A Practical Tool for Risk-Based In-use Compatibility Assessments.

In drug development, in-use compatibility studies are crucial steps to ensure that the critical quality attributes of the drug product are maintained when in contact with administration components. But once the drug is in clinical trials, unanticipated variations in these components can stretch limited resources and lengthen timelines to market, as these changes must be assessed and approved to ensure continued patient safety. It's desirable to use a science-based risk evaluation to determine the extent of data and testing needed in these situations, but there is no standard for how such evaluations are done. We have developed an Excel™-based semi-quantitative risk assessment tool to determine whether in-use testing is needed when drug delivery sites or components are changed during a clinical trial. We developed the tool based on our multi-company experience with compatibility studies for many types of drug products targeted for various geographic regions. We have employed the tool as a means to expedite decision-making and, if appropriate, reduce testing in low-risk situations. The tool can save significant time and effort (our estimate is approximately at least 6-9 months off the development cycle) and can minimize pitfalls in clinical administration. While we have designed the tool for our drug products and for use with parenteral dosing regimens, the tool can be adapted for other situations as needed. It will be especially useful for companies with more limited resources.

Assessment of compatibility of rhIGF-1/rhIGFBP-3 with neonatal intravenous medications.

BACKGROUND: Recombinant human (rh)IGF-1/IGFBP-3 protein complex, administered as a continuous intravenous infusion in preterm infants, is being studied for the prevention of complications of prematurity. METHODS: We conducted in vitro studies to evaluate the physical and chemical compatibility of rhIGF-1/IGFBP-3 with medications routinely administered to preterm neonates. In vitro mixing of rhIGF-1/IGFBP-3 drug product with small-molecule test medications plus corresponding controls was performed. Physical compatibility was defined as no color change, precipitation, turbidity, gas evolution, no clinically relevant change in pH/osmolality or loss in medication content. Chemical compatibility of small molecules was assessed using liquid chromatography (e.g., reverse-phase HPLC and ion chromatography), with incompatibility defined as loss of concentration of ≥ 10%. A risk evaluation was conducted for each medication based on in vitro compatibility data and potential for chemical modification. RESULTS: In vitro physical compatibility was established for 11/19 medications: caffeine citrate, fentanyl, fluconazole, gentamicin, insulin, intravenous fat emulsion, midazolam, morphine sulfate, custom-mixed parenteral nutrition solution (with/without electrolytes), parenteral nutrition solution + intravenous fat emulsion, and vancomycin (dosed from a 5 mg/mL solution), but not for 8/19 medications: amikacin, ampicillin, dopamine, dobutamine, furosemide, meropenem, norepinephrine, and penicillin G, largely owing to changes in pH after mixing. Small-molecule compatibility was unaffected post-mixing, with no loss of small-molecule content. For physically compatible medications, risk analyses confirmed low probability and severity of a risk event. CONCLUSION: Co-administration of rhIGF-1/rhIGFBP-3 drug product with various medications was assessed by in vitro studies using case-by-case risk analyses to determine the suitability of the products for co-administration.

Development of Protein-Specific Analytical Methodologies to Evaluate Compatibility of Recombinant Human (rh)IGF-1/rhIGFBP-3 with Intravenous Medications Co-Administered to Neonates.

The protein complex of recombinant human insulin-like growth factor-1 and insulin­like growth factor binding protein­3 (rhIGF-1/rhIGFBP-3; mecasermin rinfabate), is an investigational product for the prevention of complications of prematurity. Delivery of rhIGF-1/rhIGFBP-3 is by continuous central line intravenous infusion in preterm infants until endogenous IGF-1 production begins. Protein-specific analytical methodologies were developed to evaluate the compatibility of rhIGF-1/rhIGFBP-3 at low protein concentrations (∼2.5-10 µg/mL) expected when co-administered with other required medications in the NICU. Highly sensitive detection of the biologic potential degradants (fragments) and/or molecular modifications (oxidized species, aggregates) required the use of reversed-phase high-performance liquid chromatography and size-exclusion ultra-performance liquid chromatography coupled with mass spectrometric detection. We report on the quantification of rhIGF-1/rhIGFBP-3, its components and degradants, to a limit of quantitation of 3.1 µg/mL upon mixing with 24 commonly administered neonatal medications. Methods developed for the rhIGF-1/rhIGFBP-3 admixtures, optimized in studies with furosemide, caffeine citrate and ampicillin, demonstrated good reproducibility, linearity, and limit of detection/quantitation. Using these methods, no increase in degradation of rhIGF-1/rhIGFBP-3 components and no increase in oxidation or aggregation level was observed with caffeine citrate, while admixtures of rhIGF-1/rhIGFBP-3 with ampicillin yielded lower mass recovery of rhIGF-1/rhIGFBP-3 components, which likely resulted from adduct formation. Furosemide was found to be physically incompatible with rhIGF-1/rhIGFBP-3. Our findings support the use of these methodologies for detection of protein modifications under various clinical administration conditions, and additionally supplement physical compatibility data studies of ultra-low concentrations of rhIGF-1/rhIGFBP-3 post co-administration to preterm infants with other medications (manuscript in-preparation).

