Development of Quantitative Comparative Approaches to Support Complex Generic Drug Development.

On October 27-28, 2022, the US Food and Drug Administration (FDA) and the Center for Research on Complex Generics (CRCG) hosted a virtual public workshop titled "Best Practices for Utilizing Modeling Approaches to Support Generic Product Development." This report summarizes the presentations and panel discussions for a session titled "Development of Quantitative Comparative Approaches to Support Complex Generic Drug Development." This session featured speakers and panelists from both the generic industry and the FDA who described applications of advanced quantitative approaches for generic drug development and regulatory assessment within three main topics of interest: (1) API sameness assessment for complex generics, (2) particle size distribution assessment, and (3) dissolution profile similarity comparison. The key takeaways were that the analysis of complex data poses significant challenges to the application of conventional statistical bioequivalence methods, and there are various opportunities for using data analytics approaches for developing and applying suitable equivalence assessment method.

Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry.

Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resolution mass spectrometry (HRMS), to identify and quantify isobaric impurity ions resulting from the deletion or addition of a uracil (U) or cytosine (C) nucleotide from or to the full-length sequence. These impurities may each encompass multiple sequence variants arising from various deletion or addition sites. The method utilizes a full or targeted MS analysis to measure accurate isotopic distributions that are chemical formula dependent but nucleotide sequence independent. This characteristic enables the quantification of isobaric impurity ions involving sequence variants, a capability typically unavailable in sequence-dependent MS/MS methods. Notably, this approach does not rely on standard curves to determine isobaric impurity compositions in test samples; instead, it utilizes the individual isotopic distributions measured for each impurity standard. Moreover, in cases where specific impurity standards are unavailable, the measured isotopic distributions can be adequately replaced with the theoretical distributions (calculated based on chemical formulas of standards) adjusted using experiment-specific correction factors. In summary, this streamlined approach overcomes the limitations of LC-MS analysis for coeluting isobaric impurity ions, offering a promising solution for the in-depth profiling of complex impurity mixtures in synthetic oligonucleotide therapeutics.

Topical pharmacokinetics of brinzolamide suspensions in rabbits and variability analysis for sample size and design considerations.

Identifying critical attributes for complex locally acting ophthalmic formulations and establishing in vitro-in vivo correlations can facilitate selection of appropriate thresholds for formulation changes that reflect lack of impact on in vivo performance. In this study the marketed antiglaucoma product Azopt® (1% brinzolamide suspension) and five other brinzolamide formulations varying in particle size distributions and apparent viscosities were topically administered in rabbits, and their ocular pharmacokinetics was determined in multiple ocular tissues. Statistical evaluation with ANOVA showed no significant differences between the formulations in the peak drug concentration (Cmax) in the aqueous humor and iris-ciliary body. As a post-hoc analysis, the within animal and total variability was determined for Cmax in the aqueous humor and iris-ciliary body. Based on the observed variability, we investigated the sample size needed for two types of study designs to observe statistically significant differences in Cmax. For the sample size calculations, assuming both 25% and 50% true differences in Cmax between two formulations, two study designs were compared: paired-eye dosing design (one formulation in one eye and another formulation in the other eye of the same animal at the same time) versus parallel-group design. The number of rabbits needed in the paired-eye dosing design are much lower than in the parallel-group design. For example, when the true difference in aqueous humor Cmax is 25%, nine rabbits are required in the paired-eye design versus seventy rabbits (35 per treatment) in the parallel-group design to observe a statistically significant difference with a power of 80%. Therefore, the proposed paired-eye dosing design is a viable option for the design of pharmacokinetic studies comparing ophthalmic products to determine the impact of formulation differences.

Mechanistic modeling of ophthalmic, nasal, injectable, and implant generic drug products: A workshop summary report.

For approval, a proposed generic drug product must demonstrate it is bioequivalent (BE) to the reference listed drug product. For locally acting drug products, conventional BE approaches may not be feasible because measurements in local tissues at the sites of action are often impractical, unethical, or cost-prohibitive. Mechanistic modeling approaches, such as physiologically-based pharmacokinetic (PBPK) modeling, may integrate information from drug product properties and human physiology to predict drug concentrations in these local tissues. This may allow clinical relevance determination of critical drug product attributes for BE assessment during the development of generic drug products. In this regard, the Office of Generic Drugs of the US Food and Drug Administration has recently established scientific research programs to accelerate the development and assessment of generic products by utilizing model-integrated alternative BE approaches. This report summarizes the presentations and panel discussion from a public workshop that provided research updates and information on the current state of the use of PBPK modeling approaches to support generic product development for ophthalmic, injectable, nasal, and implant drug products.

