Current perspectives on stability of protein drug products during formulation, fill and finish operations

Commercialization of protein-based therapeutics is a challenging task in part due to the difficulties in maintaining protein solutions safe and efficacious throughout the drug product development process, storage, transportation and patient administration. Bulk drug substance goes through a series of formulation, fill and finish operations to provide the final dosage form in the desired formulation and container or delivery device. Different process parameters during each of these operations can affect the purity, activity and efficacy of the final product. Common protein degradation pathways and the various physical and chemical factors that can induce such reactions have been extensively studied for years. This review presents an overview of the various formulation-fill-finish operations with a focus on processing steps and conditions that can impact product quality. Various manufacturing operations including bulk freeze-thaw, formulation, filtration, filling, lyophilization, inspection, labeling, packaging, storage, transport and delivery have been reviewed. The article highlights our present day understanding of protein instability issues during biopharmaceutical manufacturing and provides guidance on process considerations that can help alleviate these concerns.     

Review physical and chemical aspects of stabilization of compounds in silk

The challenge of stabilization of small molecules and proteins has received considerable interest. The biological activity of small molecules can be lost as a consequence of chemical modifications, while protein activity may be lost due to chemical or structural degradation, such as a change in macromolecular conformation or aggregation. In these cases, stabilization is required to preserve therapeutic and bioactivity efficacy and safety. In addition to use in therapeutic applications, strategies to stabilize small molecules and proteins also have applications in industrial processes, diagnostics, and consumer products like food and cosmetics. Traditionally, therapeutic drug formulation efforts have focused on maintaining stability during product preparation and storage. However, with growing interest in the fields of encapsulation, tissue engineering, and controlled release drug delivery systems, new stabilization challenges are being addressed; the compounds or protein of interest must be stabilized during: (1) fabrication of the protein or small molecule-loaded carrier, (2) device storage, and (3) for the duration of intended release needs in vitro or in vivo. We review common mechanisms of compound degradation for small molecules and proteins during biomaterial preparation (including tissue engineering scaffolds and drug delivery systems), storage, and in vivo implantation. We also review the physical and chemical aspects of polymer-based stabilization approaches, with a particular focus on the stabilizing properties of silk fibroin biomaterials.     

Sustained release of an anti-glaucoma drug: demonstration of efficacy of a liposomal formulation in the rabbit eye

Topical medication remains the first line treatment of glaucoma; however, sustained ocular drug delivery via topical administration is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Currently, daily topical administration for lowering the intra-ocular pressure (IOP), has many limitations, such as poor patient compliance and ocular allergy from repeated drug administration. Poor compliance leads to suboptimal control of IOP and disease progression with eventual blindness. The delivery of drugs in a sustained manner could provide the patient with a more attractive alternative by providing optimal therapeutic dosing, with minimal local toxicity and inconvenience. To investigate this, we incorporated latanoprost into LUVs (large unilamellar vesicles) derived from the liposome of DPPC (di-palmitoyl-phosphatidyl-choline) by the film hydration technique. Relatively high amounts of drug could be incorporated into this vesicle, and the drug resides predominantly in the bilayer. Vesicle stability monitored by size measurement and DSC (differential scanning calorimetry) analysis showed that formulations with a drug/lipid mole ratio of about 10% have good physical stability during storage and release. This formulation demonstrated sustained release of latanoprost in vitro, and then tested for efficacy in 23 rabbits. Subconjunctival injection and topical eye drop administration of the latanoprost/liposomal formulation were compared with conventional daily administration of latanoprost eye drops. The IOP lowering effect with a single subconjunctival injection was shown to be sustained for up to 50 days, and the extent of IOP lowering was comparable to daily eye drop administration. Toxicity and localized inflammation were not observed in any treatment groups. We believe that this is the first demonstration, in vivo, of sustained delivery to the anterior segment of the eye that is safe and efficacious for 50 days.     

Challenges and solutions for the delivery of biotech drugs--a review of drug nanocrystal technology and lipid nanoparticles

Biotechnology allows tailor-made production of biopharmaceuticals and biotechnological drugs; however, many of them require special formulation technologies to overcome drug-associated problems. Such potential challenges to solve are: poor solubility, limited chemical stability in vitro and in vivo after administration (i.e. short half-life), poor bioavailability and potentially strong side effects requiring drug enrichment at the site of action (targeting). This review describes the use of nanoparticulate carriers, developed in our research group, as one solution to overcome such delivery problems, i.e. drug nanocrystals, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and lipid-drug conjugate (LDC) nanoparticles, examples of drugs are given. As a recently developed targeting principle, the concept of differential protein adsorption is described (PathFinder Technology) using as example delivery to the brain.     