Freeze-thaw characterization process to minimize aggregation and enable drug product manufacturing of protein based therapeutics.

Physical instabilities of proteins in the form of protein aggregation continue to be a major challenge in the development of protein drug candidates. Aggregation can occur during different stages of product lifecycle such as freeze-thaw, manufacturing, shipping, and storage, and can potentially delay commercialization of candidates. A lack of clear understanding of the underlying mechanism(s) behind protein aggregation and the potential immunogenic reactions renders the presence of aggregates in biotherapeutic products undesirable. Understanding and minimizing aggregation can potentially reduce immunogenic responses and make protein therapeutics safer. Therefore, it is imperative to identify, understand, and control aggregation during early formulation development and develop reliable and orthogonal analytical methodologies to detect and monitor levels of aggregation. Freezing and thawing are typical steps involved in the manufacturing of drug product and could result in complex physical and chemical changes, which in turn could potentially cause protein aggregation. This study provides a systematic approach in understanding and selecting the ideal freeze-thaw conditions for manufacturing of protein-based therapeutics. It identifies the importance of balancing different excipients with an overall goal of sufficiently reducing or eliminating aggregation and developing a stable and scalable formulation. The results demonstrated that the freeze-thaw damage of mAb-1 in aqueous solutions was significantly reduced by identification of optimal freeze-thaw conditions using first a small-scale model with subsequent at-scale verifications. The work provides a framework for successful transfer of drug product manufacturing process from small-scale to the manufacturing scale production environment especially for molecules that are susceptible to freeze-thaw induced degradations.

Self-buffering capacity of a human sulfatase for central nervous system delivery.

Direct delivery of therapeutic enzymes to the Central Nervous System requires stringent formulation design. Not only should the formulation design consider the delicate balance of existing ions, proteins, and osmolality in the cerebrospinal fluid, it must also provide long term efficacy and stability for the enzyme. One fundamental approach to this predicament is designing formulations with no buffering species. In this study, we report a high concentration, saline-based formulation for a human sulfatase for its delivery into the intrathecal space. A high concentration formulation (≤ 40 mg/mL) was developed through a series of systematic studies that demonstrated the feasibility of a self-buffered formulation for this molecule. The self-buffering capacity phenomenon was found to be a product of both the protein itself and potentially the residual phosphates associated with the protein. To date, the self-buffered formulation for this molecule has been stable for up to 4 years when stored at 5 ± 3 °C, with no changes either in the pH values or other quality attributes of the molecule. The high concentration self-buffered protein formulation was also observed to be stable when exposed to multiple freeze-thaw cycles and was robust during in-use and agitation studies.

Self-association of human beta-galactocerebrosidase: Dependence on pH, salt, and surfactant.

Krabbe disease, also known as globoid cell leukodystrophy, is a rare genetic neurodegenerative disease caused by a deficiency of the galactocerebrosidase enzyme. To understand the association status of human beta-galactocerebrosidase (hGALC) in solution, we employed analytical ultracentrifugation (AUC). Our AUC results show that hGALC has a tendency for reversible self-association. Self-association decreases as the concentration of sodium chloride increases from 50 to 500 mM. This indicates that ionic interactions are involved in the association. The association is also dependent on pH, and high order oligomerization decreases as the pH increases from 4.5 to 7.5. Taken together, our results indicate that hGALC has the highest tendency for oligomerization at physiological ionic strength and pH (lysosomal lumen). This is the first report describing the self-associating property of hGALC in solution.

Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles.

