Critical Quality Attributes of Lipid Nanoparticles and In Vivo Fate

Science-based bioprocess design for filamentous fungi

Use of Raman spectroscopy and PLS for the quantification of critical quality attributes in biopharmaceutical products.

The traditional drug manufacturing process involves numerous qualitative attributes that directly impact product quality. The Process Analytical Technology approach considers that to enhance process control, identifying critical process parameters and critical quality attributes that affect the manufacturing process is very much necessary. The Ayurvedic drug manufacturing process is more driven by fuzzy qualitative attributes. The present study was executed to identify the Critical Process Parameters and Critical Quality Attributes in the manufacturing process of a herbo-mineral formulation, viz. Arogyavardhini Rasa. Fuzzy set Qualitative Comparative Analysis (fsQCA) methodology was adopted to observe and identify the critical parameters. The study was executed in three steps, viz. Data collection, Data arrangement, and Analysis. The raw data collected was arranged and analyzed in the software R studio using the package QCA in four steps, viz. Calibration, Analysis of Necessity and Sufficiency, Truth Table construction, and Minimization. The results obtained show that Size reduction, Mardana, and Drying are the identified Critical Process Parameters that, in combination, lead to the outcome, i.e., good product quality. Thus, this study proves that Fuzzy Set Qualitative Comparative Analysis can be used as an efficient tool for the identification and measurement of the Critical Process Parameters that affect the Critical Quality Attributes and, thereby, the product quality in the manufacturing of Arogyavardhini Rasa.

The number of nucleicacid therapeutics is set to growwithin thepharmaceutical industry sector, deploying nanocarrier-based deliverysystems as drug products. Poly­(A) is a widely used model sequenceused in lipid nanoparticle (LNP) formulations for which there areno reported critical quality attributes (CQAs) such as molecular weight,chain length, and impurity profile. In this study, we analyze Poly­(A)from three different vendors to measure any existing differences intheir CQAs. Poly­(A) from these brands was encapsulated in SM102 LNPsusing microfluidics to produce three branded Poly­(A)-based LNPs. Weutilized an orthogonal analytical pipeline approach for both Poly­(A)drug substance and LNP drug product CQA evaluation, which includeda combination of dynamic light scattering and flow field flow fractionationmultiplexed with inline UV, dynamic, and multiangle light-scatteringdetectors. Similar purity (260/280) values of >3 were obtainedacrossall three brands of Poly­(A); however, distinct differences in molecularweight and chain length distributions were identified across Poly­(A)brands, with this study representing the first to apply EAF4 methodologyfor in-depth characterization of model RNA drug substances. BrandA produced a smaller and broader molecular weight distribution, followedby Brand B, and then Brand C produced the largest molecular weightspecies and the most uniform molecular weight distribution. On encapsulationin LNPs, differences seen in Poly­(A) CQAs did not translate to CQAdifferences in resultant LNPs. We show that a deeper understandingof drug substance CQAs and their subsequent impact on resultant overalldrug product characteristics is needed on a case-by-case basis. Weshow correlations between analytical pipelines, with future work investigatingthe impact of RNA molecular weight in LNP formulations with differentlipid compositions and using these correlations in AI or machine learningto further enhance our knowledge of the correlation between drug substanceand resultant drug product CQAs.

Characterization of lipid nanoparticles using macro mass photometry: Insights into size and mass.