Product Quality Research for Developing and Assessing Regulatory Submissions for Generic Cyclosporine Ophthalmic Emulsions.

Approval of the first generic 0.05% cyclosporine ophthalmic emulsion (COE) in the U.S. represents a milestone achievement of the science and research program in the U.S. Food and Drug Administration's Center for Drug Evaluation and Research (CDER). COE is a locally acting complex drug product indicated to increase tear production in patients whose production is presumed to be suppressed due to ocular inflammation associated with keratoconjunctivitis sicca. The path to approval required overcoming numerous scientific challenges to determining therapeutic equivalence to the reference listed drug. Researchers in CDER's Office of Pharmaceutical Quality and Office of Generic Drugs developed a quality by design approach to understand the effects of process and formulation variables on the product's critical quality attributes, including globule size distribution (GSD), turbidity, viscosity, zeta potential, surface tension, and osmolality. CDER researchers explored multiple techniques to perform physicochemical characterization and analyze the GSD including laser diffraction, nanoparticle tracking analysis, cryogenic transmission electron microscopy, dynamic light scattering, asymmetric field flow fractionation, and two-dimensional diffusion ordered spectroscopy nuclear magnetic resonance. Biphasic models to study drug transfer kinetics demonstrated that COEs with qualitative and quantitative sameness and comparable GSDs, analyzed using earth mover's distance, can be therapeutic equivalents. This body of research facilitated the review and approval of the first U.S. generic COE. In addition, the methods and fundamental understanding developed from this research may support the development and assessment of other complex generics. The approval of a generic COE should improve the availability of this complex drug product to U.S. patients.

Characterizing how size distribution and concentration affect echogenicity of ultrasound contrast agents.

We investigated the effects of UCA gas bubble size distribution and concentration on the generated ultrasound echogenicity signal. Gas bubble size characterization using Coulter Counter and cryogenic-SEM revealed the hollow structure and rare presence of microbubbles >10 µm in a commercial UCA product, Lumason™. Volume-weighed size and concentration were observed to be more sensitive to changes in UCA bubble stability than number-weighted size and concentration. Size distribution measurements showed that the force (e.g., shaking/agitation energy) used to redisperse the sample did not affect the size distribution, concentration, or echogenicity of the UCA sample. The ultrasound backscattering coefficient (BSC) of size fractionated and serial diluted microbubbles showed that the echogenicity signal correlates most with UCA bubble concentration, especially volume-weighted concentration. Findings from this study may be used to support demonstrating the equivalence of a generic UCA product to the reference listed drug.

Physicochemical surrogates for in vitro toxicity assessment of liposomal amphotericin B.

Pharmaceutical toxicity evaluations often use in vitro systems involving primary cells, cell lines or red blood cells (RBCs). Cell-based analyses ('bioassays') can be cumbersome and typically rely on hard-to-standardize biological materials. Amphotericin B (AmB) toxicity evaluations are primarily based on potassium release from RBCs and share these limitations. This study evaluates the potential substitution of two physicochemical AmB toxicity approaches for the bioassay: Ultraviolet-visible spectroscopy (UV-vis) and in vitro drug release kinetics. UV-vis spectral analyses indicated that liposomal AmB's (L-AmB) main peak position (λmax) and peak ratio (OD346/OD322) are potential toxicity surrogates. Similarly, two first-order release parameters derived from USP-4 in vitro drug release analyses also provided linear relationships with toxicity. These were the initial, overall drug release rate and the ratio of loose to tight AmB pools. Positive slopes and high correlation coefficients (R2 > 0.9) characterized all interrelations between physicochemical parameters and toxicity. These tests converted the manufacturing variables' nonlinear (i.e., curvilinear) relationships with in vitro toxicity to linear responses. Three different toxicity attenuation approaches (2 manufacturing, 1 formulation), covering formulation composition and process aspects, support this approach's universality. These data suggest that one or more spectral and kinetic physicochemical tests can be surrogates for L-AmB in vitro toxicity testing.

Amphotericin B release rate is the link between drug status in the liposomal bilayer and toxicity.

