Salting-Out crystallization of glycopeptide Vancomycin: Phase behavior study to control the crystal habit
Graphical abstract
Introduction
Crystallization is a classical unit operation widely used in the purification of both small-molecule pharmaceuticals and large-molecule biologics (e.g., therapeutic proteins, monoclonal antibody, nucleic acids) (Pu and Hadinoto, 2020). In addition to crystal size, crystal habit (or external shape) has profound influences on the filterability (Bourcier et al., 2016), bulk density (Kim and Koo, 2019), adhesion tendency, which in turn affects flowability (Shah et al., 2014), and compactibility (Pudasaini et al., 2017) of the crystals produced. These properties greatly influence the downstream processing efficiencies in steps such as filtration, centrifugation, drying, tableting, as well as storage and handling (Azad et al., 2021). Moreover, crystal habit also influences the solubility, dissolution, and consequently the bioavailability and therapeutic activity of pharmaceuticals and biologics (Modi et al., 2013, Phan et al., 2021, Ren et al., 2019, Yin et al., 2008).
Needle habit as one of the more commonly produced habits in pharmaceutical crystallization (Cote et al., 2020) is notorious for causing several downstream processing issues and difficult handling. For example, needle crystals tend to align with the flow of the mother liquor, thus blocking the filter pores and they are prone to fracture during filtration creating unwanted fines (MacLeod and Muller, 2012, Steenweg et al., 2022). Aqueous dispersions of needle crystals also exhibit a high viscosity requiring a higher energy requirement to transport them to filtration unit (Wood, 2001). Needle crystals are brittle, prone to solvent inclusion (Meekes et al., 2003), and typically associated with low bulk density, poor tablet quality, and difficulty in loading the required dose into capsules (Black, 2019, Ghazi et al., 2019). For this reason, the design of industrial crystallization processes has been geared towards avoiding needle crystal formation (Lovette and Doherty, 2013).
Crystal habit is governed by the relative growth rates of individual faces of the crystals. Understanding crystal growth mechanism of a compound is essential in order to control its crystal habit to avoid the needle habit formation. The crystal growth rates are governed by both internal crystal structure and crystallization conditions (e.g., supersaturation, temperature, pH) (Lovette et al., 2008). Crystal morphological modellings based on crystal growth theories (e.g., Bravais-Friedel-Donnay-Harker (BFDH), periodic bond chain (PBC), and slice attachment energy theories) have been carried out to predict the crystal habit (Civati et al., 2021, Li et al., 2006). The crystal growth theories are typically coupled with kinetic models to incorporate the effect of crystallization conditions (Nayhouse et al., 2013, Winn and Doherty, 2002). From the crystal morphological modelling, organic small molecules can be categorized as either persistent (largely unavoidable), or controllable in their needle habit formation tendency (Civati et al., 2021). Moreover, the crystal morphological modelling has enabled researchers to develop guidelines on solvent selection to avoid the needle habit formation, which have been validated with experimental data (Lovette and Doherty, 2013, Taulelle et al., 2006, Tilbury et al., 2016).
Besides crystal morphological modelling, various crystal habit modification techniques have been explored experimentally (Hadjittofis et al., 2018). Solvent selection via its influence on the drug solubility has been the most widely investigated, particularly for small-molecule pharmaceuticals (Li et al., 2016). For example, the aspect ratio of anticholesterol drug lovastatin crystals could be lowered (less needle-like) by using less polar solvents (e.g., hexane, methylcyclohexane, ethyl acetate), in place of water–acetone mixture, or methanol used in the industrial crystallization of lovastatin (Hatcher et al., 2020, Turner et al., 2019). An opposite trend was observed in the crystallization of anti-inflammatory drug ibuprofen, where the use of low-polarity solvents resulted in needle crystals formation (Rasenack and Müller, 2002).
The crystal habit can also be modified by adding a growth inhibiting agent that is adsorbed onto the fast-growing crystal face. For example, the addition of hydrophobic polymers resulted in the formation of plate-like lovastatin crystals, in place of needle crystals produced without additives (Hatcher et al., 2020). The needle habit formation was suppressed by adding Polysorbate-80 surfactant and poly(sebacic anhydrite) in the crystallization of antihypertensive drug nifedipine (Kumar et al., 2015) and antifungal drug griseofulvin (Jarmer et al., 2005), respectively.
Another widely used experimental approach to modify the crystal habit is by precise manipulation of the supersaturation level via temperature cycling. For example, the aspect ratio of needle crystals of aspirin was successfully modified by multiple cycles of heating and cooling (Neugebauer et al., 2018). Successful crystal habit modifications by temperature cycling were also demonstrated in the crystallization of paracetamol (Lovette et al., 2012) and a proprietary active pharmaceutical ingredient (Eren et al., 2021). The needle habit formation can also be suppressed by simultaneous controls of multiple crystallization process variables (e.g., temperature, stirring rate, cooling rate, and seeding) as demonstrated in the crystallization of painkiller celecoxib (Banga et al., 2010).
Successful crystal habit modifications have also been demonstrated for bioactive macromolecules, particularly proteins, albeit they have not been as extensively studied as small-molecule pharmaceuticals. For example, needle habit formation of antimicrobial lysozyme could be avoided by (1) performing the crystallization under acidic condition and low temperature (Yu et al., 2015), (2) addition of ionic liquid as the habit modifier (Judge et al., 2009, Yu et al., 2019), and (3) use of crosslinked polymers as seeds (Grzesiak and Matzger, 2008). Needle habit formation of ovalbumin and catalase, on the other hand, was promoted at lower pH (Dumetz et al., 2008). In general, the aspect ratio of protein crystals was found to be most affected by pH and temperature (Dumetz et al., 2008, Liang et al., 2013).