Introduction . Quality, along with efficacy and safety, are the key characteristics of a drug. Therefore, it is important in frame of pharmaceutical development to lay the foundation for obtaining a quality product and achieving the desired product characteristics. One of the tools used for this is the «Quality by design» approach (QbD) - quality through development. The ICH Q8 «Pharmaceutical development» manual defines it as a systematic approach to development that starts with pre-defined goals and focuses on understanding the product and process, as well as managing the process based on reliable scientific data and quality risk management. Aim . The aim of the research is to develop the composition of tablets based on clover meadow grass phytosubstance using the QbD tool with application of mathematical model that links the composition of tablets (CMA) with its quality attributes (CQA). Materials and methods . The research was based on the concept of quality by design/quality through development. The main method for conducting the development process was design of experiments method/experiment planning with creating of individual design. The experiment planning was performed in the software package JMP Pro 14 (ver. 14. 3. 0), SAS Institute Inc., USA. The method of risk analysis-failure mode and effects analysis (FMEA)/analysis of the types and consequences of failures was used for risk assessment in research. As methods of analysis of the tablet mixture and the tablets were used the following the tests: disintegration of tablets, crushing strength of tablets, determination of hygroscopicity, determination of Carr's and Hausner index. Results and discussion . The composition of tablets based on the clover meadow grass phytosubstance obtained by direct pressing was developed. In order to develop the composition of tablets in accordance with the ICH Q8 guidelines, the first step was to create a quality target product profile. To ensure the properties specified in the quality target product profile, the component composition of the developed tablets was selected based on the selected production technology and key characteristics of both the tablet mixture and the finished product. The risk assessment for the product composition determined that the quantitative content of 4 excipients are considered as critical quality attributes of materials (CMA). CMAs affect the critical quality attributes (CQA) of the dosage form, which determine the effectiveness of the composition. Based on the initial data analysis, optimal content boundaries for each component were established. Potentially critical qualitative characteristics of the drug under development (CQA) were identified. A mathematical model linking critical quality parameters and tablet composition was obtained and analyzed. Conclusion . Based on a mathematical model, the optimal composition of tablet mixture and tablets obtained from it was determined. The tablets quality indicators corresponding to the selected composition were calculated, and the adequacy of the obtained model was evaluated by comparing calculated and real indicators. It is shown that the calculation error does not exceed 10 %, and the proposed optimization algorithm and the model derived from it can be successfully used to optimize the composition of tablets.

The influence of citrate buffer molarity on mRNA-LNPs: Exploring factors beyond general critical quality attributes

Lipid nanoparticles (LNPs), most commonly recognised for their role in COVID-19 mRNA vaccines, are important delivery vehicles for nucleic acid (mRNA, siRNA) therapies. The physicochemical attributes, such as size, nucleic acid encapsulation and electric charge, may have a significant impact on the efficacy of these medicines. In this study, adjustments to aqueous to lipid phase ratios were assessed for their impact on LNP size and other critical quality attributes (CQAs). It was observed that minor adjustments of aqueous-to-organic lipid phase ratios can be used to precisely control the size of ALC-0315-formulated LNPs. This was then used to evaluate the impact of phase ratio and corresponding size ranges on the in vitro and in vivo expression of these LNPs. In HEK293 cells, larger LNPs led to higher expression of the mRNA cargo within the LNPs, with a linear correlation between size and expression. In THP-1 cells this preference for larger LNPs was observed up to 120 d.nm after which there was a fall in expression. In BALB/c mice, however, LNPs at the lowest phase ratio tested, >120 d.nm, showed reduced expression compared to those of range 60–120 d.nm, within which there was no significant difference between sizes. These results suggest a robustness of LNP expression up to 120 d.nm, larger than those <100 d.nm conventionally used in medicine.

A robust manufacturing process and the relationship between intermediate quality attributes (IQAs), critical quality attributes (CQAs), and critical process parameters (CPPs) for high-shear wet granulation was determined in this study. Based on quality by the design (QbD) approach, IQAs, CQAs, and CPPs of a telmisartan tablet prepared by high-shear wet granulation were determined and then analyzed with multivariate analysis (MVA) to evaluate mutual interactions between IQAs, CQAs, and CPPs. The effects of the CPPs on the IQAs and CQAs were quantitatively predicted with empirical models of best fit. The models were used to define operating space, and an evaluation of the risk of uncertainty in model prediction was performed using Monte Carlo simulation. MVA showed that granule size and granule hardness were significantly related to % dissolution. In addition, granule FE (Flow Energy) and Carr’s index had effects on tablet tensile strength. Using the manufacture of a clinical batch and robustness testing, a scale-up from lab to pilot scale was performed using geometric similarity, agitator torque profile, and agitator tip speed. The absolute biases and relative bias percentages of the IQAs and CQAs generated by the lab and pilot scale process exhibited small differences. Therefore, the results suggest that a risk reduction in the manufacturing process can be obtained with integrated process parameters as a result of the QbD approach, and the relationship between IQAs, CQAs, and CPPs can be used to predict CQAs for a control strategy and SUPAC (Scale-Up and Post-Approval Guidance).