This study investigated the root cause behind an observed free fatty acid particle formation and resulting Polysorbate 20 (PS20) loss for a sulfatase drug product upon long-term storage at 5 ± 3°C. Reversed- phase chromatography with mass spectrometric analysis as well as charged aerosol detection was used to characterize the peaks associated with the intact and degraded PS20. Additionally, a proteomics study was undertaken to identify the residual host cell proteins in the sulfatase drug substance. PS20 stability studies were conducted in the presence of sulfatase, a sulfatase inhibitor, putative phospholipase B-like 2, and mock drug substance produced using a null cell line vector under experimental conditions optimized for PS20 degradation. This study provides the first published evidence where the residual host cell protein present in the drug substance was identified and experimentally shown to catalyze the breakdown of PS20 in a protein formulation over time, resulting in free fatty acid particles and PS20 loss. This study demonstrates the importance of early detection of potential impurities in the protein drug substance that may contribute to polysorbate degradation to make a judicious selection of the surfactant and its optimized concentration for the final drug product.

DB-PABP: a database of polyanion-binding proteins.

The interactions between polyanions (PAs) and polyanion-binding proteins (PABPs) have been found to play significant roles in many essential biological processes including intracellular organization, transport and protein folding. Furthermore, many neurodegenerative disease-related proteins are PABPs. Thus, a better understanding of PA/PABP interactions may not only enhance our understandings of biological systems but also provide new clues to these deadly diseases. The literature in this field is widely scattered, suggesting the need for a comprehensive and searchable database of PABPs. The DB-PABP is a comprehensive, manually curated and searchable database of experimentally characterized PABPs. It is freely available and can be accessed online at http://pabp.bcf.ku.edu/DB_PABP/. The DB-PABP was implemented as a MySQL relational database. An interactive web interface was created using Java Server Pages (JSP). The search page of the database is organized into a main search form and a section for utilities. The main search form enables custom searches via four menus: protein names, polyanion names, the source species of the proteins and the methods used to discover the interactions. Available utilities include a commonality matrix, a function of listing PABPs by the number of interacting polyanions and a string search for author surnames. The DB-PABP is maintained at the University of Kansas. We encourage users to provide feedback and submit new data and references.

A network-based analysis of polyanion-binding proteins utilizing human protein arrays.

The existence of interactions between many cellular proteins and various polyanionic surfaces within a cell is now well established. The functional role of such interactions, however, remains to be clearly defined. The existence of protein arrays, with a large selection of different kinds of proteins, provides a way to better address a number of aspects of this question. We have therefore investigated the interaction between five cellular polyanions (actin, tubulin, heparin, heparan sulfate, and DNA) and approximately 5,000 human proteins using protein microarrays in an attempt to better understand the functional nature of such interaction(s). We demonstrate that a large number of polyanion-binding proteins exist that contain multiple positively charged regions, are often disordered, are involved in phosphorylation processes, and appear to play a role in protein-protein interaction networks. Considering the crowded nature of cellular interiors, we propose that polyanion-binding proteins interact with a wide variety of polyanionic surfaces in cells in a functionally significant manner.

A network-based analysis of polyanion-binding proteins utilizing yeast protein arrays.

The high affinity of certain cellular polyanions for many proteins (polyanion-binding proteins (PABPs)) has been demonstrated previously. It has been hypothesized that such polyanions may be involved in protein structure stabilization, stimulation of folding through chaperone-like activity, and intra- and extracellular protein transport as well as intracellular organization. The purpose of the proteomics studies reported here was to seek evidence for the idea that the nonspecific but high affinity interactions of PABPs with polyanions have a functional role in intracellular processes. Utilizing yeast protein arrays and five biotinylated cellular polyanion probes (actin, tubulin, heparin, heparan sulfate, and DNA), we identified proteins that interact with these probes and analyzed their structural and amino acid sequence requirements as well as their predicted functions in the yeast proteome. We also provide evidence for the existence of a network-like system for PABPs and their potential roles as critical hubs in intracellular behavior. This investigation takes a first step toward achieving a better understanding of the nature of polyanion-protein interactions within cells and introduces an alternative way of thinking about intracellular organization.

The use of mucoadhesive polymers in buccal drug delivery.

