both respiratory illnesses caused by SARS-CoV-2 infection. Five therapeutic and vaccine candidates against COVID-19. synthetic-peptide candidates already are being tested in However, development of peptide therapeutics or vaccines patients with COVID-19: four candidates are in phase 2 and in response to COVID-19 needs to be rapid to get such one is in phase 1. The pipeline also includes one products through all clinical phases to market while ensuring investigational new drug (IND)/clinical trial application (CTA) the highest levels of safety and efficacy. That can be filed candidate, two preclinical-stage candidates, and six achieved only through the application of validated analytical discovery-stage candidates. methods. The need for specialist, bespoke analytical To infect cells, the virus uses its spike protein to attach to approaches to understand all critical quality attributes and the angiotensin-converting enzyme 2 (ACE2) receptor, a build strategic quality control programs never has been protein on the surfaces of many types of human cells. more important. Compounds that target the interactions of proteins required interchangeability faces additional regulatory hurdles adequately characterizing peptide products for ANDA because a biosimilar peptide manufacturer needs to submission. However, because of developments in provide sufficient data to demonstrate the technologies for peptide synthesis and pharmacokinetic, pharmacodynamic, efficacy, and characterization, the FDA now believes that an ANDA safety profiles of a biosimilar peptide with the applicant can demonstrate pharmaceutical reference product at recommended doses. equivalence with data that show that the active The European Medicines Agency (EMA) ingredient in a proposed generic synthetic peptide is distinguishes peptides based on the manufacturing the same as that in the RLD that is of rDNA origin. technology used (whether they are derived through rDNA technology or chemically synthesized) rather Scientific Considerations than size. All applications for marketing authorization An important factor in determining whether a for products derived from biotechnology or considered generic drug product meets statutory requirements to be potentially innovative are required to follow a for approval is whether its active ingredient is the centralized procedure. That solid framework, which “same” as that in the RLD. Physicochemical has pioneered the regulation of biosimilars, allows characterization and biological evaluation can be approval to be awarded centrally through the EMA performed to establish the sameness of the active rather than nationally. Marketing authorization ingredient in a proposed generic synthetic peptide. holders (MAHs) can market their products and make Comparative testing of a proposed generic synthetic them available to patients throughout the European peptide and RLD product also is recommended. Union (EU) based on a single marketing authorization. Critical attributes (Table 1) to assess include the For the FDA to approve an ANDA, an applicant following: must demonstrate that a proposed generic drug has • primary sequence and physicochemical the “same” active ingredient, conditions of use, properties dosage form, route of administration, strength, and • secondary structure labeling as the reference listed drug (RLD) and that • oligomer/aggregation states it is bioequivalent. The analytical methods used • biological activities (by in vitro or animal must be adequate to ensure and preserve identity, studies). strength, quality, and purity of a peptide. In the Regulatory agencies encourage an orthogonal past, analytical methods were not always capable of approach to characterize those properties to ensure

4 BioProcess International 18(9)si September 2020 SPONSORED To meet development milestones and comply with regulatory requirements, HIGH-QUALITY bioanalytical and analytical data are needed to aid informed decision-making and identify sources of risk.

different structural conformations. And because Regulatory agencies recommend the application of peptides typically do not have a folded globular sensitive and high-resolution analytical procedures. structure, they are susceptible to immune-system targeting and can be be destroyed easily by precision, selectivity, and confidence in results proteases. Thus, the objective of peptide generated. formulations is to preserve a stable equilibrium of The level of impurities also is a critical factor to the peptide, rather than to achieve and preserve the measure. A proposed generic synthetic peptide most stable conformation — as is the case with larger should not contain impurities at levels greater than proteins that have tertiary and quaternary those found in the RLD. All impurities, including structures. As such, the focus of biophysical new ones, should be justified to help ensure that a characterization for peptides is on the assessment of generic drug does not pose a safety risk with respect stability liabilities caused by changes in formulation to immunogenicity. Impurities to evaluate include that could affect secondary structure and self- the following: association, which could lead to formation of • peptide-related impurities irreversible and/or reversible species in solution. • host-cell–related impurities Heterogeneity and the associated formation of • other (non–peptide-related) impurities. subvisible species can complicate formulation, Regulatory agencies recommend the application characterization, release testing, and stability of sensitive and high-resolution analytical assessment. So robust assessment and thorough procedures (e.g., ultra–high-performance liquid understanding of the relationship between chromatography with high-resolution mass formulation parameters and biophysical properties spectrometry, UHPLC-HRMS) to detect and are critical to ensure that a high-quality peptide characterize peptide-related impurities in a proposed drug product is produced. generic peptide for comparison with the RLD. Higher-Order Structure: Structural characterization of peptides can be performed by Analytical Techniques using techniques such as circular dichroism (CD), for Quality Control Assurance Fourier transform infrared (FTIR) spectroscopy, and To meet development milestones and comply with nuclear magnetic resonance (NMR) spectroscopy. regulatory requirements, high-quality bioanalytical Each technique has limitations, and its use depends and analytical data are needed to aid informed on the system being studied. If the intention simply decision-making and identify sources of risk. Safety is to examine a peptide’s structure, NMR provides assessment is key because slight changes in the details at the atomic level. However, if a quick structure, physicochemical properties, stability, and analysis of secondary structural features is required, impurity profile of a peptide can provoke an adverse then CD and FTIR are better techniques for analysis. immune response in its recipients. Some key Fluorescence spectroscopy (with either intrinsic or analytical techniques required as part of a robust extrinsic fluorescence) is another technique that can peptide quality control program are detailed below. provide a quick evaluation of tertiary structure and Biophysical characterization for development of solution stability. Because some peptides do not have safe and robust peptide formulations can be secondary structures, certain techniques — challenging. Because of their small size, peptides particularly CD, FTIR, and fluorescence — might not typically have a shallow equilibrium between be applicable for analysis. Because peptides are

