Pharmaceutical Development of Liposomes Using the QbD Approach

*Alina Porfire, Marcela Achim, Cristina Barbalata, Iulia Rus, Ioan Tomuta and Cecilia Cristea*

## **Abstract**

Quality by Design (QbD) is a systematic, risk-based approach to pharmaceutical product and manufacturing development, which uses quality-improving scientific methods upstream in the research, development, and design phases, in order to assure that quality and safety are designed into product at as early stage as possible. This work focuses on the state-of-the-art applications of the QbD principles in the development of liposomes. The QbD approach has recently been proposed as a useful tool to obtain higher-quality liposomal products, as their development is a challenging task, involving intricate formulation and manufacturing processes. Thus, the current strategies to define the relationship between the critical material attributes or process parameters and product critical quality attributes and to establish the design space are overviewed. Additionally, the current characterization methodologies are described, as part of the control strategy required within the QbD paradigm.

**Keywords:** liposomes, Quality by Design, critical quality attributes, design space, quality control

#### **1. Introduction**

In the recent years, the Quality by Design (QbD) concept has gained importance in drug development and drug manufacturing. QbD is recommended by the drug regulatory agencies (FDA—Food and Drug Administration and EMA—European Medicines Agency) to improve the quality of pharmaceutical products.

For the pharmaceutical products, quality is regarded as a mandatory topic and must be assured for all. Pharmaceutical products' quality is ensured through the ability to get the therapeutic benefit mentioned on the label and through the absence of contamination [1, 2]. Product quality refers to performance, robustness, trustworthiness, and has to be built into it [3]. To ensure the quality of a product, scientific approaches such as QbD can be implemented. The concept of QbD was first defined a few decades ago by Dr. Joseph M. Juran, a well-known pioneer of quality, and emphasizes the design of a product and manufacturing process to reach a certain predefined quality [1, 2, 4]. According to Dr. Juran, quality should be built into a product, and the way in which a product is designed is accountable for most

issues related to its quality. Dr. Juran believed that the quality of a product could be planned and the most quality inadequacies originate from the way in which the quality of the product was planned [4, 5].

In the pharmaceutical field, there are several regulatory guidelines developed by ICH, FDA, and EMA, which offer the necessary information in understanding how the QbD concept may be implemented [2, 3, 6–8]. In ICH Q8—Pharmaceutical Development guideline, QbD is defined as "a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management" [9]. Such a scientific, risk-based, and pro-active approach from the development to manufacturing of a pharmaceutical product will provide the necessary knowledge and information to minimize the risk and ensure a predefinite quality of the product [10]. So, QbD provides the tool to understand the way in which the quality of a pharmaceutical product is influenced by formulation, input materials' characteristics, and process variables; therefore, the quality of the product can be ensured by controlling the formulation input materials and the manufacturing process key variables [2, 4, 11]. It involves designing a formulation and manufacturing process in such a way to obtain a pharmaceutical product with predetermined quality specifications [12]. QbD identifies characteristics that are critical to quality of the product, translates them into attributes that the drug should possess, and establishes how the critical formulation and process parameters can be varied to constantly produce a drug product with the desired characteristics [13, 14]. The goal of the QbD approach is in-depth understanding of the formulation and process variables, and of the relationship between them, in order to obtain a drug product with consistent desired characteristics [15, 16]. Practically, QbD helps establishing the critical quality attributes (CQAs) of the product and how the critical material attributes (CMAs) and critical process parameters (CPPs) can be modified to deliver a product with predetermined quality specifications [12].

A QbD approach includes several key steps, as follows: (1) defining the quality target product profile (QTPP); (2) performing a risk assessment, in order to identify which formulation, material, or process parameters can potentially influence the product's quality attributes (CQAs); (3) studying the impact of the formulation variables, material attributes, and process parameters on the critical quality attributes (CQAs) of the drug product, and finding which of them are critical material attributes (CMAs) and critical process parameters (CPPs); (4) establishing a design space that ensures desired product specifications; and (5) designing and implementing a control strategy in order to ensure a continuous improvement [9, 17, 18]. In order to study the relationship between the CMAs and CPPs and their impact on CQAs in a mathematical form, the Design of Experiments (DoE) strategy is used. This method also allows to establish the design space by running a minimum number of experiments [2, 11, 19]. The design space for liposome preparation may be established by implementing the QbD strategy as a systematic approach in liposome development, in order to improve the product quality, by understanding and controlling formulation, materials, and manufacturing variables. The strategy is recommended by the drug regulatory agencies for the development of betterquality products and may be used in liposomal drug product development.