Buccal delivery of the desired drug using mucoadhesive polymers has been the subject of interest since the early 1980s. Advantages associated with buccal drug delivery have rendered this route of administration useful for a variety of drugs. This review highlights the use of mucoadhesive polymers in buccal drug delivery. Starting with a review of the oral mucosa, mechanism of drug permeation, and characteristics of the desired polymers, this article then proceeds to cover the theories behind the adhesion of bioadhesive polymers to the mucosal epithelium. Additionally, we focus on the new generation of mucoadhesive polymers such as thiolated polymers, followed by the recent mucoadhesive formulations for buccal drug delivery.

Current strategies used to enhance the paracellular transport of therapeutic polypeptides across the intestinal epithelium.

The intent of this paper is to update the reader on various strategies which have been utilized to increase the paracellular permeability of protein and polypeptide drugs across the intestinal epithelium. Structural features of protein and polypeptide drugs, together with the natural anatomical and physiological features of the gastrointestinal (GI) tract, have made oral delivery of this class of compounds extremely challenging. Interest in the paracellular route for the transport of therapeutic proteins and polypeptides following oral administration has recently intensified and continues to be explored. The assumption that molecules with a large molecular weight are not able to diffuse through the tight junctions of the intestinal membrane has been challenged by current research, along with an increased understanding of tight junction physiology.

A randomly coiled, high-molecular-weight polypeptide exhibits increased paracellular diffusion in vitro and in situ relative to the highly ordered alpha-helix conformer.

PURPOSE: The current investigation was conducted to examine the effect of secondary structure of model polypeptides on their hindered paracellular diffusion. METHODS: Poly-D-glutamic acid (PDGlu) was selected as one of the model polypeptides because of its ability to form two secondary structures; a negatively charged random coil and an alpha-helix with partial negative charge at pH 7.4 and 4.7, respectively. Poly-D-lysine (PDL) was selected as a positively charged random coil conformation at pH 7.4. Transport experiments were conducted across both a Caco-2 cell monolayer and the intestinal membrane of Sprague-Dawley rats. Additionally, using NMR, an estimation for the diffusion coefficient and the equivalent hydrodynamic radius for each model polypeptide was obtained. RESULTS: PDGlu in the randomly coiled conformation exhibited greater paracellular transport when compared to either the same polypeptide having an alpha-helix secondary structure or the positively charged, randomly coiled PDL. CONCLUSIONS: Randomly coiled PDGlu was able to permeate through the negatively charged tight junctions of both biological membranes to a greater extent than PDGlu having an alpha-helix structure and suggests that molecular flexibility associated with the random coil conformation may play a more important role than overall charge and hydrodynamic radius on its hindered paracellular diffusion.

An attempt to modulate the microporous diffusion of a model polypeptide by altering its secondary structure.

The purpose of the present study was to determine whether intentional alteration of the secondary structure of a model polypeptide, conantokin-G, influenced the rate and extent of aqueous pore diffusion across a synthetic microporous membrane. Use of a microporous synthetic membrane allowed for analysis of polypeptide transport without the confounding variables of protein binding, acid- and/or enzyme-mediated degradation, endocytotic uptake, and enzymatic inactivation associated with a biological membrane. Conantokin-G was intentionally changed from its native random coil structure to the alpha-helix structure using calcium, and both structures were verified using circular dichroism. The alpha-helix structure of conantokin-G was retained even after additional free calcium was removed by equilibrium dialysis. Over the concentration range of 1.25 to 20 mM, there was a linear relationship between the solution calcium concentration and the percent of the alpha-helix conformer present. The apparent permeability, the apparent aqueous diffusion coefficient with and without inclusion of the Renkin function, and the hydrodynamic radii estimated by diffusion and a computer-software program were calculated for the random coil and alpha-helix structures of conantokin-G. Calcium-mediated conversion of conantokin-G to its alpha-helix structure did not significantly (p >.05) change its apparent permeability across a microporous membrane. It is suggested that perhaps complete conversion to the alpha-helix structure of only a fraction of the conantokin-G molecules (only 0.45 or 45% of the molecules can be converted to the alpha-helix structure at Ca(2+) concentrations >or= 20 mM) may have limited the extent of transport of the alpha-helix conformer.

Poly(L-lysine) as a model drug macromolecule with which to investigate secondary structure and microporous membrane transport, part 2: diffusion studies.

