Pegylation and formulation strategy of Anti-Microbial Peptide (AMP) according to the quality by design approach

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Abstract

Antimicrobial resistance is one of the main global threats according to the World Health Organization's (WHO) report (World Health Organization 2014), therefore there is a need for the development of other agents, such as antimicrobial peptides (AMPs). Although AMPs are considered as major candidates for next-generation antibiotics, several challenges including low bioavailability, high manufacturing cost and toxicity are still to be solved for their practical use in therapeutic applications. Novel chemical modification approaches as well as strategies for their delivery offer several opportunities to overcome these barriers and develop more stable and cost-effective synthetic peptides with efficient delivery to the target site.

The integration of the Quality by Design (QbD) approach in the early pharmaceutical developments supports researchers in optimizing the targeted product by a risk based manner. Peptide modifications and formulation of peptide delivery systems are challenging tasks and hide several risks. Understanding and evaluating the cause - effect relations within the initial Risk Assessment (RA) step in case of all attributes give the basis for the experimental design as the next step, and aids the formulation development in order to get the final product in the targeted quality range.

This study presents a Quality by Design based antimicrobial peptide modification and formulation design. Analyses the potential risks in the AMP PEGylation process through the example of PGLa. The QbD based initial RA screened and evaluated the risk factors in this AMP modification procedure. The critical quality and process related factors were defined and their ranking was performed due to their estimated critical effect on the PEGylated AMP. This pre-formulation design study highlights the critical risk factors as decision points for the further steps.

Introduction

Increased development of antimicrobial resistance to many available antibiotics is one of the biggest challenges in the global health sector together with advancements in biotechnology, genetic engineering and synthetic chemistry lead scientist to focus on alternative substitutes such as antimicrobial peptides (AMPs). AMPs are small molecules with less than 50 amino acids, having activity against a wide range of microorganisms and showing less immunogenicity compared to recombinant proteins and antibodies (Boge et al., 2019; Ghosh et al., 2019, World Health Organization 2014). Recent researches have demonstrated that in addition to the antimicrobial functions of AMPs, these peptides also play an important role in the complex pathogenesis of several inflammatory diseases (Zaiou, 2007; Roby and Di Nardo, 2013). Beside the previously mentioned advantages, AMPs have also limitations such as low bioavailability, high manufacturing cost and toxicity which still need to be faced in order to be able to use these peptides in therapeutic applications (Marr et al., 2006).

These initial barriers are being increasingly overcome with new chemical modification strategies such as N- and C-modifications, incorporation of non-natural or d-amino acids, cyclization and the attachment of the polyethylene glycol polymer to peptides (PEGylation). These approaches allowed several researchers to enhance the bioavailability of AMPs and improve their bio-distribution and rate of clearance. The proteolytic degradation of peptides can be decreased by protecting their C- and N-terminus with acetylation or amidation. Also, modifying the sequences of peptides by substitution of natural l-amino acids for their D enantiomers, α/β-substituted α-amino acids or even β- amino acids are other similar approaches that result in overcoming peptide hydrolysis. d-amino acid substitution in a peptide may influence not only the peptide's stability but also its secondary structure and therefore its ability to incorporate into membranes (Gomes et al., 2018; Castro et al., 2017; Sun et al., 2017; Hamamoto et al., 2002). The attachment of PEG molecules to proteins and peptides provide steric interference and thus protects peptides from proteolysis and offers several functional advantages for AMPs such as prolonged plasma half-lives, improved water solubility, stability, resistance, biocompatibility, minimal toxicity and immunogenicity (Hamley, 2014). After approval of the first PEGylated protein drug product by the FDA in 1990, several PEGylated protein drug products have become part of the pharmaceutical market (Pinholt et al., 2011). However non-specific covalent attachment of a large PEG molecule to AMP is a risky factor which can change AMP structure and thus its antimicrobial activity (Christian et al., 2009); complexity of the peptide structure itself may lead to decreased reproducibility (Wei et al., 2012). Moreover, PEG molecules may mask the binding (active) site of AMP and therefore cause loss of antimicrobial activity of AMP. Too low molecular weight (Mw) of PEG can reduce specificity and conjugate activity while too high Mw can sterically shield the bioactive domain of the peptide and reduce its biological activity (Turecek et al., 2016). The experimental conditions of PEGylation reaction (i.e. pH, temperature, reaction time, overall cost of the process and molar ratio between PEG derivative and peptide) also have an impact on the stability of the final PEGylated AMP (González-Valdez et al., 2012). For AMPs with membrane disruptive mechanism of action another important risk of PEGylation is the possibility of reducing membrane affinity and interference of PEG molecule with the mechanism of action of AMPs; the attachment of PEG molecule cause AMP to lose its positive charges and thus suppress bacterial lysis resulting in reduction of biological activity (Singh et al., 2014). To overcome the abovementioned barriers, different strategies such as changing Mw of PEG, site of PEGylation and type of linkage of PEG molecule were offered by several researchers (Zhang et al., 2008; Obuobi et al., 2018). This research article mostly describes the risks that may occur during PEGylation process of AMPs. Nevertheless, risk related to PEGylated therapeutics (risks that may occur after PEGylation) must also be considered. One of the common risks reported by numerous research groups is the production of antibodies by immune system. These antibodies specifically bind PEG and thus cause “accelerated blood clearance” of PEGylated therapeutics (Yang and Lai, 2015). An improved understanding of the mechanisms of anti-PEG immunity, monitoring patients before and during PEGylated drug treatment and less immunogenic delivery approaches are needed as strategies to overcome the challenge of PEG-specific immunity (Zhang et al., 2016).

