Study of the Stability of Sandoz Rituximab Biosimilar Rixathon®/Riximyo® When Subjected for up to 21 Days to Ambient Storage.

AIM: The purpose of this study was to evaluate the extended physicochemical and biological stability of Sandoz Rixathon®/Riximyo® (SDZ-RTX) after exposure to out-of-fridge (OOF) conditions. MATERIALS AND METHODS: The impact of the short-term temperature excursion on stability parameters of SDZ-RTX was simulated by subsequently exposing the three batches of SDZ-RTX (100 and 500 mg) to OOF conditions, (I) 25 ± 2 °C/60 ± 5% relative humidity (RH) and (II) 30 ± 2 °C/65 ± 5% RH, for up to 21 days after more than the claimed 36-month shelf-life storage in long-term conditions (5 ± 3 °C). Analytical methods used included the cation exchange chromatography (CEX), size exclusion chromatography (SEC), and non-reducing capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS), as well as biological activity by complement-dependent cytotoxicity (CDC)-bioactivity as well as further methods, for example, related to identity and pharmacopoeia test methods. RESULTS: No notable changes were observed across all batches with respect to identity (charge and primary structure), pharmaceutical tests (clarity, visible and subvisible particles analytics, container appearance, degree of coloration, pH, osmolality, extractable volume, and container closure integrity testing), protein content by UV and microbiological parameters (sterility and bacterial endotoxins) under both OOF conditions. Only minor changes were observed for parameters evaluated via SEC, CEX, and nrCE-SDS. For potency (CDC-bioactivity) only one of the batches showed a relevant change. Even for these stability-indicating test methods, all analyzed parameters complied with the shelf-life specifications. CONCLUSION: SDZ-RTX is safe for use even under worst-case conditions, for example, after subjecting it for up to 21 days at OOF conditions (25 ± 2 °C/60 ± 5% RH or 30 ± 2 °C/65 ± 5% RH) after the batches had reached an age that was already beyond the claimed shelf-life.

Statistically Significant versus Practically Relevant Trend in Stability Data.

How to decide whether a statistically significant trend is of practical relevance? In the context of stability data of pharmaceuticals, this publication provides a way forward to use different measures of (method) variability to compare to the observed changes over time. A panel of analytical experts assessed whether statistically significant trends were of practical relevance or not. For different types of assessing variability, recommendations for decision criteria were derived that best matched these assessments, i.e., finding a suited balance between not detecting a relevant trend and between flagging a trend wrongly as relevant. For this purpose, more than 60 data sets from Biosimilar projects of Sandoz/Novartis were leveraged. Hence, this article provides a scientific way to assess whether a statistically significant trend is of practical relevance or not, and a case study is presented and discussed.

Can Cross-Linked Siliconized PFS Come to the Rescue of the Biologics Drug Product?

Silicone can present a challenge during the development of a biologics drug product particularly in pre-filled syringe (PFS). Due to silicone related challenges, substantial changes in components and manufacturing of the PFS are being sought. Cross-linking of the silicone being one of them, can help reduce mobilization of the silicone into drug product whilst retaining its functionality of lubrication during injection. In this work, we systematically compare the stability of a fusion protein and monoclonal antibody formulation in conventionally siliconized and cross-linked siliconized PFS available from commercial manufacturers. The two types of syringes did not influence the aggregation profile of proteins as determined by HP-SEC. Compared to conventionally siliconized PFS, a cross-linked siliconized PFS can have a favorable or indifferent impact (depending on vendor) on the sub-visible particles profile as assessed by light obscuration and microflow imaging. The different PFS after 24 months of long-term storage showed comparable functionality attributes like break-loose/gliding force and silicone oil distribution. Cross-linked siliconized PFS can offer an incremental advantage over conventionally siliconized PFS for the moderately concentrated protein solutions, however the differences in the quality of these PFS amongst manufacturers is an important aspect that needs to be considered as shown in this study.

Investigation of drug product and container-closure interactions: A case study of diluent containing prefilled syringe.

Prefilled syringes (PFS) constitute a widely used medical device for drug delivery particularly for the drugs of biological origin. Interactions between the product contents and the components of the PFS play a critical role in determining the suitability of selected PFS. A diluent (with benzyl alcohol/BzOH as a preservative) containing PFS used for reconstitution of the lyophilized product revealed a systematic decrease in the BzOH content during accelerated and stress stability program. Investigation was carried out to understand and identify the underlying causes of this phenomenon. BzOH has a varying propensity to bind to the rubber components (stopper and tip-cap) of the PFS. Vapor permeation behavior across the tip-cap of the PFS was studied via headspace-gas chromatography-mass spectroscopy (HS-GC-MS) enabled analysis. Depending on the properties of the rubber components, BzOH can not only bind but also traverse across them, resulting in a systematic loss during the course of the stability. PFS can allow not only water vapor permeation across the tip-cap as shown in previous studies, but also molecules like benzyl alcohol. This phenomenon stresses the need for careful selection of the components of the primary packaging and also provides an opportunity to deploy novel tools like HS-GC-MS in the early selection of the optimal primary packaging configuration.

Structural insights into a wildtype domain of the oncoprotein E6 and its interaction with a PDZ domain.

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.

NMR assignment of a PDZ domain in complex with a HPV51 E6 derived N-terminally pyroglutamic acid modified peptide.

The resonance assignment of an amino-terminal pyroglutamic acid containing peptide derived from the E6 protein of human papillomavirus (HPV) type 51 in complex with PDZ domain 2 of hDlg/SAP-97 is reported. The assignments include (1)H, (13)C and (15)N resonances for the protein and peptide in the complex and all of the peptide's pyroglutamic acid nuclei.