Protein adsorption and displacement at lipid layers determined by total internal reflection fluorescence (TIRF)

In many drug delivery systems such as liposomes, the adsorption of interstitial proteins upon administration can have a huge effect on the elimination, release, and stability of the delivery system. For example, it is assumed that PEGylated liposomes prevent the adsorption of opsonins and thereby prolong the circulation time in vivo, and EMEA guidelines recommend that more than 80% of the protein antigen is adsorbed in the formulation of adjuvant systems. However, few methods exist to elucidate this protein adsorption. The present study indicates that total internal reflection fluorescence (TIRF) is a possible method to examine the adsorption and exchange of proteins at lipid surfaces. In the TIRF set-up, a lipid layer can be formed [exemplified with dimethyldioctadecylammonium bromide (DDA) and D-(+)-trehalose 6,6’-dibehenate (TDB)] whereafter protein (i.e., ovalbumin or an antigen, Ag85B-ESAT-6) is adsorbed, and these proteins can subsequently be displaced by the abundant interstitial protein (i.e., serum albumin).     

Harnessing the therapeutic potential of anticancer drugs through amorphous solid dispersions

The treatment of cancer is still a major challenge. But tremendous progress in anticancer drug discovery and development has occurred in the last few decades. However, this progress has resulted in few effective oncology products due to challenges associated with anticancer drug delivery. Oral administration is the most preferred route for anticancer drug delivery, but the majority of anticancer drugs currently in product pipelines and the majority of those that have been commercially approved have inherently poor water solubility, and this cannot be mitigated without compromising their potency and stability. The poor water solubility of anticancer drugs, in conjunction with other factors, leads to suboptimal pharmacokinetic performance. Thus, these drugs have limited efficacy and safety when administered orally. The amorphous solid dispersion (ASD) is a promising formulation technology that primarily enhances the aqueous solubility of poorly water-soluble drugs. In this review, we discuss the challenges associated with the oral administration of anticancer drugs and the use of ASD technology in alleviating these challenges. We emphasize the ability of ASDs to improve not only the pharmacokinetics of poorly water-soluble anticancer drugs, but also their efficacy and safety. The goal of this paper is to rationalize the application of ASD technology in the formulation of anticancer drugs, thereby creating superior oncology products that lead to improved therapeutic outcomes.     

Reactive Impurities in Excipients: Profiling,Identification and Mitigation of Drug–Excipient Incompatibility

Reactive impurities in pharmaceutical excipients could cause drug product instability, leading to decreased product performance, loss in potency, and/or formation of potentially toxic degradants. The levels of reactive impurities in excipients may vary between lots and vendors. Screening of excipients for these impurities and a thorough understanding of their potential interaction with drug candidates during early formulation development ensure robust drug product development. In this review paper, excipient impurities are categorized into six major classes, including reducing sugars, aldehydes, peroxides, metals, nitrate/nitrite, and organic acids. The sources of generation, the analytical method for detection, the stability of impurities upon storage and processing, and the potential reactions with drug candidates of these impurities are reviewed. Specific examples of drug–excipient impurity interaction from internal research and literature are provided. Mitigation strategies and corrective measures are also discussed.     

Differential Scanning Calorimetry (DSC): a tool to study the thermal behavior of lipid bilayers and liposomal stability

Thermodynamical techniques are applied for determining the thermal stress of medicinal compounds of the excipients as well as their interactions during the formulation process. The physicochemical properties and the stability of the medicinal products could be measured as a function of temperature or time using thermal analysis. Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique for determining the purity, the polymorphic forms and the melting point of a sample in the Pharmaceutical Industry. It is also considered as a tool to study the thermal behavior of lipid bilayers and of lipidic drug delivery systems, like liposomes by measuring thermodynamic parameters (i.e. DeltaH and Tm), which affect the stability of the liposomal suspension under given storage conditions.     

Animal-free production of ccc-supercoiled plasmids for research and clinical applications

The topological structure of plasmid DNA can be characterized by capillary gel electrophoresis (CGE analysis)-an important tool for quality control and stability assessments in DNA storage or application. Hence, a large-scale manufacturing process was developed that allows the removal of undesired open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA, proteins as well as lipopolysaccharides (endotoxins) and results in obtaining supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form without using any animal-derived substances. Using CGE, the development and in-line monitoring for pharmaceutical plasmid production starting from fermentation control throughout the whole manufacturing process including the formulated and filled product can be performed the first time in a way conforming to good manufacturing practices (GMP). Plasmid stability data were obtained from analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection). The physical stability of plasmid DNA is for the first time evaluated in DNA storage experiments on the level of different plasmid forms.     