### **2. Defining the QTPP for liposomal products and identification of the CQAs**

The most important element in using QbD concept to assist formulation and process design is to predefine the desired final product quality profile [9, 14, 20].

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*Pharmaceutical Development of Liposomes Using the QbD Approach*

**QTPP elements Target**

Biocompatibility Microbiological quality Bacterial endotoxins

osmolarity, appearance) Drug identification Drug content *In vivo* stability Drug release Degradation products Residual solvents

Nanoformulation For targeted delivery Parenteral

scientific research

Sterile Free

Lack of hemolytic activity

Must meet the standards resulted from the specifications of similar approved products or from the current

According to the definition of ICH, the QTPP is "a prospective summary of the quality characteristics of a drug product that ideally will be achieved to ensure the desired quality, taking into account safety and efficiency of the drug product" [9]. In order to establish the QTPP, the following considerations must be taken into account: route of administration, dosage form, dosage strength, delivery system, attributes affecting pharmacokinetic characteristics, stability, sterility, and drug

Physical attributes (particle size and morphology, viscosity, zeta potential,

QTPP is established based on prior scientific knowledge and appropriate *in vivo* relevance. According to the current flux of literature, a QTPP for liposomal prod-

When designing a nanoformulation, the efficiency of the final product will be directly related to particle size. Particle size is the most important factor influencing biodistribution, circulation half-life, and cellular uptake. Due to their small size and large surface area, pharmaceutical nanosystems show enhanced bioavailability and additional ability to cross the biological membranes. Furthermore, in cancer therapy, smart nanoparticles deliver the drug into the tumor tissue and avoid normal tissues and organs [22]. After intravenous administration, liposomes accumulate in tumors by a passive or active targeting, for determining higher therapeutic efficiency and less side effects [23]. As a parenteral dosage form, liposomal products must be sterile, pyrogen free, and well tolerated [24]. The lack of hemolytic activity

The second step in a QbD approach is the identification of CQAs. According to ICH Q8 definition, a CQA is a "physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality." They are either derived from the QTPP or defined based on regulatory requirements and review of the literature [9]. Based on these recommendations, for the liposomal products the following CQAs are usually identified: mean particle size, size distribution, and zeta potential (as physical CQAs); drug content (as chemical CQAs); *in vivo* stability and drug

a.Generally, for all nanoparticles, the mean particle size and particle size distribution are major CQAs, which play an important role in determining their *in vivo*

release appropriate for the intended final product [14, 20].

is also mentioned as requirement for liposome products [25].

release (as biological CQAs); sterility (as microbiological CQA).

distribution, drug loading, drug release, and targeting ability.

ucts is presented in **Table 1** along with the targets for each element [21].

*DOI: http://dx.doi.org/10.5772/intechopen.85374*

Dosage form Dosage design Administration route

**Table 1.**

Quality attributes of the liposomal product

*QTPP of liposomal products.*

*Pharmaceutical Development of Liposomes Using the QbD Approach DOI: http://dx.doi.org/10.5772/intechopen.85374*


#### **Table 1.**

*Liposomes - Advances and Perspectives*

quality of the product was planned [4, 5].

issues related to its quality. Dr. Juran believed that the quality of a product could be planned and the most quality inadequacies originate from the way in which the

deliver a product with predetermined quality specifications [12].