Amphotericin B (AmB) is an amphiphilic drug commonly formulated in liposomes and administered intravenously to treat systemic fungal infections. Recent studies on the liposomal drug product have shed light on the AmB aggregation status in the bilayer, which heat treatment (curing) modifies. Although toxicity was found related to aggregation status - loose aggregates significantly more toxic than tight aggregates - the precise mechanism linking aggregation and toxicity was not well understood. This study directly measured drug release rate from various AmB liposomal preparations made with modified curing protocols to evaluate correlations among drug aggregation state, drug release, and in vitro toxicity. UV-Vis spectroscopy of these products detected unique curing-induced changes in the UV spectral features: a ∼25 nm blue-shift of the main absorption peak (λmax) in aqueous buffer and a decrease in the OD346/OD322 ratio upon thermal curing, reflecting tighter aggregation. In vitro release testing (IVRT) data showed, by applying and fitting first-order release kinetic models for one or two pools, that curing impacts two significant changes: a 3-5-fold drop in the overall drug release rate and a ten-fold decrease in the ratio between the loosely aggregated and the tightly aggregated, more thermodynamically stable drug pool. The kinetic data thus corroborated the trend independently deduced from the UV-Vis spectral data. The in vitro toxicity assay indicated a decreased toxicity with curing, as shown by the significantly increased concentration, causing half-maximal potassium release (TC50). The data suggest that the release of AmB requires dissociation of the tight complexes within the bilayer and that the reduced toxicity relates to this slower rate of dissociation. This study demonstrates the relationship between AmB aggregation status within the lipid bilayer and drug release (directly measured rate constants), providing a mechanistic link between aggregation status and in vitro toxicity in the liposomal formulations.

Characterization of Complex Drug Formulations Using Cryogenic Scanning Electron Microscopy (Cryo-SEM).

The physicochemical properties of complex drug formulations, including liposomes, suspensions, and emulsions, are important for understanding drug release mechanisms, quality control, and regulatory assessment. It is ideal to characterize these complex drug formulations in their native hydrated state. This article describes the characterization of complex drug formulations in a frozen-hydrated state using cryogenic scanning electron microscopy (cryo-SEM). In comparison to other techniques, such as optical microscopy or room-temperature scanning electron microscopy, cryo-SEM combines the advantage of studying hydrated samples with high-resolution imaging capability. Detailed information regarding cryo-fixation, cryo-fracture, freeze-etching, sputter-coating, and cryo-SEM imaging is included in this article. A multivesicular liposomal complex drug formulation is used to illustrate the impact of different cryogenic sample preparation conditions. In addition to drug formulations, this approach can also be applied to biological samples (e.g., cells, bacteria) and soft-matter samples (e.g., hydrogels). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cryo-fixation to preserve the native structure of samples using planchettes Alternate Protocol: Cryo-fixation to preserve the native structure of biological samples on sapphire disks Basic Protocol 2: Sample preparation for cross-sectional cryo-SEM imaging Basic Protocol 3: Cryo-SEM imaging and microanalysis.

Impact of particle flocculation on the dissolution and bioavailability of injectable suspensions.

Injectable suspensions occasionally exhibit variations in dissolution and bioavailability, which may impact the clinical outcome of the drug product. Here, variation in the injection method (i.e., applied shear) for triamcinolone acetonide (TA) injectable suspension (40 mg/mL) altered the flocculation state of the particles and subsequently their dissolution. Notably, TA suspensions contained primary particles of approximately 2 µm and secondary flocculates of tens of microns. The conversion between flocculated and deflocculated particles was rapid, reversible and highly shear dependent. As such, changing shear rates during laser diffraction (LD) measurement like stirring rate, sonication, and sample introduction method (micropipette vs 25-gauge needle) may result in variability in particle size distributions (PSD) that have the potential to alter drug dissolution. Furthermore, a non-sink, discriminatory in vitro release testing (IVRT) method was developed, which combined in-situ fiber optic UV with LD to simultaneously monitor the dissolution and changing PSD of the suspension. The simultaneously measured dissolution and PSD data showed that flocculated and deflocculated particles followed different dissolution pathways. Importantly, deflocculated particles dissolved up to six times faster than the flocculated particles. Similar shear-induced changes during injection could occur in a clinical setting and have implications for drug bioavailability.

Adaptive perfusion: An in vitro release test (IVRT) for complex drug products.