PGLa as a model AMP in this study is an 21-residue amphipathic antimicrobial peptide-amide (GMASKAGAIAGKIAKVALKAL-NH2), isolated from the African clawed frog Xenopus leavis, that can destroy bacteria by interacting with their lipid membrane (Bechinger et al., 1998; Hartmann et al., 2010). The folded structure of PGLa displays the positively charged lysine sidechains on one side and hydrophobic residues on the opposite side (Strandberg et al., 2006). It is shown to be helical between residues 6 and 21 when associated with detergent micelles by multidimensional solution nuclear magnetic resonance (NMR) spectroscopy and the helix axis is parallel to the plane of the bilayers. NMR spectroscopy indicates that the amino-terminal residues are highly mobile and that the fluctuations of backbone sites decrease from Ala6 toward the carboxyl terminus (Bechinger et al., 1998). PGLa is known to decrease the antibiotic resistance level of resistant bacteria when co-administered as an adjuvant (Lázár et al., 2018). The exact mechanism of action is not known, but it is believed to form pores upon interacting with the bacterial membrane, and induces membrane permeability even at sub-MIC conditions (Hartmann et al., 2010).

There are two possible ways of its PEGylation: PEGylation in solvent phase or during the solid-phase synthesis, both carried out through the amino groups. Solvent-phase reactions are quite common in protein ligation techniques where only limited chemical modifications are possible during the synthesis. PEGylation can be carried out easily in the solvent phase, but either all amino groups are PEGylated with a high excess of reagent, or the PEG groups are randomly distributed when using sub-equimolar amounts. In case of AMPs however, PEGylation during solid-phase synthesis is possible, which allows selective modifications at desired sites. N-terminal PEGylation can be achieved after removing the final amino protecting group, and coupling an amine-reactive PEG derivative before cleaving the peptide from the resin.

For PEGylation at specific sites of the peptide, we have to use alternative sidechain protecting groups for the selected lysine residues which can be removed before the cleavage of the peptide. Selective removal of these protecting groups such as methyltrityl allows the coupling of the PEG chain at the selected position, followed by the removal of the N-terminal protecting group and the cleavage of the peptide.

PEG-Linker-Drug strategy is another possibility to increase the half-life of PGLa. By using a linker which is degraded by the bacteria itself, the risk of decreased antimicrobial activity by the PEGylation can be circumvented and there is no need to site-specific modifications. Bacterial enzymes which play roles in antibiotic resistance (such as β-lactamase) are the most promising candidates, as the overexpression of the enzymes would facilitate the release of the drug.

Solid-phase FMOC /tBu strategy is a common strategy designed and developed by using protected amino acids as building blocks (Tsubery et al., 2004; Lu et al., 2009). The controlled synthesis of peptides and formation of amide bonds requires the use of reversible ion of the amino group. Common amino protecting groups are: tert‑Butoxycarbonyl (tBoc), 9-Fluorenylmethyloxycarbonyl (Fmoc) and N-Allyloxycarbonyl (Alloc). It is also necessary to reversibly mask reactive side chain functional groups. The peptide remains anchored to an insoluble solid resin support. Resins commonly used are composed of polystyrene. The excess reagents and soluble byproducts will be removed after each reaction cycle.

As shown in Fig. 1, in this approach the first protected amino acid is attached to the resin through its carboxyl group (Coupling) (the addition of activating agent). Then the protecting group is removed (deprotection) under a mildly basic condition. This exposes a free α-amino group to react with the next incoming protected amino acid. Then again deprotection step is repeated (To confirm that the protecting groups are removed, a Kaiser-test is performed). The process is repeated through a cycle of deprotection, coupling and washing until the peptide is completely synthesized. The synthesized peptide is usually cleaved from the resin by trifluoroacetic acid (TFA), which removes the side chain protection groups at the same time. The purification steps usually includes the precipitation from the cleavage reaction mixture by ice-cold diethyl ether. Further purification can be achieved by gel-filtration, ion exchange chromatography and reversed-phase HPLC (Lu et al., 2009).