Tocilizumab is an immunoglobulin G1 monoclonal antibody targeting the interleukin-6 receptor (IL-6R). BAT1806/BIIB800 (tocilizumab-bavi) has been developed as a biosimilar to the reference product tocilizumab (TCZ). The objective of this study was to demonstrate physicochemical and functional similarity between BAT1806/BIIB800 and TCZ in a comprehensive comparability exercise. A comprehensive panel of over 20 methods was used to generate datasets comparing critical and non-critical product quality attributes for 10 BAT1806/BIIB800 lots and 44 TCZ lots (16 sourced from China, 16 from the EU, and 12 from the US). Primary structure, higher-order structure, and physicochemical properties were assessed using liquid chromatography, mass spectrometry, various spectroscopy techniques/methods, capillary electrophoresis, and thermoanalytical techniques. Fragment antigen-binding (Fab)- and fragment crystallizable (Fc)-mediated biological properties were assessed using cell-based assays, immunoassays, flow cytometry, and kinetic binding assays. BAT1806/BIIB800 and TCZ (irrespective of source) were shown to be similar in terms of structural and functional properties. No differences were observed in terms of the most critical quality attributes, that is, soluble-IL-6R binding and inhibition of IL-6-mediated cell proliferation. BAT1806/BIIB800 and TCZ demonstrated similarity in terms of Fab- and Fc-mediated binding and biological activity. Minor differences were observed in glycosylation (afucosylation and sialylation), glycation, aggregation, and charge variants, which were demonstrated to be not clinically relevant. BAT1806/BIIB800 and TCZ were highly similar for all critical quality attributes. Where differences were observed in less critical quality attributes, additional analytical assessments and clinical study results determined these to be not clinically meaningful.

Biosimilars and haemophilia.

During the development of Critical Embedded Systems (CES), quality attributes that are critical for them (e.g., correctness, security, etc.) must be guaranteed. However, this often leads to complex quality trade-offs, since non-critical qualities (e.g., reusability, understandability, etc.) may be compromised. In this study, we aim at empirically investigating the existence of quality trade-offs, on the implemented architecture, among versions of open source CESs, and compare them with those of systems from other application domains. The results of the study suggest that in CES, non-critical quality attributes are usually compromised in favor of critical quality attributes. On the contrary, we have not observed compromises of critical qualities in favor of non-critical ones in either CES or other application domains. Furthermore, quality trade-offs are more frequent among critical quality attributes, compared to trade-offs among non-critical quality attributes. Our study has implications for both practitioners when making trade-offs in practice, as well as researchers that investigate quality trade-offs.

Aim. To determine the potential risk factors associated with the critical quality indicators of the combined medicine “Neuronucleos” in the capsule dosage form for the treatment of polyneuropathies using the general risk assessment methodology while planning the drug quality at the stage of pharmaceutical development (PD). Materials and methods. The series of the combined medicine “Neuronucleos” in the capsule dosage form, critical indicators of the drug quality, the flowchart of the drug production, critical control points of the drug manufacturing process were developed. The method of causality was used. The quantitative assessment of risk factors was carriedout using the FMECA methodology. Results and discussion. The main objective of this study was to apply the Quality-by-Design (QbD) approach to PD of the combined medicine in the capsule dosage form based on uridine-5 monophosphate disodium salt (UMP), cytidine-5-monophosphate disodium salt (CMF), vitamin B6, thioctic acid and magnesium lactate dihydrate. For better patient compliance and the product quality, a target quality profile as a base for PD planning, as well as critical quality attributes (CQA) related to the product safety and efficacy were determined. The criticality of each CQA was assessed using a special scale. It was shown that “Quantitative content”, “Uniformity of dosage units”, “Dissolution”, “Impurities” were defined as the most CQA due to the minimum amount of UMF and CMF in the capsule and the possibility of their decomposition and increase in the quantity of impurities. The critical attributes of materials (CMA) were identified, and the characteristics required to control them were determined in order to ensure the expected product quality. The primary assessment of the quality indicator risks of the active ingredients was performed. It was found that the particle size affected the homogeneity, the quantitative content of API and dissolution in FPP, and it was shown that the solubility of active substances had a high risk when performing the “Dissolution” test. To determine the potential factors with a significant impact on the drug quality the maximum number of factors was found, and the Ishikawa diagram was constructed. The risk factors associated with the quality and compatibility of active substances and excipients, the quality of primary packaging, production conditions, the drug quality control and the technological process were identified. These factors are the causes of risk and can lead to a situation with negative consequences for the quality of a medicinal product. The FMECA process assessment allowed us to determine the impact of the manufacturing process on the CQA. Conclusions. At the stage of PD for the combined medicine in the capsule dosage form the potential critical indicators of the drug quality have been determined. The critical parameters of the quality of the initial components and the properties of the product have been assessed; the most probable risks for the drug quality have been identified, analyzed and assessed. Further research is advisable to focus on studying the effect of process parameters on critical parameters of the product quality and assessing risks for quality and creating a validation plan and its implementation.

