Characterization of Microbial Growth Potential in Antibody Drug IV Admixtures by Microbial Challenge.

Monoclonal antibody (mAb) drug products (DP) for IV administration are commonly diluted in a diluent such 0.9% sodium chloride (saline) or 5% dextrose (D5W) injection yielding IV admixtures before infusion or injection. During dose preparation, storage, and administration, the sterility of IV admixtures must be maintained to ensure patient safety. However, the introduction of adventitious microorganisms may occur during dose preparation, and microbial proliferation may take place during IV admixture storage. Sterility testing of IV admixtures prior to administration is not feasible in clinic due to its destructive nature. Instead, microbial growth potential assessment could be performed to ensure patient safety. To assess microbial growth potential of IV admixtures, microbial challenge studies, which evaluate the ability of IV admixtures supporting or not supporting microorganism proliferation, are often recommended. Since the initial introduction of microbial challenge studies 2009, there has been very limited data published on microbial challenge studies for IV admixtures. In this publication, data from independent microbial challenge studies for IV admixtures prepared from 10 monoclonal antibodies (mAb) were generated, pooled, and analyzed together for microbial growth trends. The results indicated that major factors impacting the microbial growth in mAb IV admixtures include temperature and time as well as protein and excipient concentration. No microbial growth was observed for IV admixtures stored at 2-8 °C for up to 14 days. At room temperature, no microbial growth was observed for 12 h in IV admixture with protein concentration ≤32 mg/mL. Growth of E. coli, P. aeruginosa, and K. pneumoniae are commonly observed in IV admixtures stored for 16-48 h at room temperature. The study results provided input for designing effective challenge studies to maximize IV admixtures in-use time as well as for potential regulatory guidance development to facilitate the drug development while ensuring patient safety.

An Intercompany Perspective on Compatibility and In-use Stability Studies to Enable Administration Of Biopharmaceutical Drug Products.

In-use stability and compatibility studies are often used in biotherapeutic development to assess stability and compatibility of biologic drugs with diluents and/or administration components at relevant conditions for the target route of administration (commonly intravenous, subcutaneous or intramuscular), to assure that patient safety and product efficacy are maintained during clinical use. To gain an understanding of current industry approaches for in-use stability and compatibility studies, the Formulation Workstream of the BioPhorum Development Group (BPDG), an industry-wide consortium, conducted an inter-company collaboration exercise, which included five bench-marking surveys around in-use stability and compatibility studies of biologic drugs. The results of this industry collaboration provide insights into the practicalities of these studies and how they are being used to support administration of biologics from early clinical programs to marketed products. The surveys queried topics including regulatory strategies and feedback; clinical in-use formulation, patient and site considerations; clinical blinding, masking and placebo approaches; study setup, execution and reporting; and clinical in-use stability and compatibility testing to provide a comprehensive picture of the range of common industry practices. This paper discusses the survey results and presents various approaches which can be used to guide the strategy and design of an in-use stability and compatibility program based on clinical and biomolecule needs.

Characterization of Proteinaceous Particles in Monoclonal Antibody Drug Products Using Mass Spectrometry.

In recent years, monoclonal antibodies (mAb) have become one of the most important classes of therapeutic proteins. Among many of the quality attributes monitored and controlled throughout therapeutic antibody development, particulate matter is one of the critical quality attributes (CQAs) for drug products. Visible and subvisible particulates in drug products may pose safety and immunogenicity risks to patients and therefore are tightly controlled and regulated. Characterization of the particle composition in drug products is essential to understand the origin of particulates and their mechanism of formation. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) based method and integrated it into the typical particulate characterization workflow to identify and quantify the composition of proteinaceous particles isolated from a therapeutic mAb drug product. The LC-MS workflow provides a useful tool to study particle formation and monitor the protein composition of particulates during therapeutic mAb development.

An Industry Perspective on Compatibility Assessment of Closed System Drug-Transfer Devices for Biologics.