In this work, adaptive perfusion, a pressure-driven separation method based on the principle of tangential flow filtration (TFF) was developed for investigating the rate and extent of drug release from drug products containing particulates, such as emulsions, suspensions, liposomes, drug-protein complexes. The TFF filters were pre-conditioned with unique conditioning solutions and processes to improve the fiber reproducibility and robustness. The adaptive perfusion method achieved size-based separation of the particulates with simultaneous analysis of the released drug as well as remaining drug. By contrast to conventional dialysis methods, the adaptive perfusion method can be used to measure the rate and extent of the drug release from drug solution, drug loaded micelles and nanoemulsions via adjustment of the filter molecular weight cutoff, feed flow rate or back-pressure. Notably, the adaptive perfusion method provided discriminatory drug release profiles for drug in solution, in micelles, and in small, medium, and large globule size nanoemulsions. The drug release profile obtained using adaptive perfusion method was found significantly faster (e.g., minutes rather than hours) and higher (e.g., >60%) than the release obtained using dialysis method. The IVRT method presented here is free from the constraints of rate-limiting factors, such as diffusion through dialysis membrane, and has potential to be extended further to examine the impact of manufacturing process on drug distribution and release characteristics of other challenging complex drug products.

Analyzing ophthalmic suspension particle size distributions using laser diffraction: Placebo background subtraction method.

The current study demonstrated that the presence of excipients can interfere with the measurement of particle size distribution (PSD), a critical quality attribute of ophthalmic suspensions, by laser diffraction (LD) and that a placebo background subtraction approach can eliminate the impact of excipients on the PSD measurement. Commercially available loteprednol etabonate and brinzolamide ophthalmic suspensions were used as model suspensions. The impact of excipients in these formulations on the LD measurements was determined using a one-factor-at-a-time experimental design approach, using National Institute of Standards and Technology (NIST) traceable polystyrene particle size standards as references. Among the evaluated excipients, polymers containing polyacrylic acid were found to interfere with the PSD analysis by creating the LD signals correspond to particles ranging from a few micrometers to a hundred micrometers in size. As a result, the measured PSD of active pharmaceutical ingredient (API) particles in the formulation overlapped with or superimposed on the excipient PSD signal, leading to erroneous interpretation of the API particle size. Additionally, dispersion of brinzolamide particles in unsaturated solutions led to rapid dissolution of brinzolamide particles during the measurement, resulting in underestimation of the particle size range. Here, a placebo background subtraction approach was developed to eliminate the interference of the excipients. This newly developed LD method was also evaluated using orthogonal methods, including polarized light microscopy and scanning electron microscopy (SEM). The strategy used in this study to eliminate the interference of excipients may also be useful for other heterogeneous dispersions where excipient interference may be of concern.

Regulating Generic Ophthalmologic Drug Bioequivalence-Envisioning Accessibility for Patients.

New, brand-name, ophthalmology drug products are developed, investigated, and submitted for marketing approval through premarket interactions with the Food and Drug Administration (FDA). These drug applications for novel drugs are reviewed by FDA for safety and effectiveness before being allowed on the market. Many brand-name drugs are allowed a period of marketing exclusivity and/or have patent protections that can delay generic competition. When these exclusivity periods or patents expire or are challenged (in the case of patents), generic competitors may then market equivalent products, as allowed by U.S. law (eg, Drug Price Competition and Patent Term Restoration Act, often referred to as "the Hatch-Waxman Act"). To be approved as a therapeutic equivalent, a generic product must demonstrate that it is both pharmaceutically equivalent and bioequivalent to the brand-name drug product, which can involve innovative analytical methods and study designs. To facilitate generic drug assessment and approval, the FDA has negotiated the Generic Drug User Fee Amendments (GDUFA) program that funds a rigorous generic drug development program that includes pre-Abbreviated New Drug Application (pre-ANDA) correspondence and meetings, targeted bioequivalence research, and publication of product-specific guidances (PSGs) to support generic drug research and development for manufacturers interested in developing generic drugs for the U.S. market. FDA's regulatory practices include the monitoring of quality and postapproval adverse events of all marketed products, including those for use in and around the eyes.

In vitro physicochemical characterization and dissolution of brinzolamide ophthalmic suspensions with similar composition.