Differential Scanning Calorimetry (DSC): A Tool to Study the Thermal Behavior of Lipid Bilayers and Liposomal Stability

Thermodynamical techniques are applied for determining the thermal stress of medicinal compounds of the excipients as well as their interactions during the formulation process.

The physicochemical properties and the stability of the medicinal products could be measured as a function of temperature or time using thermal analysis.

Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique for determining the purity, the polymorphic forms and the melting point of a sample in the Pharmaceutical Industry. It is also considered as a tool to study the thermal behavior of lipid bilayers and of lipidic drug delivery systems, like liposomes by measuring thermodynamic parameters (i.e. ΔH and Tm), which affect the stability of the liposomal suspension under given storage conditions.     

Omega-3 fatty acid-based self-microemulsifying drug delivery system (SMEDDS) of pioglitazone: Optimization,in vitro and in vivo studies

Pioglitazone (PGL) is an effective insulin sensitizer, however, side effects such as accumulation of subcutaneous fat, edema, and weight gain as well as poor oral bioavailability limit its therapeutic potential for oral delivery. Recent studies have shown that combination of both, PGL and fish oil significantly reduce fasting plasma glucose, improve insulin resistance, and mitigate pioglitazone-induced subcutaneous fat accumulation and weight gain. Nevertheless, developing an effective oral drug delivery system for administration of both medications have not been explored yet. Thus, this study aimed to develop a self-micro emulsifying drug delivery system (SMEDDS) for the simultaneous oral administration of PGL and fish oil. SMEDDS was developed using concentrated fish oil,Tween® 80, and Transcutol HP and optimized by central composite design (CCD). The reconstituted, optimized PGL-SMEDDS exhibited a globule size of 142 nm, a PDI of 0.232, and a zeta potential of −20.9 mV. The in-vitro drug release study of the PGL-SMEDDS showed a first-order model kinetic release and demonstrated remarkable 15-fold enhancement compared to PGL suspension. Additionally, following oral administration in fasting albino Wistar rats, PGL-SMEDDS exhibited 3.4-fold and 1.4-fold enhancements in the AUC_0–24h compared to PGL suspension and PGL marketed product. The accelerated stability testing showed that the optimized SMEDDS formulation was stable over a three-month storage period. Taken together, our findings demonstrate that the developed fish oil-based SMEDDS for PGL could serve as effective nanoplatforms for the oral delivery of PGL, warranting future studies to explore its synergistic therapeutic potential in rats.     

Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

Background  

Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.     

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.     

Advanced protein formulations

It is well recognized that protein product development is far more challenging than that for small‐molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long‐term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site‐specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze‐dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution.     

Determinants of the overall response to furosemide: pharmacokinetics and pharmacodynamics

After an oral or i.v. dose of furosemide, there is considerable interindividual variability in the amount of unchanged drug delivered into the urine. On the average, approximately half as much reaches the intraluminal site of action with an oral compared with an i.v. dose. However, the natriuretic response to the same dose administered by either route is virtually the same. Similarly, after pretreatment with probenecid, the same total amount of furosemide in urine causes a greater overall response. It has been presumed that this paradox is accounted for by differences in rate of delivery of furosemide to the active site such that after an oral dose or after pretreatment with probenecid, amounts of drug are at the "steep" portion of the dose-response curve for longer periods of time. Our analysis shows that this is not the case. For furosemide, the amount of diuretic delivered into the urine that is maximally efficient (21.5 micrograms/min) is considerably less than the amount causing half-maximal response (69.8 micrograms/min). Oral administration or pretreatment with probenecid maintains drug close to this maximally efficient amount more persistently than does i.v. administration. By so doing, total response to an oral dose approaches that of i.v. dosing despite delivering half the amount of drug to the active site, and after probenecid an i.v. dose causes a greater response than i.v. dosing alone despite delivering the same amount of drug to the active site. These data emphasize the importance of the time course of delivery of drug to the active site as an independent determinant of overall response.     

