Analytical considerations for characterization of generic peptide product: A regulatory insight.

The Peptide therapeutics market was evaluated to be around USD 45.67 BN in 2023 and is projected to witness massive growth at a CAGR of around 5.63 % from 2024 to 2032 (USD 80.4 BN). Generic peptides are expected to reach USD 27.1 billion by 2032 after the patent monopoly of the pioneer peptides expires, and generic peptides become accessible. The generic manufacturers are venturing into peptide-based therapeutics for the aforementioned reasons. There is an abundance of material accessible regarding the characterization of peptides, which can be quite confusing for researchers. The FDA believes that an ANDA applicant may now demonstrate that the active component in a proposed generic synthetic peptide drug product is the "same" as the active ingredient in a peptide of rDNA origin that has previously been approved. To ensure the efficacy, safety, and quality of peptide therapies during development, regulatory bodies demand comprehensive characterization utilizing several orthogonal methodologies. This article elaborates the peptide characterization by segmenting into different segments as per the critical quality attribute from identification of the peptide to the physicochemical property of the peptide therapeutics which will be required to demonstrate the sameness with reference product based on the size of the peptide chain and molecular weight of the peptides. Article insights briefly on each individual technique and the orthogonal techniques for each test were explained. The impurities requirements in the generic peptides as per the regulatory requirement were also discussed.

A comprehensive guide for secondary structure and tertiary structure determination in peptides and proteins by circular dichroism spectrometer.

Secondary structure refers to highly regular local sub-structures formed by the polypeptide backbone through hydrogen bonding. The two main types of secondary structures are α-helices and ß-strands (which can form ß-sheets). The development of a robust circular dichroism (CD) method for structural analysis of biomolecules requires careful consideration of several key factors. Solvent selection plays a crucial role in maintaining the native or desired conformation of the sample while ensuring transparency in the relevant wavelength regions. Aqueous buffers are often preferred for studying proteins in their native state. Optimizing the sample concentration and path length is essential to achieve an optimal absorbance range and maximize the signal-to-noise ratio. Typical concentrations for far-UV CD measurements range from 0.1 to 1 mg/ml, with shorter path lengths (1 mm) allowing for higher concentrations and longer path lengths (5 mm) suitable for dilute solutions. Instrumental parameters, such as scanning speed, accumulations, and nitrogen flow rate, significantly impact the quality and reliability of the acquired CD spectra. Data processing is a critical step in obtaining accurate and interpretable CD spectra. Baseline correction, smoothing, and conversion to mean residue ellipticity are essential for reliable secondary structure analysis.

Evaluation of effects of temperature and humidity on the secondary structure of Ganirelix in an injectable formulation and comparison with Orgalutran® using circular dichroism spectroscopy.

Ganirelix, a drug used in in vitro fertilization (IVF), prevents ovulation in women who are not ready to have children by inhibiting a gene that produces gonadotropin. Peptides are macromolecules that are able to preserve a predetermined shape while carrying out the structural and regulatory roles for which they were originally intended. Peptide structures can be altered in the production and storage processes. Therapeutic peptides' biological activity can be drastically altered by even small modifications in their primary and secondary structures. The molecules' secondary structures can be monitored by subjecting them to different processing or storage conditions. In our investigation, we used circular dichroism (CD) spectroscopy with two different software programs for secondary structure evaluation to look at how environmental factors like temperature and humidity affected the secondary structure of Ganirelix in an injectable formulation. The CD results revealed that the alpha-helical (regular and distorted), beta-sheet, beta-strands (regular and distorted), beta-turn, and random coil structures of temperature and humidity stressed generic drug products are comparable to reference-listed drug.

Systematic Selection of Impurities, Development, and Validation of Related Substance Methods for Estradiol and Progesterone in a Combination Drug Product.