Besides proteins, another clinically important class of bioactive macromolecules is peptides, whose molecular weight (MW) typically lies between small-molecule pharmaceuticals and proteins (i.e., 1 MW 5 kDa) (Lau and Dunn, 2018). Unlike proteins, crystallization of peptides for purification purposes has not been widely employed (Yu et al., 2020). Ultrafiltration membrane and chromatography, which pose problems of low throughput and high operational costs, remain predominantly employed in industrial purification of bioactive peptides (de Castro and Sato, 2015). The few studies on peptide crystallization were mostly aimed at crystal structure determination (Guo et al., 2021, Karle et al., 2003, Schäfer et al., 1996). To the best of our knowledge, modification of crystal habit in peptide crystallization had not been investigated before.
In the present work, we investigated the feasibility of avoiding needle habit formation in peptide crystallization using vancomycin hydrochloride (Van) - a glycopeptide antibiotic - as the model peptide. Van (MW 1.45 kDa) is widely used to treat infections caused by MRSA (methicillin-resistant Staphylococcus aureus), penicillin-resistant pneumococci, and to treat infections in patients who are allergic to penicillin and cephalosporins (Bruniera et al., 2015). Purification of Van, which is industrially produced by bacterial fermentation, requires multicycles of ion-exchange chromatography with pH adjustments, followed by antisolvent/salting-out crystallization as the final purification step (Lee et al., 2006).
While octahedral Van crystals were produced by hanging drop vapor diffusion crystallization (Schäfer et al., 1996), bulk crystallization of Van resulted in the undesirable needle crystals (Lee et al., 2010). Not unlike protein crystallization, the significant influences of pH and temperature were demonstrated in bulk Van crystallization performed in stirred vessels, where a narrow workable range of pH and temperature existed with pH 2.5 and 10 °C determined as the optimal condition (Lee et al., 2010). Bulk Van crystallization was slow needing 24 h to reach 95 % yield, despite combined cooling/salting-out/antisolvent (with acetone) crystallizations were employed. Even though the bulk crystallization rate could be enhanced by the addition of ionic liquid (Ha and Kim, 2015) and polymer seeds (Kim et al., 2011), needle crystals remained the predominant habit.
The first objective of the present work was to carry out a phase behavior study to determine crystallization conditions in which the needle habit could be avoided and to identify the predominant non-needle crystal habit produced. Recognizing the significant influences of solvent, pH, and temperature on the resultant crystal habit, we carried out the phase behavior study in conditions distinct from the ones employed in Lee et al. (2010). Specifically, we employed salting-out crystallization at room temperature using acetate buffer solution as the solvent, in contrast to simultaneous cooling/salting-out/antisolvent crystallization pursued in Lee et al. (2010). Phase behaviors of Van crystallization at different (i) pH, (ii) concentrations of Van and salt, and (iii) incubation time were examined in a high-throughput l-scale crystallization setup.
The second objective of the present work was to carry out batch crystallization of the predominant non-needle crystal habit identified in the phase behavior study (i.e., octahedral crystals). The octahedral Van crystals from the batch crystallization were characterized in terms of the (1) production yield and capacity, (2) purity, (3) crystal size distribution, (4) thermal stability, (5) interfacial water content, (6) dissolution characteristics, and lastly (7) antibiotic activity. The characteristics of the octahedral crystals were evaluated relative to the needle crystals, which prior to the present study had been the predominant crystal habit produced from batch crystallization of Van.
Section snippets
Materials
Van (United States Pharmacopeia (USP) grade, ≥ 900 μg/mg) was purchased from Duly Biotech Co. ltd. (Nanjing, China). Glycine (≥99 %), glacial acetic acid, potassium dihydrogen phosphate (KH2PO4), sodium hydroxide (NaOH), sodium acetate (≥99 %), ethanol (≥99.5 %), and paraffin oil (puriss., Ph. Eur.) were purchased from Sigma Aldrich (Singapore). Hydrochloric acid (HCl, 37 %) was purchased from VWR (Singapore). Mueller Hinton Broth (MHB) and phosphate buffered saline (PBS, pH 6.8) were purchased
Van solubility in buffer solutions of different pH and ionic strength
Prior to the phase behavior study, the solubility of Van in buffer solutions of different pH and NaCl concentrations ([NaCl]) was characterized to determine the supersaturation level in the phase behavior study. Van is a basic glycopeptide with pI of 8.1 (Horká et al., 2014). For basic proteins, several studies had advocated crystallization at pH below the pI to enhance the crystallization propensity (Charles et al., 2006, Kirkwood et al., 2015, Zhang et al., 2013). Furthermore, Van exhibited
Conclusions
The phase behavior study revealed the O crystals as the predominant crystal habit in salting-out room-temperature crystallization of Van. The O crystals were produced across the range of pH (2.6 to 5.6), Van (15 to 50 mg/mL), and NaCl concentrations (0.4 to 1.0 M) investigated. The needle crystal formation was largely avoided except at pH 5.6 and at NaCl concentrations in which the salting-in event took place (0.1 to 0.5 M). Increasing the incubation time beyond 24 h resulted in a wider range
CRediT authorship contribution statement
Siyu Pu: Writing – original draft, Writing – review & editing, Methodology, Data curation, Methodology, Investigation, Visualization. Kunn Hadinoto: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
The authors would like to acknowledge the research funding from Ministry of Education Singapore (Grant No. AcRF Tier 1 RG82/20).
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