Demonstration of Physicochemical and Functional Similarity of Biosimilar Pegfilgrastim-cbqv to Pegfilgrastim.

BACKGROUND: Pegfilgrastim-cbqv/CHS-1701 (UDENYCA®) (hereafter referred to as pegfilgrastim-cbqv) was approved in 2018 by the US Food and Drug Administration as a biosimilar for pegfilgrastim (Neulasta®) (hereafter referred to as pegfilgrastim). Both pegfilgrastim-cbqv and pegfilgrastim are conjugates of recombinant human granulocyte colony stimulating factor (r-metHuG-CSF) with a 20 kDa polyethylene glycol (PEG) indicated to decrease the incidence of infection, as manifested by febrile neutropenia, in patients receiving myelosuppressive anticancer drugs. The demonstration of analytical similarity for PEG-protein conjugates presents unique challenges since both the protein and PEG attributes must be characterized. OBJECTIVE: The current study demonstrates the analytical similarity of pegfilgrastim-cbqv and the reference product, pegfilgrastim. In addition to the physicochemical and functional characterization of the protein, the study assessed attributes specific to PEGylation including PEG size and polydispersity, site of attachment, linker composition, and PEGylation process-related variants. METHODS: The structural, functional, and stability attributes of pegfilgrastim-cbqv and pegfilgrastim were compared using state-of-the-art analytical methods. For the protein, the primary structure, disulfide structure, and secondary and tertiary structures were assessed using traditional protein characterization techniques such as mass spectrometry (MS), circular dichroism (CD), intrinsic fluorescence, and differential scanning calorimetry (DSC), as well as more advanced techniques such as two-dimensional (2D) nuclear magnetic resonance (NMR) and hydrogen deuterium exchange (HDX). For the PEG moiety, the site of attachment, occupancy, linker composition, size and polydispersity were compared using mass spectrometry (both intact and after endoprotease digestion), multiangle light scattering detection (MALS), and Edman degradation. Purity assessments included the assessment of both protein variants and PEGylation variants using chromatographic and electrophoretic analytical separation techniques. The functional similarity between pegfilgrastim-cbqv and pegfilgrastim was compared using both a cell-based bioassay and surface plasmon resonance (SPR). The degradation rates and stability profiles were compared under accelerated and stressed conditions. RESULTS: Biosimilarity was demonstrated by a thorough assessment of physiochemical and functional attributes, as well as comparative stability, of pegfilgrastim-cbqv relative to pegfilgrastim. These studies demonstrated identical primary structure and disulfide structure, highly similar secondary and tertiary structure, as well as functional similarity. The impurity profile of pegfilgrastim-cbqv was comparable to that of pegfilgrastim with only minor differences in PEGylation variants and a slight offset in the PEG molar mass. These differences were not clinically relevant. The degradation profiles were qualitatively and quantitatively similar under accelerated and stress conditions. CONCLUSION: The structural, functional, and stability data demonstrate that pegfilgrastim-cbqv is highly similar to the reference product, pegfilgrastim.

Rational Selection, Criticality Assessment, and Tiering of Quality Attributes and Test Methods for Analytical Similarity Evaluation of Biosimilars.

Leading regulatory agencies recommend biosimilar assessment to proceed in a stepwise fashion, starting with a detailed analytical comparison of the structural and functional properties of the proposed biosimilar and reference product. The degree of analytical similarity determines the degree of residual uncertainty that must be addressed through downstream in vivo studies. Substantive evidence of similarity from comprehensive analytical testing may justify a targeted clinical development plan, and thus enable a shorter path to licensing. The importance of a careful design of the analytical similarity study program therefore should not be underestimated. Designing a state-of-the-art analytical similarity study meeting current regulatory requirements in regions such as the USA and EU requires a methodical approach, consisting of specific steps that far precede the work on the actual analytical study protocol. This white paper discusses scientific and methodological considerations on the process of attribute and test method selection, criticality assessment, and subsequent assignment of analytical measures to US FDA's three tiers of analytical similarity assessment. Case examples of selection of critical quality attributes and analytical methods for similarity exercises are provided to illustrate the practical implementation of the principles discussed.

Statistical Approaches to Assess Biosimilarity from Analytical Data.

Protein therapeutics have unique critical quality attributes (CQAs) that define their purity, potency, and safety. The analytical methods used to assess CQAs must be able to distinguish clinically meaningful differences in comparator products, and the most important CQAs should be evaluated with the most statistical rigor. High-risk CQA measurements assess the most important attributes that directly impact the clinical mechanism of action or have known implications for safety, while the moderate- to low-risk characteristics may have a lower direct impact and thereby may have a broader range to establish similarity. Statistical equivalence testing is applied for high-risk CQA measurements to establish the degree of similarity (e.g., highly similar fingerprint, highly similar, or similar) of selected attributes. Notably, some high-risk CQAs (e.g., primary sequence or disulfide bonding) are qualitative (e.g., the same as the originator or not the same) and therefore not amenable to equivalence testing. For biosimilars, an important step is the acquisition of a sufficient number of unique originator drug product lots to measure the variability in the originator drug manufacturing process and provide sufficient statistical power for the analytical data comparisons. Together, these analytical evaluations, along with PK/PD and safety data (immunogenicity), provide the data necessary to determine if the totality of the evidence warrants a designation of biosimilarity and subsequent licensure for marketing in the USA. In this paper, a case study approach is used to provide examples of analytical similarity exercises and the appropriateness of statistical approaches for the example data.