The Formulation Workstream of the BioPhorum Development Group (BPDG), an industry-wide consortium, has identified the increased use of closed system drug-transfer devices (CSTDs) with biologics, without an associated compatibility assessment, to be of significant concern. The use of CSTDs has increased significantly in recent years due to the recommendations by NIOSH and USP that they be used during preparation and administration of hazardous drugs. While CSTDs are valuable in the healthcare setting to reduce occupational exposure to hazardous compounds, these devices may present particular risks that must be adequately assessed prior to use to ensure their compatibility with specific types of drug products, such as biologic drugs, which may be sensitive. The responsibility of ensuring quality of biologic products through preparation and administration to the patient lies with the drug product sponsor. Due to the significant number of marketed CSTD systems, and the large variety of components offered for each system, a strategic, risk-based approach to assessing compatibility is recommended herein. In addition to traditional material compatibility, assessment of CSTD compatibility with biologics should consider additional parameters to address specific CSTD-related risks. The BPDG Formulation Workstream has proposed a systematic risk-based evaluation approach as well as a mitigation strategy for establishing suitability of CSTDs for use.

Alginate-zinc (II) phthalocyanine conjugates: Synthesis, characterization and tumor-associated macrophages-targeted photodynamic therapy.

Tumor-associated macrophages (TAMs)-targeted photodynamic therapy (PDT) has dual-selectivity and hence is promising in cancer treatment. Since the scavenger receptor-A (SR-A) on TAMs can recognize polyanions, two molecular-weight sodium alginates (SA1, 41.2 kDa; SA2, 1231.5 kDa) were herein respectively conjugated with 1-[4-(2-aminoethyl) phenoxy] zinc (II) phthalocyanine (1) and two novel conjugates were obtained, characterized and evaluated for their TAMs-targeted PDT efficacy. The SA introduction makes 1 water-soluble, less aggregated and capable of emitting considerable fluorescence in water. Compared with 1, both conjugates, especially 1-SA1, can give higher selectivity and photocytotoxicity to SR-A-positive macrophages J774A.1 than SR-A-negative HepG2 cells. The in vivo biodistribution evaluation shows both conjugates can selectively accumulate at the tumor site and 1-SA1 owns higher tumor accumulation. 1-SA1 can achieve an 87 % tumor inhibition rate without observable systemic toxicity. These results reveal the great potential of SA as a carrier for conjugating anti-tumor drugs and 1-SA1 for TAMs-targeted PDT.

A non-aggregated silicon(IV) phthalocyanine-lactose conjugate for photodynamic therapy.

To develop a highly efficient photosensitizer for photodynamic therapy (PDT), we have designed and synthesized a phthalocyanine-lactose conjugate (Pc-Lac) through axial modification of silicon(IV) phthalocyanine with lactose moieties. With the lactose substituents, Pc-Lac is highly hydrophilic and non-aggregated with efficient reactive oxygen species (ROS) generation in aqueous media. With these desirable properties, Pc-Lac shows high photocytotoxicity and cellular uptake toward HepG2 cells. In addition, in vivo fluorescence imaging shows that Pc-Lac could selectively remain at tumor site, leading to its enhanced photodynamic efficacy against H22 tumor-bearing mice. Therefore, Pc-Lac shows a great potential as a highly efficient molecular photosensitizer for PDT.

Size-Tunable Targeting-Triggered Nanophotosensitizers Based on Self-Assembly of a Phthalocyanine-Biotin Conjugate for Photodynamic Therapy.

Self-assembled phototheranostic nanomaterials used for photodynamic therapy (PDT) have attracted increasing attention owing to their several advantages. Herein, we developed a novel strategy for size-tunable self-assembled nanophotosensitizers for PDT through a simple method. A series of switchable self-assembled nanophotosensitizers (NanoPc90, NanoPc40, NanoPc20, and NanoPc10) of different particle sizes were readily prepared based on an amphiphilic silicon(IV) phthalocyanine (SiPc)-biotin conjugate by regulating the amount of the Cremophor EL surfactant used. The photoactivities, including fluorescence and reactive oxygen species (ROS), of the self-assemblies could be regulated by the particle size. The self-assemblies could be specifically disassembled by tumor-overexpressing biotin receptors, leading to the recovery of quenched photoactivities. Demonstrated by the competitive assay, the self-assemblies were able to enter HepG2 cells through a biotin-receptor-mediated pathway, followed by biotin-receptor-triggered fluorescence recovery at the cellular level. Moreover, the particle size could also affect the in vitro and in vivo PDT effects and tumor targeting. The photocytotoxicity of NanoPc20 against HepG2 cells was more potent compared to that of NanoPc90 because of its strong intracellular fluorescence, higher intracellular ROS generation, and different subcellular localization. In addition, NanoPc20 showed higher in vivo tumor targeting and photodynamic therapeutic efficacy than NanoPc90. This work would provide a valuable reference for the development of self-assembled nanophotosensitizers for cancer diagnosis and therapy.