The Quality by Design (QbD) approach is a holistic, systematic, knowledge and risk based methodology of pharmaceutical developments, which focuses on the profound preliminary design (Soravia et al., 1988) considering all of the influencing parameters from the industry, the regulatory body and from the user (eg. patient, doctor). The application of the QbD method in the industrial development and manufacturing is forced by the regulatory authorities but it has also many benefits in the early phase of the developments (Pallagi et al., 2018; Zerweck et al., 2017), as brings scientific results closer to the practical requirements and has a facilitating effect on industrial scale up and product transfer to the market.

The QbD has several steps, described in the guidelines of the International Council of harmonization (ICH Q8 (R2), ICH Q9, ICH Q10) (Werle and Bernkop-Schnürch, 2006; Bahar and Ren, 2013; Santos et al., 2018). The main steps are: (1) the definition of the Quality Target Product Profile (QTPP), (2) the identification of the quality attributes and the selection of the Critical Quality Attributes (CQAs) related to the target product, (3) the prior selection of the production method and the identification of the Critical Process Parameters (CPPs) as well as the Critical Material Attributes (CMAs), (4) performing of the initial Risk Assessment (RA). RA is a systematic process of organizing information to support a risk decision and is the key activity in this model. The results of the RA will be the ranking of the CQAs and CPPs according to their calculated risk severity. RA results help to aim attention on the most critical influencing factors and avoid profitless efforts in later phases of the development process. The following steps of the QbD approach are: (5) the Design of the Experiments (DoE) which namely means the planning of the practical tasks by the RA results, (6) the performing of the experiments in practice and establishment of the Design Space (DS). These are followed by the (7) compilation of the Control Strategy which is the monitoring of the factors with highly risk potential in the process. The whole QbD guided process should be designed and performed by considering the possibilities of the (8) Continuous Improvement. In this thinking, generally the RA is the most accentual element which it is especially advantageous in the case of complex and sensitive drugs like peptides.

This paper aims to take steps in the enhancement of AMPs’ properties for pharmaceutical use and evaluate the risks of AMP PEGylation such as loosing antimicrobial activity of peptides, used PGLa as a model AMP. This study analyses the potential risks in the antimicrobial peptide PEGylation process by means of the RA method within the QbD approach of early pharmaceutical development.

Section snippets

Materials

PGLa (H-Gly-Met-Ala-Ser-Lys-Ala-Gly-Ala-Ile-Ala-Gly-Lys-Ile-Ala-Lys-Val-Ala-Leu-Lys-Ala-Leu-NH2) is 21-residue amphipathic antimicrobial peptide amide. Its net charge is +5 at physiological pH. It has good water solubility, and shows only limited haemolytic activity (Soravia et al., 1988).

Knowledge space development

The collection and systemic organization of the related scientific literature and experience from the previous studies means the “knowledge space development” (Zerweck et al., 2017). After the analysis of the

Results

The basis for initial RA was an evaluation of the present knowledge in the different limitations of PEGylation and how these barriers can lead to risks and how these risks can be overcome by novel opportunities offered by chemistry or biochemistry for achieving desirable bioactive AMP (Table 1.).

The initiative step of the RA process of the preparation of the PEGylated PGLa by the solid-phase FMOC/tBu strategy was the construction of the Ishikawa diagram (Fig. 2), where the different factors and

Discussion

The main focus in this study was on evaluating the risk factors and the required decision points in PEGylation process of PGLa. From the proposed structure and mechanism of action of PGLa, we suggested two possible ways of PEGylation process. N-terminal PEGylation or PEGylation at specific positions. The second approach is worth to try, since it can slow the degradation process and increase bioactivity of PGLa. However the attachment of PEG molecule in different positions can cause PGLa to lose

Conclusion

In this study the risk factors that influence the PEGylation process of PGLa were investigated by the application of the Quality by Design (QbD) concept. This approach is resulted in identifying the critical factors with the highest effect on the quality of a final modified AMP [44,45]. The priority ranking of these factors is as following: its final size, its conjugate activity (increased) and its specificity (increased). On the other hand, the following critical influencing factors during

Authors Contributions

The University of Szeged, Faculty of Pharmacy, Doctoral School of Pharmaceutical sciences and the Institute of Pharmaceutical Technology and Regulatory Affairs made possible the scientific work.

Declaration of competing interest

None

Acknowledgement

This project was supported by the Ministry of Human Capacities, Hungary grant 20391-3/2018/FEKUSTRAT.

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