A QbD approach includes several key steps, as follows: (1) defining the quality target product profile (QTPP); (2) performing a risk assessment, in order to identify which formulation, material, or process parameters can potentially influence the product's quality attributes (CQAs); (3) studying the impact of the formulation variables, material attributes, and process parameters on the critical quality attributes (CQAs) of the drug product, and finding which of them are critical material attributes (CMAs) and critical process parameters (CPPs); (4) establishing a design space that ensures desired product specifications; and (5) designing and implementing a control strategy in order to ensure a continuous improvement [9, 17, 18]. In order to study the relationship between the CMAs and CPPs and their impact on CQAs in a mathematical form, the Design of Experiments (DoE) strategy is used. This method also allows to establish the design space by running a minimum number of experiments [2, 11, 19]. The design space for liposome preparation may be established by implementing the QbD strategy as a systematic approach in liposome development, in order to improve the product quality, by understanding and controlling formulation, materials, and manufacturing variables. The strategy is recommended by the drug regulatory agencies for the development of betterquality products and may be used in liposomal drug product development.

**2. Defining the QTPP for liposomal products and identification of the** 

The most important element in using QbD concept to assist formulation and process design is to predefine the desired final product quality profile [9, 14, 20].

In the pharmaceutical field, there are several regulatory guidelines developed by ICH, FDA, and EMA, which offer the necessary information in understanding how the QbD concept may be implemented [2, 3, 6–8]. In ICH Q8—Pharmaceutical Development guideline, QbD is defined as "a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management" [9]. Such a scientific, risk-based, and pro-active approach from the development to manufacturing of a pharmaceutical product will provide the necessary knowledge and information to minimize the risk and ensure a predefinite quality of the product [10]. So, QbD provides the tool to understand the way in which the quality of a pharmaceutical product is influenced by formulation, input materials' characteristics, and process variables; therefore, the quality of the product can be ensured by controlling the formulation input materials and the manufacturing process key variables [2, 4, 11]. It involves designing a formulation and manufacturing process in such a way to obtain a pharmaceutical product with predetermined quality specifications [12]. QbD identifies characteristics that are critical to quality of the product, translates them into attributes that the drug should possess, and establishes how the critical formulation and process parameters can be varied to constantly produce a drug product with the desired characteristics [13, 14]. The goal of the QbD approach is in-depth understanding of the formulation and process variables, and of the relationship between them, in order to obtain a drug product with consistent desired characteristics [15, 16]. Practically, QbD helps establishing the critical quality attributes (CQAs) of the product and how the critical material attributes (CMAs) and critical process parameters (CPPs) can be modified to

**72**

**CQAs**

*QTPP of liposomal products.*

According to the definition of ICH, the QTPP is "a prospective summary of the quality characteristics of a drug product that ideally will be achieved to ensure the desired quality, taking into account safety and efficiency of the drug product" [9]. In order to establish the QTPP, the following considerations must be taken into account: route of administration, dosage form, dosage strength, delivery system, attributes affecting pharmacokinetic characteristics, stability, sterility, and drug release appropriate for the intended final product [14, 20].

QTPP is established based on prior scientific knowledge and appropriate *in vivo* relevance. According to the current flux of literature, a QTPP for liposomal products is presented in **Table 1** along with the targets for each element [21].

When designing a nanoformulation, the efficiency of the final product will be directly related to particle size. Particle size is the most important factor influencing biodistribution, circulation half-life, and cellular uptake. Due to their small size and large surface area, pharmaceutical nanosystems show enhanced bioavailability and additional ability to cross the biological membranes. Furthermore, in cancer therapy, smart nanoparticles deliver the drug into the tumor tissue and avoid normal tissues and organs [22]. After intravenous administration, liposomes accumulate in tumors by a passive or active targeting, for determining higher therapeutic efficiency and less side effects [23]. As a parenteral dosage form, liposomal products must be sterile, pyrogen free, and well tolerated [24]. The lack of hemolytic activity is also mentioned as requirement for liposome products [25].

The second step in a QbD approach is the identification of CQAs. According to ICH Q8 definition, a CQA is a "physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality." They are either derived from the QTPP or defined based on regulatory requirements and review of the literature [9].

Based on these recommendations, for the liposomal products the following CQAs are usually identified: mean particle size, size distribution, and zeta potential (as physical CQAs); drug content (as chemical CQAs); *in vivo* stability and drug release (as biological CQAs); sterility (as microbiological CQA).

a.Generally, for all nanoparticles, the mean particle size and particle size distribution are major CQAs, which play an important role in determining their *in vivo* distribution, drug loading, drug release, and targeting ability.