Highly efficient miniaturized coprecipitation screening (MiCoS) for amorphous solid dispersion formulation development.

Microprecipitated bulk powder (MBP) is a novel solid dispersion technology to manufacture amorphous formulations of poorly soluble compounds that cannot be processed by spray drying or melt extrusion. An efficient high-throughput screening method has been developed to aid the selection of polymer type, drug loading and antisolvent to solvent ratio for MBP formulation development. With a 96-well platform, the miniaturized coprecipitation screening (MiCoS) includes mixing of drug and polymer in dimethylacetamide, controlled precipitation to generate MBP, filtration/washing, drying and high throughput characterization. The integrated MiCoS approach has been demonstrated with a model compound, glybenclamide. Based on the solid state stability and kinetic solubility of the MBP, hydroxypropylmethylcellulose acetate succinate polymer with 40% or lower drug loading, and antisolvent (0.01 N HCl) to solvent (dimethylacetamide) ratio of 5:1 or higher were selected to make glybenclamide MBP. MiCoS can be applied to both early and late stage formulation processing. In early stage research programs, the system can be used to enable efficacy, pharmacokinetics or mini-toxicology studies for poorly water soluble molecules using minimal amount of drug substance (2-10mg). In late stage development programs, MiCoS can be used to optimize MBP formulation by expanding the experimental design space to include additional formulation variants.

Evaluation of chemical labeling strategies for monitoring HCV RNA using vibrational microscopy.

Raman and coherent anti-Stokes Raman scattering (CARS) microscopies have the potential to aid in detailed longitudinal studies of RNA localization. Here, we evaluate the use of carbon-deuterium and benzonitrile functional group labels as contrast agents for vibrational imaging of hepatitis C virus (HCV) replicon RNA. Dynamic light scattering and atomic force microscopy were used to evaluate the structural consequences of altering HCV subgenomic replicon RNA. Modification with benzonitrile labels caused the replicon RNA tertiary structure to partially unfold. Conversely, deuterium-modified replicon RNA was structurally similar to unmodified replicon RNA. Furthermore, the deuterated replicon RNA provided promising vibrational contrast in Raman imaging experiments. The functional effect of modifying subgenomic HCV replicon RNA was evaluated using the luciferase gene as a genetic reporter of translation. Benzonitrile labeling of the replicon RNA prevented translation in cell-based luciferase assays, while the deuterated replicon RNA retained both translation and replication competency. Thus, while the scattering cross-section for benzonitrile labels was higher, only carbon-deuterium labels proved to be non-perturbative to the function of HCV replicon RNA.

Synthesis and characterization of CN-modified protein analogues as potential vibrational contrast agents.

A recombinant VH single-domain antibody recognizing staphylococcal protein A was functionalized on reactive lysine residues with N-hydroxysuccimidyl-activated 4-cyanobenzoate. Surface plasmon resonance analysis of antibody-antigen binding revealed that modified and unmodified antibodies bound protein A with similar affinities. Raman imaging of the modified antibodies indicated that the benzonitrile group provides vibrational contrast enhancement in a region of the electromagnetic spectrum that is transparent to cellular materials. Thus, the modified single-domain antibody may be amenable to detecting protein A from samples of the human pathogen Staphylococcus aureus using vibronic detection schemes such as Raman and coherent anti-Stokes Raman scattering. The generality of this labeling strategy should make it applicable to modifying an array of proteins with varied structure and function.