SPONSORED September 2020 18(9)si BioProcess International 5 broad mass range. Size-exclusion chromatography (SEC) has been used for peptide characterization and has become even more versatile because of the addition of new-generation detectors such as multiangle light scattering (MALS). As a result of those new advancements, the sensitivity of the method has increased many fold. Dynamic light scattering (DLS) provides a means for rapid analysis of peptides in different formulations, but it is limited in its ability because of a lack of resolution and a need for high-concentration formulations (because of the small size of peptides). However, DLS can detect very small amounts of aggregates in cases for which other Analytical ultracentrifugation (AUC) provides a noninvasive medium for aggregation techniques would struggle to detect them. NMR analysis for peptides. provides direct structural information in either one- or two-dimensional mode, thereby providing designed to target specific receptors or molecules, information about specific molecular interactions. the above techniques also can be used for assessing Because aggregates usually are intermolecular β-sheet protein–ligand interactions. That is particularly true structures, FTIR spectroscopy also can be used to of fluorescence, in which case anisotropy detect them through the presence of a band in the experiments can be used to determine association/ amide I region that is not seen in native molecules. dissociation kinetics. Fluorescence spectroscopy can provide important Aggregation Profiling: In peptide-based information about conformational stability and pharmaceutical development, peptide molecules are aggregation state. Specifically, extrinsic fluorescence present in different formulations. So in most cases, can be used with external dyes such as solution conditions are not physiological. In such 8-anilinonaphthalene-1-sulfonic acid (ANS), thioflavin nonnative conditions, both thermodynamic and T/S, or Nile red to provide relevant information. kinetic stabilities of molecules can be affected, Disulfide-Bond Analysis: Disulfide bonds are one thereby changing their oligomeric behavior and determinant of the structure of a peptide. Some leading to aggregation. Those aggregates can be disulfide bonds also are known to be key to a caused by either completely nonspecific association molecule’s activity. Thus, it is important to know the of different polypeptide chains (amorphous disulfide bonding present within a biotherapeutic aggregates) such as insulin at high temperatures, or and control associated heterogeneity. Some short they can follow specific association pathways cyclic peptides such as linaclotide are cysteine rich leading to structured aggregates — cross-β-sheet and therefore particularly challenging. structures such as amyloid-like fibrils, as seen with LC-MS and MS/MS are key technologies for glucagon and salmon calcitonin (5). determination of disulfide bonding in peptides. Because peptides aggregate nonspecifically in Depending on the size and sequence of the peptides most cases due to a general lack of structure, before LC-MS, an analyte might be digested, with the multiple approaches can be adopted for analysis aim to generate fragments that each contain a single (keeping in mind that all techniques have disulfide bond. High-resolution mass spectra then limitations). Care needs to be taken when selecting are used to assign those disulfide bonds with MS/MS techniques because an aggregate is not always a data used when required. For some molecules that large conglomeration of molecules. It could be as are either cysteine rich or display cysteine-rich small as a dimer or a trimer. Multiple analytical domains, MS and LC parameters can be optimized to tools are available and have been used for induce limited reduction during analysis, enabling investigating such nonnative oligomeric species. the binding partners of specific cysteines to be Analytical ultracentrifugation (AUC) provides a resolved. noninvasive medium for peptide analysis (often of A challenge in all disulfide-bond mapping products in their original formulations). The main exercises is to minimize scrambling of bonds advantage AUC has over other orthogonal methods is induced by the analytical approach. The level of a high resolution and detection of molecules over a artifactual scrambling can be reduced by optimizing