The biodistribution of nanoparticles circulating in the blood stream is considerably influenced by particle size. Smaller particles, of 20–30 nm, are eliminated by kidneys, while larger particles, of up to 300 nm, are taken up by reticuloendothelial system (RES) [26]. By decreasing particle size below 100 nm, RES uptake can be reduced [27]. According to other studies, the ideal nanocarriers should have particle size ranging between 10 and 100 nm in order to evade the kidney filtration and capture by the liver [28]. For example, in tumor targeting, the particles need to be smaller than the cut-off of the fenestrations in the tumor neovasculature [29]. The literature reveals that for an efficient enhanced permeability and retention (EPR) effect, the particle size must be, generally, between 50 and 100 nm, depending on the tumor type, its environment, and its localization [28]. Particle size less than 200 nm is also beneficial for the sterility of the liposomal product, since this size allows aseptic preparation and sterile filtration of the final product [30].

Particle size also influences the kinetics of drug release. As particle size gets smaller, their surface area-to-volume ratio gets larger. This would imply that more of the drug is closer to the surface of the particle compared to a larger particle. Being at or near the surface would lead to a faster drug release [23].

Polydispersity index, reflecting particle size distribution of liposomal products, is a physical parameter for which the target is to have reduced values, indicating a good homogeneity of the dispersion. Generally, polydispersity index values below 0.5 are reported to be acceptable [31].


High encapsulation efficiency is very important for both manufacturers and patients. A higher percentage of drug encapsulation could reduce the manufacturing cost and increase drug concentration in the final formulation allowing greater flexibility in dosing. Depending on the pharmacokinetics, higher drug concentration can result in increased dosing intervals and hence improved patient compliance [30].

**75**

*Pharmaceutical Development of Liposomes Using the QbD Approach*

Drug retention inside liposomes is also very important. Since liposomes are intended to deliver the drug to the target site, there should be no drug leakage

d.In order to have a prolonged drug release and an efficient tumor accumulation by passive targeting, *in vivo* stability is essential. *In vivo* behavior of liposomes after intravenous administration is directly linked to their interaction with blood components, which depends on the hydrophobic/hydrophilic character of liposome surface. The more hydrophobic a nanoparticle is, the more likely it is to be cleared by phagocytosis, due to higher binding of blood proteins (opsonization) [22]. The improved surface properties are associated with the steric hindrance effect offered by hydrophilic polymers, which can prevent the surface-modified

liposomes from being rapidly eliminated by RES ("stealth liposomes"). Hydrophilization of liposome surface prolongs circulation time (long-circulating liposomes), enhancing the therapeutic potential of the entrapped drug [33].

**3. The critical material attributes and critical process parameters:** 

be reconsidered during different stages of the drug development [37].

To establish the CMAs, the main components of liposomes are evaluated, that is the active substance, the lipids, and others like buffer solution [38]. Each active substance has different physiochemical properties and can influence the desired CQAs [9, 39]. Depending on its solubility, it can be entrapped in the aqueous core

All the material attributes (MAs) and/or process parameters (PPs) that can affect the desired critical quality attributes (CQAs) of the final/intermediate product are identified through risk assessment. After risk identification, risk analysis evaluates the impact of the identified MAs and PPs on the CQAs. Further, through risk evaluation, a qualitative or quantitative scale is used for risk estimation of each identified factor on the desired CQA [35]. After the risk evaluation of potential MAs and PPs, only few of them will become potentially critical for quality attributes of the final drug product. In this case, identified MAs will become critical material attributes (CMAs), which must possess certain characteristics or should be chosen in an appropriate range to assure CQAs of the intermediate/final drug product [21]. Critical process parameters (CPPs) are those PPs that should be monitored and controlled in order to obtain the desired CQAs of the intermediate/final drug product [9]. Many tools are used for risk assessment, but the most used ones are Ishikawa or fish-bone diagram for risk identification and failure mode and effect analysis for risk evaluation [36]. Risk assessment should be done in the first step of the drug product development, and analysis of these risk factors is recommended to