BACKGROUND: A kind of estrogen called estradiol is a female sex hormone that regulates several body processes. In hormone replacement therapy for women, the endogenous steroid progesterone is employed. The soft gel capsule medication, which contains a mix of 1 mg estradiol and 100 mg progesterone, is used to treat moderate to severe menopause symptoms, such as hot flashes, in women who have uteruses, as well as feelings of warmth in the face, neck, and chest. OBJECTIVE: To establish the stability of drug products in accordance with the ICH Harmonized Tripartite Guideline, Stability Testing of New Drug Substances and Products (Q1A), this study aims to develop a reverse-phase chromatographic method, the necessary analytical methodology for identifying the drug degradation profiles. METHOD: The procedures were optimized using a C18 column (250 × 4.6 mm, 5 µ) as the stationary phase and conventional solvents such as water-acetonitrile mixtures as the mobile phase. A quantification technique had been an HPLC combined with a UV/PDA and fluorescence detector. RESULTS: The developed methods for both estradiol and progesterone were capable of all components of each active individually by overcoming the extremely difficult challenges of a combination product with similar structural compounds, having 24 impurities in all, and having complex excipients used for soft gel formulation. CONCLUSIONS: The method has been successfully created, validated for use in compliance with regulatory specifications, and determined to be accurate, linear, specific, and stability-indicating in nature. HIGHLIGHTS: To quantify the associated impurities of both actives in the presence of a highly complex matrix, the performance of the HPLC approach considerably possesses a higher degree of selectivity.

Structural Elucidation of Novel Degradation Impurity and Development, Validation of a Single HPLC Method for all Putative Impurities of Clobetasol Propionate in a Foam Drug Product.

Clobetasol Propionate is a highly strong corticosteroid that is used in a variety of topical medication formulations, including foam, ointment, lotion, spray and shampoo; with a dosage strength of 0.05% (w/w). The goal of this research was to identify and characterize a substantial unknown impurity (UK) detected during the stability testing of a Clobetasol Propionate foam pharmaceutical product in accelerated conditions (40°C and 75% relative humidity). Developing a single, robust and accurate HPLC method that is LC-MS compatible for quantifying all 14 potential Clobetasol Propionate impurities in therapeutic drugs is another goal. Preparative column chromatography was used to separate the impurity, and spectroscopic techniques like IR, NMR and MS were used to characterize its structure. The structure of isolated UK was effectively characterized and defined using spectroscopic data evaluation. The chromatographic method has also been validated according to the International Conference on Hominization's Q (2) quality guidelines. The isolated and characterized impurity had the same equivalence as the impurity found during stability testing. Precision, accuracy, linearity, robustness and ruggedness are all met in the method validation data.

Assessment of the Water Sorption Capacity of Rifaximin Using the Dynamic Vapor Sorption Technique for Optimization of the Choice of Excipients and the Manufacturing Environment of Rifaximin Tablets.

BACKGROUND: Rifaximin, a medication of the rifamycin family with two distinct strengths of 200 mg and 550 mg in tablet form, is useful for the treatment of travelers' diarrhea. It has a solid yellow hue and is very hygroscopic in nature. It exhibits a variety of polymorphic forms such as α, ß, γ, δ, and ε depending on bonded moisture. These polymorphs' varying chemical and physical characteristics, such as solubility and water content, may have a big impact on in vivo absorption, which in turn affects efficacy and safety. Therefore, understanding the polymorphic stability of rifaximin is crucial for formulating rifaximin tablets. OBJECTIVE: The current effort focuses on the understanding of water vapor sorption properties to control the polymorphic stability of rifaximin in the tablet formulation using the appropriate selection of excipients and manufacturing process. METHODS: The dynamic vapor sorption method in the range of 0-90% relative humidity at 25°C is used for understanding the sorption properties of drug substances and drug excipient mixtures; the state-of-the-art techniques of the X-ray diffraction method are used to identify polymorph conversions; and dissolution procedures are used for in vitro correlation studies. RESULTS: The sorption study data reveals that rifaximin is highly unstable at relative humidity conditions above 36%. When using excipients that have a low tendency to adsorb water in the formulation, the polymorphic results do not show any change in their intended form, and the in vitro dissolution data show an equivalency with the reference drug product. CONCLUSION: The study prompted a successful outcome-oriented development of the formulation processing environment conditions design to develop a test formulation that has adequate polymorphic stability and also similarity in in-vitro dissolution profiles, with the reference product with highest similarity. HIGHLIGHTS: The overall study described here is useful for swiftly gaining insight into the sorption characteristics of rifaximin, and it contributes to the widespread acceptance of rifaximin tablets as a treatment option.