Tyrosine (Tyr) is crucial to the maintenance of the monoclonal antibody (mAb) titers and quality attributes in fed-batch cultures of recombinant Chinese hamster ovary (rCHO) cells. However, the relation between tyrosine and these aspects is not yet fully defined. In order to further elucidate such a relation, two groups of fed-batch experiments with high tyrosine (H-T) or low tyrosine (L-T) additions producing an IgG1 monoclonal antibody against CD20 were implemented to investigate the intracellular and extracellular effects of tyrosine on the culture performance. It was found that the scarcity of tyrosine led to the distinctive reduction in both viable cell density and antibody specific production rate, hence the sharply reduced titer, possibly related to the impaired translation efficiency caused by the substrate limitation of tyrosine. In addition, alterations to the critical quality attributes were detected in the L-T group, compared to those in the H-T condition. Notable decrease in the contents of intact antibody was found under the L-T condition because of the elevated reductive level in the supernatant. Moreover, the aggregate content in the L-T condition was also reduced, probably resulting from the accumulation of extracellular cystine. In particular, the lysine variant content noticeably increased with tyrosine limitation owing to the downregulation of two carboxypeptidases, i.e., CpB and CpH. Overall, understanding the role of tyrosine in these aspects is fundamental to the increase of product titers and control of critical quality attributes in the monoclonal antibody production of rCHO cell fed-batch cultures. KEY POINTS: • Tyrosine is essential in the maintenance of product titers and the control of product qualities in high cell density cultivations in rCHO cell. • This study revealed the bottleneck of decreased qmAbupon the deficiency of tyrosine. • The impact of tyrosine on the critical product qualities and the underlying mechanisms were also thoroughly assessed.

The pharmaceutical industry continues to face challenges in the seamless manufacturing of ibuprofen tablets, despite decades of commercial production. Key issues include the drug’s inherent properties—such as its low melting point (70°C), which causes sticking during compression—as well as solubility and in vitro release challenges due to its BCS Class II classification. Additionally, modifying API properties and approved formulations involves significant regulatory and cost constraints under SUPAC Level 2 changes. While Quality by Design (QbD) approaches have primarily focused on Critical Material Attributes (CMAs) and formulation-based Design of Experiments (DOE), understanding the impact of process variability on Critical Process Parameters (CPPs) remains crucial for ensuring consistent product quality. The integration of Artificial Intelligence and Machine Learning (AIML) in Pharma 4.0 offers transformative potential by enabling predictive analytics, real-time monitoring, and automated decision-making for CPP optimization. Key benefits include precise process control, predictive deviation management, and continuous improvement through data-driven insights. A structured approach involving statistical analysis, machine learning, and process rationalization is essential to minimize variability and align with quality attributes. By leveraging AIML, pharmaceutical manufacturers can enhance efficiency, reduce downtime, and ensure consistent production of high-quality ibuprofen tablets, paving the way for advanced, data-driven pharmaceutical manufacturing. Objective: Identify the optimal Critical Process Parameters (CPPs) for the manufacture of ibuprofen tablets (600mg). Determine the point of control within the specification and control limits to ensure process capability and reliability. Methods: PubMed and Embase databases have been searched, and related studies are compiled and summarized. Results: A designed experiment evaluated critical process parameters (CPPs)—granulation time (3–12 min), drying temperature (45–60°C), compaction force (6–18 kN), and compression speed (10–25 RPM) on tablet quality. Physical, disintegration, and dissolution tests were conducted. Statistical analysis (Jupiter Notebook) revealed correlations between CPPs and critical quality attributes (CQAs), particularly disintegration time (DT) and dissolution %. Conclusion: This study established key correlations between critical process parameters (CPPs) and quality attributes: compression speed/force and granulation/drying times significantly affect disintegration time (DT), while DT shows an inverse relationship with dissolution%. Regression analysis revealed limitations in predictive modeling, emphasizing the need for comprehensive CPP evaluation combined with physical testing. The identified CPP control ranges (9 min granulation, 50°C drying, 14 kN compaction, 16 RPM speed) enable targeted optimization of DT and dissolution%, ensuring therapeutic efficacy. These findings provide a science-based framework for quality-by-design in tablet manufacturing, though continued validation through physical testing remains essential for robust quality assurance.