The quality of an ophthalmic suspension is crucial for its in vivo performance, and often impact product's effectiveness. An in-depth understanding of critical quality attributes (CQAs) of ophthalmic suspensions such as particle size distribution (PSD) and rheology, as well as the impact of these CQAs on product performance are important for successful product development, quality control, and regulatory approval. This study employed brinzolamide ophthalmic suspension, 1%, as a model ophthalmic product, and six batches were manufactured using an innovative planetary centrifugal milling (PCM) process. Three batches were manufactured to have distinctly different PSD. These three batches had qualitatively (Q1) and quantitatively (Q2) the same composition as the model drug product (i.e., Azopt), while the differences in PSD were introduced by changing only the manufacturing process parameters. On the other hand, changes in rheology were introduced by altering the input level of the viscosity enhancing polymer in the formulation. A systematic approach was employed to understand the relation between manufacturing process parameters, CQAs, and in vitro product performance. Among the evaluated CQAs, PSD, rheology, surface tension, and drug dissolution were found more sensitive to the changes in the manufacturing processes. Most notably, we developed a rapid dissolution method (completed within minutes) employing in-situ fiber optic UV dissolution system. This novel dissolution method mimics the environmental conditions of the eye such as dissolution under "non-sink" condition and under high shear (from blinking). The method was highly discriminatory to differences in the PSD in the suspension. This study also revealed an important relation between the PSD of the suspension and its rheology which originated as a result of an interaction at the molecular level between the solid drug particles and the viscosity enhancing polymers. These findings underscore the need to evaluate CQAs of the ophthalmic suspensions in concert rather than separately when comparing ophthalmic drug products and product performance.

Evaluating drug distribution and release in ophthalmic emulsions: Impact of release conditions.

The purpose of this study is to investigate the process of drug distribution and mechanism of drug release of ophthalmic emulsions in the context of factors associated with the drug release. Cyclosporine and difluprednate emulsions were chosen as model systems. A kinetic method was used to quantitatively evaluate the drug distribution within a simplified biphasic (emulsion) system. The impacts of release associated factors were investigated, including the amount of sodium dodecyl sulfate (SDS), ethanol, and ionic strength in the release medium as well as the temperature. SDS and ethanol were found to significantly enhance both rate and extent of drug diffusion from oil to aqueous phase for both cyclosporine and difluprednate emulsions. The ionic strength was found to decrease the rate and extent of cyclosporine transfer from oil to aqueous phase but had little impact on the transfer of difluprednate between phases. Diffusion of cyclosporine to aqueous phase exhibited a decreasing trend with increasing temperature due to its atypical temperature dependent solubility in water. Based on our previous method to investigate the impact of formulation variables on drug diffusion and the findings in the current study, a biphasic release model for emulsions is proposed and discussed. Lastly, the underlying relationship of three key quality attributes (i.e., globule size distribution, drug distribution, and release characteristics) and their effect on product quality and performance were discussed. This study provides a fundamental insight into the drug distribution and release in complex emulsion systems. It also elucidates the critical variables for the development of in vitro release method to support regulatory assessment of ophthalmic emulsions and formulation development.

Correction to: Equivalence Testing of Complex Particle Size Distribution Profiles Based on Earth Mover's Distance.

On page 5, in the second paragraph, the authors inadvertently included inaccurate information for the description of the analytical method.

Distinguishing Pharmacokinetics of Marketed Nanomedicine Formulations Using a Stable Isotope Tracer Assay.

The pharmacokinetics of nanomedicines are complicated by the unique dispositional characteristics of the drug carrier. Most simplistically, the carrier could be a solubilizing platform that allows administration of a hydrophobic drug. Alternatively, the carrier could be stable and release the drug in a controlled manner, allowing for distribution of the carrier to influence distribution of the encapsulated drug. A third potential dispositional mechanism is carriers that are not stably complexed to the drug, but rather bind the drug in a dynamic equilibrium, similar to the binding of unbound drug to protein; since the nanocarrier has distributional and binding characteristics unlike plasma proteins, the equilibrium binding of drug to a nanocarrier can affect pharmacokinetics in unexpected ways, diverging from classical protein binding paradigms. The recently developed stable isotope tracer ultrafiltration assay (SITUA) for nanomedicine fractionation is uniquely suited for distinguishing and comparing these carrier/drug interactions. Here we present the the encapsulated, unencapsulated, and unbound drug fraction pharmacokinetic profiles in rats for marketed nanomedicines, representing examples of controlled release (doxorubicin liposomes, Doxil; and doxorubicin HCl liposome generic), equilibrium binding (paclitaxel cremophor micelle solution, Taxol generic), and solubilizing (paclitaxel albumin nanoparticle, Abraxane; and paclitaxel polylactic acid micelle, Genexol-PM) nanomedicine formulations. The utility of the SITUA method in differentiating these unique pharmacokinetic profiles and its potential for use in establishing generic nanomedicine bioequivalence are discussed.

Factors affecting the particle size distribution and rheology of brinzolamide ophthalmic suspensions.

