Stress Factors in Primary Packaging, Transportation and Handling of Protein Drug Products and Their Impact on Product Quality.

Protein-based biologic drugs encounter a variety of stress factors during drug substance (DS) and drug product (DP) manufacturing, and the subsequent steps that result in clinical administration by the end user. This article is the third in a series of commentaries on these stress factors and their effects on biotherapeutics. It focuses on assessing the potential negative impact from primary packaging, transportation, and handling on the quality of the DP. The risk factors include ingress of hazardous materials such as oxidizing residuals from the sterilization process, delamination- or rubber stopper-derived particles, silicone oil droplets, and leachables into the formulation, as well as surface interactions between the protein and packaging materials, all of which may cause protein degradation. The type of primary packaging container used (such as vials and prefilled syringes) may substantially influence the impact of transportation and handling stresses on DP Critical Quality Attributes (CQAs). Mitigations via process development and robustness studies as well as control strategies for DP CQAs are discussed, along with current industry best practices for scale-down and in-use stability studies. We conclude that more research is needed on postproduction transportation and handling practices and their implications for protein DP quality.

An Investigation to Examine the Effect of the Elastomeric Surface Treatment on Protein Stability.

Various kinds of treatments on the surface of the elastomeric components can have negative impacts on the quality of protein therapeutics. We compared the effects of bare (non-siliconized and nonlaminated), siliconized, and fluoropolymer-laminated elastomeric components on the stability of ß-lactoglobulin, human serum albumin, adalimumab, abatacept, and immunoglobulin antibodies. The study was conducted in two main parts. Part I was to evaluate the stability of proteins under agitation-induced stress. Protein aggregate formation, turbidity, and protein recovery were analyzed using dynamic FI, absorbance at 350 nm, and size-exclusion high-performance liquid chromatography, respectively. Proteins were found to be more stable with laminated stoppers as compared with bare or siliconized stoppers. Part II was to identify the chemical modifications when the proteins were stored in contact with the same three stoppers. Capillary isoelectric focusing analysis of the adalimumab samples showed formation of acidic variants in siliconized and bare stoppers. Reverse-phase high-performance liquid chromatography suggested chemical changes to the human serum albumin. Analysis of tryptic digest of human serum albumin by liquid chromatography/mass spectrometry/mass spectrometry indicated that the amino acids most susceptible to oxidation (cysteine, tryptophan, and methionine) were also the ones that were modified. Part III of this study investigated the barrier property of the fluoropolymer film with no drug product. Our results were consistent with the suggestion that the fluoropolymer lamination provides a barrier that prevents leachables from the elastomeric components into the protein therapeutics. Our work provided an in-depth understanding of the effects of elastomeric surface treatments on the biophysical and chemical stability of protein drugs.

A Streamlined Bioanalytical Approach to Select a Compatible Primary Container System Early in Drug Development: A Toolbox for the Biopharmaceutical Industry.

To ensure drug efficacy and patient safety, the importance of interaction between primary container and a biological drug product must not be ignored. The United States Food and Drug Administration guidance on development and manufacturing of combination products (e.g., the biologic and the primary container) encourages careful selection and stability testing of the drug and its performance in the marketed primary container. With various options available for primary container type (vials, prefilled syringes, and cartridges), material (e.g., glass or plastic), and lubricants/coatings, we designed a platform consisting of several bioanalytical methods that can simplify selection of a compatible primary container. We tested the stability of a commercially available monoclonal antibody (mAb) in 3 syringe types under 3 conditions: cold storage, high temperature, and agitation induced stress, respectively. Under each condition, dynamic fluid imaging was sensitive enough to differentiate mAb stability as measured by aggregate formation in different syringe systems, followed by size exclusion-high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis but only at high temperature. With this platform, we identified a primary container that provided higher mAb stability under cold storage as well as stress conditions. We recommend that such an approach should be applied early in drug development stage to identify a superior primary container system to maintain drug stability and quality.

Model Protein Adsorption on Polymers Explained by Hansen Solubility Parameters.

Hansen solubility parameters (HSP) theory has been successful in explaining the wettability of organic solvents on polymer surfaces and miscibility of different polymers. Here, we demonstrate that the amount of bovine serum albumin (BSA) protein adsorption on different polymer surfaces can also be explained by HSP. Interestingly, the HSP of the adsorbed BSA proteins calculated from the protein adsorption data is different than the HSP of native BSA protein itself. The HSP of the adsorbed BSA proteins are more hydrophobic than the native BSA protein. This observation suggested adsorbed BSA proteins are partially denatured and exposed their hydrophobic core toward the polymer surfaces. These results highlight a new strategic direction to understand interaction of protein with a surface: a theoretical approach that compliments experimental approach. The model in this study could be used to predict the amount of BSA adsorption on a polymer or any other solid surface, if the HSP of that surface is known. Further, the model can serve as a prescreen method to identify surfaces that are problematic at the outset and inform subsequent empirical studies to select packaging that will have the least adsorption for the specific biologic application.

A Comparison of Protein Stability in Prefillable Syringes Made of Glass and Plastic.