Stabilization of a recombinant human epidermal growth factor parenteral formulation through freeze-drying

Development studies were performed to design a pharmaceutical composition that allows the stabilization of a parenteral rhEGF formulation in a lyophilized dosage form. Unannealed and annealed drying protocols were tested for excipients screening. Freeze-dry microscopy was used as criterion for excipients and formulation selection; as well as to define freeze-drying parameters. Excipients screening were evaluated through their effect on freeze-drying recovery and dried product stability at 50 °C by using a comprehensive set of analytical techniques assessing the chemical stability, protein conformation and bioactivity. The highest stability of rhEGF during freeze-drying was achieved by the addition of sucrose or trehalose. After storing the dried product at 50 °C, the highest stability was achieved by the addition of dextran, sucrose, trehalose or raffinose. The selected formulation mixture of sucrose and dextran could prevent protein degradation during the freeze-drying and delivery processes. The degradation rate assessed by RP-HPLC could decrease 100 times at 37 °C and 70 times at 50 °C in dried with respect to aqueous formulation. These results indicate that the freeze-dried formulation represents an appropriate technical solution for stabilizing rhEGF.     

Recent Advances in Lipid Nanoparticle Formulations with Solid Matrix for Oral Drug Delivery

Lipid nanoparticles based on solid matrix have emerged as potential drug carriers to improve gastrointestinal (GI) absorption and oral bioavailability of several drugs, especially lipophilic compounds. These formulations may also be used for sustained drug release. Solid lipid nanoparticle (SLN) and the newer generation lipid nanoparticle, nanostructured lipid carrier (NLC), have been studied for their capability as oral drug carriers. Biodegradable, biocompatible, and physiological lipids are generally used to prepare these nanoparticles. Hence, toxicity problems related with the polymeric nanoparticles can be minimized. Furthermore, stability of the formulations might increase than other liquid nano-carriers due to the solid matrix of these lipid nanoparticles. These nanoparticles can be produced by different formulation techniques. Scaling up of the production process from lab scale to industrial scale can be easily achieved. Reasonably high drug encapsulation efficiency of the nanoparticles was documented. Oral absorption and bioavailability of several drugs were improved after oral administration of the drug-loaded SLNs or NLCs. In this review, pros and cons, different formulation and characterization techniques, drug incorporation models, GI absorption and oral bioavailability enhancement mechanisms, stability and storage condition of the formulations, and recent advances in oral delivery of the lipid nanoparticles based on solid matrix will be discussed.     

Development of Oral Flexible Tablet (OFT) Formulation for Pediatric and Geriatric Patients: a Novel Age-Appropriate Formulation Platform

Development of palatable formulations for pediatric and geriatric patients involves various challenges. However, an innovative development with beneficial characteristics of marketed formulations in a single formulation platform was attempted. The goal of this research was to develop solid oral flexible tablets (OFTs) as a platform for pediatrics and geriatrics as oral delivery is the most convenient and widely used mode of drug administration. For this purpose, a flexible tablet formulation using cetirizine hydrochloride as model stability labile class 1 and 3 drug as per the Biopharmaceutical Classification System was developed. Betadex, Eudragit E100, and polacrilex resin were evaluated as taste masking agents. Development work focused on excipient selection, formulation processing, characterization methods, stability, and palatability testing. Formulation with a cetirizine-to-polacrilex ratio of 1:2 to 1:3 showed robust physical strength with friability of 0.1% (w/w), rapid in vitro dispersion within 30 s in 2–6 ml of water, and 0.2% of total organic and elemental impurities. Polacrilex resin formulation shows immediate drug release within 30 min in gastric media, better taste masking, and acceptable stability. Hence, it is concluded that ion exchange resins can be appropriately used to develop taste-masked, rapidly dispersible, and stable tablet formulations with tailored drug release suitable for pediatrics and geriatrics. Flexible formulations can be consumed as swallowable, orally disintegrating, chewable, and as dispersible tablets. Flexibility in dose administration would improve compliance in pediatrics and geriatrics. This drug development approach using ion exchange resins can be a platform for formulating solid oral flexible drug products with low to medium doses.     

Investigation of archaeosomes as carriers for oral delivery of peptides

Oral administration of peptide and protein drugs faces a big challenge partly due to the hostile gastrointestinal (GI) environment. Lipid-based delivery systems are attractive because they offer some protection for peptides and proteins. In this context, we prepared a special lipid-based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and explored its potential as an oral drug delivery vehicle. Our study demonstrates that archaeosomes have superior stability in simulated GI fluids, and enable fluorescent labeled peptides to reside for longer periods in the GI tract after oral administration. Although archaeosomes have little effect on the transport of insulin across the Caco-2 cell monolayers, the in vivo experiments indicated that archaeosomes containing insulin induced lower levels of blood glucose than a conventional liposome formulation. These data indicate that archaeosomes could be a potential carrier for effective oral delivery of peptide drugs.     

Cell culture media impact on drug product solution stability

To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf‐life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell‐free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG₄ monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998–1008, 2016