Drug particle size distribution (PSD) and dispersion viscosity are two critical quality attributes that govern the performance of topical ophthalmic suspensions, such as suspension physical stability, ocular retention, and drug release characteristics.. An in-depth knowledge of the effects of formulation and manufacturing process on these critical quality attributes may facilitate the product and process development, quality control, as well as support regulatory policy and approval. The current study has investigated the effect of formulation and process parameters on the quality attributes of brinzolamide ophthalmic suspensions. In the first step, three milling techniques (probe sonication, microfluidization, and media milling with a planetary centrifugal mixer) were evaluated for manufacturing of brinzolamide suspension. Out of the three techniques, the planetary centrifugal media milling yielded the narrowest PSD and thus was considered the most viable lab-scale technique for this purpose. In the next step, various process parameters of media milling were evaluated using a central-composite experimental design. The independent variables included bead size, agitating intensity, and process time while the PSD of drug particles (D50) was the response variable. The effect of shear rate and shear time of the homogenization process and the concentration of carbomer on the rheological properties of the suspension were studied using a Box-Behnken design. Additionally, effects of sodium chloride and mannitol concentration on the rheological properties of the suspension was also investigated. Sodium chloride was found to exert a pronounced effect on rheology of the suspension. Despite variations in the carbomer concentration, a suspension of comparable rheology could be prepared by controlling the process parameters namely the shear rate and process time.

An NMR Protocol for In Vitro Paclitaxel Release from an Albumin-Bound Nanoparticle Formulation.

The paclitaxel protein-bound particles for injectable suspension (marketed under the brand name Abraxane®) contains nanosized complexes of paclitaxel and albumin. The molecular interaction between paclitaxel and albumin within the higher-order nanostructure is analytically challenging to assess, as is any correlation of differences to differences in therapeutic effect. However, because the higher-order nanostructures may affect the paclitaxel release, a suitable in vitro assay to detect potential differences in paclitaxel release between comparator lots and products is desirable. Herein, solution NMR spectroscopy with a T2-filtering technique was developed to detect paclitaxel signal while suppressing albumin signals to follow the released paclitaxel in the NMR tube upon dilution. The non-invasive nature of NMR allows for precise measurement of a full range of dilution-induced drug release percentage from 14 to 92% without any sample extraction. The critical concentration of the drug product (DP) at 50% of release was 0.63 ± 0.04 mg/mL in PBS buffer. In addition, 2D diffusion ordered NMR spectroscopy (DOSY) results revealed that the released paclitaxel experiencing slightly slowed diffusion rates than free paclitaxel, which was attributed to paclitaxel in equilibrium with albumin-bound states. Collectively, the dilution-based NMR method offered an analytical approach to investigate physicochemical attributes of complex injectable products with minimal needed sample preparation and perturbation to nanoparticle formulation.

Optimization of the manufacturing process of a complex amphotericin B liposomal formulation using quality by design approach.

In this work, the manufacturing process of a complex liposomal amphotericin B (AmB) product was optimized using quality by design (QbD) approach. A comprehensive QbD-based process understanding and design space (DS) to the critical process parameters (CPPs) is essential to the drug development and consistent quality control. The process was based on the acid-aided formation of drug-lipid complexes in a methanol-chloroform mixture (step I) followed by spray drying (step II), hydration and liposome formation by microfluidization (step III), and lyophilization (step IV). Firstly, the risk assessment was conducted to identify the critical process parameters among the four key steps. Nine CPPs and five CQAs (API Monomer identity (absorbance main peak at 321 nm), API Aggregation identity (absorbance peak ratio, OD 415 nm/321 nm), particle size, in-vitro toxicity, and the cake quality) were determined based on their severity and occurrences with their contribution to the quality target product profile (QTPP). Based on the risk assessment results, the final screening design of experiments (DoE) was developed using fractional factorial design. Secondly, the empirical equation was developed for each CQA based on experimental data. The impact of CPPs on the CQAs was analyzed using the coefficient plot and contour plot. In addition to the effect of individual formulation parameters and process parameters, the effects of the four key separate steps were also evaluated and compared. In general, the curing temperature during microfluidization has been identified as the most significant CPP. Finally, design space exploration was carried out to demonstrate how the critical process parameters can be varied to consistently produce a drug product with desired characteristics. The design space size increased at the higher value of the curing temperature, the API to phospholipid ratio (API:PL), and the lower value of the DSPG to phospholipid ratio (PG:PL) and aspirator rate.