The development of protein therapeutics requires stabilization of these labile molecules during shipment and storage. Biologics, particularly monoclonal antibodies, are frequently packaged at high concentration in prefillable syringes traditionally made of glass. However, some biologics are unstable in glass due to sensitivity to silicone oil, tungsten, glue, or metal ions. Syringes made from the plastic cyclic olefin polymer, Daikyo Crystal Zenith® (CZ), with a Flurotec-laminated piston, have none of these issues. This study compared the stability of several proteins including biotherapeutics when stored up to 14 months at 5 °C and 25 °C in prefillable siliconized syringes made of glass or silicone oil-free CZ syringes, and when subjected to mild agitation by end-over-end rotation at room temperature. At each time point, proteins were analyzed by several techniques including turbidity, size exclusion high-performance liquid chromatography, reversed phase high-performance liquid chromatography, ion-exchange chromatography, electrophoresis, and light scattering to monitor changes in aggregation and degradation. The results show that proteins have comparable stability when stored in glass syringes or in syringes made of CZ sterilized by E-beam or autoclave. In addition, proteins stressed by agitation were generally more stable and aggregated less in syringes made of CZ than in ones made of glass.LAY ABSTRACT: Biotherapeutic protein drugs such as monoclonal antibodies are frequently packaged at high concentration in prefillable syringes, which allows the drug to be directly administered by the patient or caregiver. Protein drugs, or biologics, can be unstable, and may aggregate, particularly when shaken. These aggregates can be immunogenic, stimulating the body's immune system to produce antibodies that can reduce the drug's efficacy. Although prefillable syringes are traditionally made of glass, some biologics are unstable in glass syringes due to the presence of substances used in their manufacture, including silicone oil, which is necessary for lubricity. Syringes made from the plastic cyclic olefin polymer, Daikyo Crystal Zenith® (CZ), have none of these issues. This study compared the stability of several biotherapeutic proteins when stored up to 14 months at 5 °C and 25 °C in prefillable siliconized syringes made of glass or silicone oil-free CZ syringes, and when mildly agitated at room temperature. Proteins were analyzed by several techniques to detect changes in aggregation and degradation. The results show that biotherapeutic proteins have similar stability whether stored in syringes made of glass or CZ. In addition, proteins subjected to agitation were generally more stable and aggregated less in CZ syringes than in glass syringes.

Characterization of a cyclic olefin polymer microcentrifuge tube.

Here, we describe the properties of a prototype microcentrifuge tube made from the plastic cyclic olefin polymer (COP). This material has been used in the manufacture of primary containers including syringes and vials for the storage, shipment, and delivery of biotherapeutics, vaccines, and cell therapy products. Its low level of extractable substances and metals along with its glass-like clarity make COP an attractive material for the fabrication of microcentrifuge tubes and other consumable laboratory plasticware where contamination is an important consideration, such as in the storage and analysis of labile proteins, nucleic acids, and metabolites. We compare the performance of microcentrifuge tubes made of COP with that of several brands made of polypropylene (PP), the plastic most widely used in the manufacture of microcentrifuge tubes. Our results show COP microcentrifuge tubes perform as well as tubes made of PP, with reduced levels of compounds capable of leaching into solvents typically used in the laboratory.

Biochemical and structural characterization of a novel class of inhibitors of the type 1 insulin-like growth factor and insulin receptor kinases.

The type 1 insulin-like growth factor receptor (IGF-1R) is often overexpressed on tumor cells and is believed to play an important role in anchorage-independent proliferation. Additionally, cell culture studies have indicated that IGF-1R confers increased resistance to apoptosis caused by radiation or chemotherapeutic agents. Thus, inhibitors of the intracellular kinase domain of this receptor may have utility for the clinical treatment of cancer. As part of an effort to develop clinically useful inhibitors of IGF-1R kinase, a novel class of pyrrole-5-carboxaldehyde compounds was investigated. The compounds exhibited selectivity against the closely related insulin receptor kinase intrinsically and in cell-based assays. The inhibitors formed a reversible, covalent adduct at the kinase active site, and treatment of such adducts with sodium borohydride irreversibly inactivated the enzyme. Analysis of a tryptic digest of a covalently modified IGF-1R kinase fragment revealed that the active site Lys1003 had been reductively alkylated with the aldehyde inhibitor. Reductive alkylation of the insulin receptor kinase with one of these inhibitors led to a similarly inactivated enzyme which was examined by X-ray crystallography. The crystal structure confirmed the modification of the active site lysine side chain and revealed details of the key interactions between the inhibitor and enzyme.

Crystal structure of the Apo, unactivated insulin-like growth factor-1 receptor kinase. Implication for inhibitor specificity.

The x-ray structure of the unactivated kinase domain of insulin-like growth factor-1 receptor (IGFRK-0P) is reported here at 2.7 A resolution. IGFRK-0P is composed of two lobes connected by a hinge region. The N-terminal lobe of the kinase is a twisted beta-sheet flanked by a single helix, and the C-terminal lobe comprises eight alpha-helices and four short beta-strands. The ATP binding pocket and the catalytic center reside at the interface of the two lobes. Despite the overall similarity to other receptor tyrosine kinases, three notable conformational modifications are observed: 1) this kinase adopts a more closed structure, with its two lobes rotated further toward each other; 2) the conformation of the proximal end of the activation loop (residues 1121-1129) is different; 3) the orientation of the nucleotide-binding loop is altered. Collectively, these alterations lead to a different ATP-binding pocket that might impact on inhibitor designs for IGFRK-0P. Two molecules of IGFRK-0P are seen in the asymmetric unit; they are associated as a dimer with their ATP binding clefts facing each other. The ordered N terminus of one monomer approaches the active site of the other, suggesting that the juxtamembrane region of one molecule could come into close proximity to the active site of the other.