e.Drug release kinetics has important implication for the therapeutic activity of liposomes. The drug must be delivered to the disease site at a level within its therapeutic window, at a sufficient rate, for a sufficient period, to have optimal therapeutic activity [34]. Drug delivery to the right site is related to the identification and the interaction with the target cells. Cell surface or blood vessel surface of tumor tissues has a series of specific and overexpressed receptors, which are closely related with tumor process [33]. Targeting ligands attached on liposomes' surface bind the corresponding receptors and, as a result, the liposomes are internalized by endocytosis. These ligands can be monoclonal antibodies, fragments of antibodies, peptides, proteins, nucleic acids, carbohy-

*DOI: http://dx.doi.org/10.5772/intechopen.85374*

until cellular uptake [30].

drates, or small molecules [29].

**identification and linking to CQAs**

*Liposomes - Advances and Perspectives*

The biodistribution of nanoparticles circulating in the blood stream is considerably influenced by particle size. Smaller particles, of 20–30 nm, are eliminated by kidneys, while larger particles, of up to 300 nm, are taken up by reticuloendothelial system (RES) [26]. By decreasing particle size below 100 nm, RES uptake can be reduced [27]. According to other studies, the ideal nanocarriers should have particle size ranging between 10 and 100 nm in order to evade the kidney filtration and capture by the liver [28]. For example, in tumor targeting, the particles need to be smaller than the cut-off of the fenestrations in the tumor neovasculature [29]. The literature reveals that for an efficient enhanced permeability and retention (EPR) effect, the particle size must be, generally, between 50 and 100 nm, depending on the tumor type, its environment, and its localization [28]. Particle size less than 200 nm is also beneficial for the sterility of the liposomal product, since this size allows

aseptic preparation and sterile filtration of the final product [30].

index values below 0.5 are reported to be acceptable [31].

Particle size also influences the kinetics of drug release. As particle size gets smaller, their surface area-to-volume ratio gets larger. This would imply that more of the drug is closer to the surface of the particle compared to a larger particle. Being at or near the surface would lead to a faster drug release [23].

Polydispersity index, reflecting particle size distribution of liposomal products, is a physical parameter for which the target is to have reduced values, indicating a good homogeneity of the dispersion. Generally, polydispersity

b.Zeta potential or particle charge is an important parameter in the evaluation of colloidal system's stability. Particles with a high negative or positive zeta potential value repel each other, indicating that the colloidal system is stable [32]. On the contrary, decreasing the zeta potential value to nearly neutral leads to liposomal aggregation [20]. The charge of nanosystems influences both systemic circulation time and the interactions with the target tissue. The presence of surface charge can alter the opsonization profile of the particles, its recognition by cells in the organs of the RES, and the overall plasma circulation profile [29]. Regarding the interaction with the target, it is known that cationic liposomes have affinity for negatively charged cancer cell membrane and higher selectivity than neutral or anionic liposomes. The intracellular uptake of cationic liposomes by tumor cells can be 14-fold higher than that of normal liposomes. Consequently, cationic liposomes enhance the safety of liposomal drug. The liposome charge can also influence drug loading, cationic liposomes exhibiting higher encapsulation efficiency for negatively charged drugs [32].

c.Depending on drug distribution in liposomal dispersion, the drug content of liposomes can be expressed in three ways: drug concentration (μg/mL); entrapment efficiency (EE, the amount of drug contained inside liposomes compared with total amount of drug; %); and drug loading (the amount of drug contained relative to the amount of the lipid used; drug-to-lipid ratio) [14, 20].

High encapsulation efficiency is very important for both manufacturers and patients. A higher percentage of drug encapsulation could reduce the manufacturing cost and increase drug concentration in the final formulation allowing greater flexibility in dosing. Depending on the pharmacokinetics, higher drug concentration can result in increased dosing intervals and hence

improved patient compliance [30].

**74**

Drug retention inside liposomes is also very important. Since liposomes are intended to deliver the drug to the target site, there should be no drug leakage until cellular uptake [30].