6 BioProcess International 18(9)si September 2020 SPONSORED sample preparation and carefully analyzing test submission, consider timelines for approval, and set results to provide a high-quality comprehensive data forth the best approach to ensure timely and set for guiding product development. successful product approvals. A robust analytical Product-Related Impurity Analysis: Peptide-based program should be conducted in laboratories that therapeutics can have high degrees of complexity for comply with good manufacturing practices (GMPs) to impurities that encompass sequence variants help you establish and meet specifications and (including insertion or deletion) and degradation support your regulatory submissions. Careful products, including oxidation, isomerization, and selection of techniques to form an orthogonal deamidation. A common approach is LC-UV, which is approach is key to a successful program. Such a used to monitor impurities for small molecules, but strategy should be tailored to a peptide of interest to it is unlikely to detect all impurities present. ensure precision, selectivity, and confidence in the A study published by the FDA advises that results generated. LC-HRMS should be used to ensure that impurities not resolved by LC-UV alone can be monitored (6). References The use of LC-HRMS enables species to be quantified 1 Global Peptide Therapeutics Market, Dosage, Price down to 0.1%, including for species that are not and Clinical Trails Insight 2018–2024. ResearchandMarkets. com, September 2018; https://www.globenewswire.com/ baseline-resolved from the main product peak. news-release/2018/09/28/1586413/0/en/Global-Peptide- Because of the nature of LC-HRMS, users also get Therapeutics-Market-Dosage-Price-Clinical-Trials-Insights- qualitative confirmation of peak identity from the 2018-2024-612-Drugs-in-the-Pipeline-with-a-50-Billion- same assay. Care must be taken when methods are Opportunity.html. set up and validated for impurity analysis of 2 Sabaratnam K. Analyst Briefing: Synthetic Peptides peptides to ensure that all impurities can be Are Promising Therapeutic Candidates for COVID-19. Global Data, May 2020, Report Code: GDHC3171EI. detected. For LC-HRMS, deamidation is a concern. 3 ICH Q6A Specifications: Test Procedures and The mass shift observed for deamidation is Acceptance Criteria for New Drug Substances and New Drug consistent with that of a single carbon-13 isotope. So Products: Chemical Substance. European Medicines Agency, the MS resolution itself cannot discriminate between Amsterdam, The Netherlands, May 2000, CPMP/ deamidation and isotopes. For deamidation, a ICH/367/96. retention-time shift often is observed, but the 4 ANDAs for Certain Highly Purified Synthetic Peptide chromatographic separation achieved needs to give Drug Products That Refer to Listed Drugs of rDNA Origin: Guidance for Industry. US Food and Drug Administration: baseline resolution between analytes to ensure that Rockville, MD, October 2017; https://www.fda.gov/ quantification is not compromised. Because different media/107622/download. sites of deamidation can lead to different retention 5 Caputo N, et al. Mechanisms of Glucagon times (5), each possible site of deamidation needs to Degradation at Alkaline pH. Peptides 45, 2103: 40–47; be assessed independently, ideally with the use of https://doi.org/10.1016/j.peptides.2013.04.005. standards for comparison. 6 Zeng K, et al. Liquid Chromatography–High Resolution Mass Spectrometry for Peptide Drug Quality Control. AAPS J. 17(3) 2015: 643–651; https://doi. Strategic Quality Control Programs org/10.1208/s12248-015-9730-z. Keeping up with the changing regulatory landscape c and the different premarket requirements for peptide therapeutics can be challenging. Peptides can fall Shashank Sharma, PhD, is business development manager, and into one or more regulatory categories (as Hannah Lee, PhD, is structural characterization team leader, both at Intertek Pharmaceutical Services, Hexagon Tower, Blackley, conventional chemical molecules, biological entities, Manchester, UK M9 8GQ; 44-161-721-5247; shashank.sharma@ or biosimilars), and difficulties increase when a intertek.com; [email protected]. developer works across multiple jurisdictions. Developing a well–thought-out QC strategy is key to identifying the most efficient route to market for a peptide therapeutic, whether approval is needed in one or multiple jurisdictions. A good regulatory strategy for peptides will outline the scientific and technical data requirements for each market of interest, identify data gaps that will need to be addressed before

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