Accelerating unimolecular decarboxylation by preassociated acid catalysis in thiamin-derived intermediates: implicating Brønsted acids as carbanion traps in enzymes.

Mandelylthiamin (MT) is formally the conjugate of thiamin and benzoylformate. It is the simplified analogue of the first covalent intermediate in benzoylformate decarboxylase. Although MT is the functional equivalent of the enzymic intermediate, it is 106-fold less reactive in decarboxylation. Furthermore, upon loss of carbon dioxide, it undergoes a fragmentation reaction that is about 102-fold faster than the enzymic reaction. While Brønsted acids in general can suppress the fragmentation to some extent, they do not accelerate the decarboxylation. Surprisingly, the conjugate acid of pyridine accelerates decarboxylation; it also blocks fragmentation with particularly high efficiency. These results are consistent with the conjugate acid of pyridine acting as a "spectator" catalyst, associating with MT prior to decarboxylation. In the absence of catalyst, carbon dioxide formed upon carbon-carbon bond breaking overwhelmingly reverts to the carboxylate. Association of pyridine (and its conjugate acid) with MT permits trapping of the nascent carbanion by protonation, while nonassociated acids must arrive by the relatively slow process of diffusion. C-Alkyl pyridine acids provide similar catalysis while other acids have no effect. This suggests that an enzyme that generates an aldehyde from a 2-ketoacid should have functional Brønsted acids in their active sites that would trap the carbanion, as does benzoylformate decarboxylase. Enzymes that give nonaldehydic products from decarboxylation of thiamin diphosphate conjugates containing an associated electron acceptor or electrophilic substrate would also be able to prevent the reversal of decarboxylation.

Making thiamin work faster: acid-promoted separation of carbon dioxide.

The conjugate of thiamin and benzoylformate, mandelylthiamin (MTh), undergoes decarboxylation about 106 times slower than the analogous enzymic intermediate. It has now been discovered that the decarboxylation of MTh is accelerated by the acid component of pyridine and 4-picoline buffers. There is no role for a proton donor to stabilize the transition state for decarboxylation: catalysis must be achieved by the acid's trapping the product carbanion, preventing recarboxylation. This requires that diffusion of CO2 is rate-determining, and that protonation of the carbanion allows this to occur. This interpretation correctly predicts that the same acid components will prevent a fragmentation reaction by protonating the intermediate, which fragments only as the conjugate base.

Fragmentation of the conjugate base of 2-(1-hydroxybenzyl)thiamin: does benzoylformate decarboxylase prevent orbital overlap to avoid it?

The base-catalyzed addition of thiamin to benzaldehyde produces 2-(1-hydroxybenzyl)thiamin (HBnT), but in neutral solution HBnT undergoes base-catalyzed irreversible fragmentation into pyrimidine and thiazole derivatives. The fragmentation (rather than elimination) occurs in proportion to the extent that N1' is protonated or alkylated. Generating the conjugate base of HBnT by decarboxylation surprisingly leads to fragmentation independent of the state of N1'. Therefore, a cationic state at N1' specifically promotes removal of the C2alpha proton rather than the fragmentation process itself. It is suggested that benzoylformate decarboxylase, which generates a similar intermediate, exerts stereoelectronic control of the conformation of the carbanion, blocking fragmentation and facilitating protonation.

Reactivity of intermediates in benzoylformate decarboxylase: avoiding the path to destruction.

Benzoylformate decarboxylase forms a covalent intermediate from thiamin diphosphate (TDP) and benzoylformate, alpha-mandelylTDP. This loses carbon dioxide to form a carbanion (enamine). Protonation of the carbanion and elimination of benzaldehyde regenerate enzyme-bound TDP. We synthesized alpha-mandelylthiamin and found that the rate of the loss of carbon dioxide is one-millionth that of the enzymic reaction. Thus, the enzyme provides an environment that facilitates the unimolecular decarboxylation process. However, the resulting nonenzymic carbanion reacts very rapidly to give products that lead to the irreversible destruction of the cofactor. This contrasts with the normal process on the enzyme. Brønsted acids on the enzyme may divert the reaction to the benzaldehyde precursor, or the enzyme may block access to the pathway that leads to destruction of the cofactor.