Peptide drugs are hydrophilic in nature and so their preferred pathway of membrane transport is by the paracellular route, which primarily involves passive diffusion across intercellular pores. The objective of the present study was to investigate the effect of secondary structure on the aqueous diffusion of a model polypeptide, poly(L-lysine), through a microporous membrane. The primary aim was to systematically evaluate the variables (e.g. viscosity and/or hydrodynamic radius) that may contribute to the difference, if any, in the calculated values of the aqueous diffusion coefficient (D(aq)) for each conformer of poly(L-lysine). Variations in pH and temperature of the medium were used to induce secondary structural changes in poly(L-lysine). Transport studies were conducted for 3 h at 25 or 37 degrees C using side-by-side diffusion cells. Hydrophilic microporous polyester membranes with a 1-microm pore diameter were used to measure the free diffusion of each conformer. The values for the apparent permeability (P(app)) and D(aq) were calculated using standard equations. The viscosity of each conformer solution was determined and the hydrodynamic radius of each conformer was then estimated. At 25 degrees C, both P(app) and D(aq) of the alpha-helix conformer were approximately the same as those of the random coil conformer. In contrast, at 37 degrees C, the P(app) and the D(aq) of the beta-sheet conformer were significantly (P < 0.05) less than those of the random coil conformer. At 25 degrees C, the solutions containing primarily either the random coil or the-helix conformers had approximately the same viscosity. On the other hand, at 37 degrees C, the solutions containing the beta-sheet conformer had a significantly (P < 0.05) higher viscosity than when this conformer was absent. The random coil and the alpha-helix conformers appeared to have comparable sizes, whereas the hydrodynamic radius estimated for the beta-sheet conformer was significantly (P < 0.05) larger than those for the other two conformers. In summary, changing the secondary structure of poly(L-lysine) from the random coil to the alpha-helix did not affect its P(app) and intrinsic D(aq). On the other hand, appearance of the beta-sheet conformer significantly decreased the values of P(app) and D(aq). The differences appeared to result from the significantly higher solution viscosity as well as the extended structure associated with the beta-sheet conformer of poly(L-lysine). This strategy may represent a potential mechanism to sustain the delivery of therapeutic peptide drugs from a controlled drug delivery device.

Shape imposed by secondary structure of a polypeptide affects its free diffusion through liquid-filled pores.

The purpose of the present study was to investigate the effect of secondary structure of three model polypeptides on their apparent permeability (P(app)) across a synthetic, microporous membrane. Poly-L-lysine (PL), poly-L-glutamate (PGlu), and poly-L-lysine-L-phenylalanine (1:1) (PLP) were selected because a solution environment in which their predominant secondary structure is random coil (RC), alpha-helix, and beta-sheet, respectively, is easily achieved. The conformation of each polypeptide was verified by circular dichroism (CD). Diffusion studies were conducted under sink conditions at 25 degrees C across a microporous polyester membrane using a donor concentration of 0.02 mM for each model polypeptide. NMR was utilized to obtain a second estimation of the diffusion coefficient for each polypeptide. The equivalent hydrodynamic radii (R(e)) of the three model polypeptides were calculated using the values of the diffusion coefficient obtained by both NMR and the classic in vitro diffusion studies. The viscosity of each polypeptide solution was also determined to investigate the effect of viscosity on the aqueous diffusion coefficient. Statistical analysis demonstrated a significant (P < 0.05) difference in both P(app) and the aqueous diffusion coefficient (D(aq)), as well as the calculated R(e) values, between all three model polypeptides and there was no significant (P > 0.05) difference in the viscosity of the polypeptide solutions. Values of D(aq) and R(e) calculated from the diffusion studies were in relatively close agreement to those obtained using NMR. The logarithm of P(app) was highly correlated (r = -0.961) with the values of R(e) calculated from NMR (R(e (NMR))) rather than the mw of the polypeptides (r = 0.681). Values of the Perrin or shape factor which deviate substantially from unity are suggestive of a non-spherical or ellipsoid shape and were 1.22 +/- 0.20, 1.55 +/- 0.11, and 2.38 +/- 0.20 for PGlu, PL, and PLP, respectively. In conclusion, the observed difference in the membrane transport/diffusion of the three model polypeptides is suggested to be due to the shape associated with the secondary structure of each macromolecule, rather than the polypeptide's mw or the viscosity of the dilute polypeptide solution.