Phase I/II study to assess the clinical pharmacology and safety of single ascending and multiple subcutaneous doses of PF-06881894 in women with non-distantly metastatic breast cancer.

PURPOSE: To evaluate the pharmacodynamics (PD), pharmacokinetics (PK), and safety of single and multiple doses of PF-06881894 (pegfilgrastim-apgf; Nyvepria™), a biosimilar to reference pegfilgrastim (Neulasta®), in women with non-distantly metastatic breast cancer. METHODS: In Phase I (Cycle 0) of this Phase I/II study, the PD response (absolute neutrophil count [ANC]; CD34 + count), PK profile, and safety of a single 3- or 6-mg subcutaneous dose of PF-06881894 were assessed in chemotherapy-naïve patients before definitive breast surgery. In Phase II (Cycles 1-4), the PD response (duration of severe neutropenia [DSN, Cycle 1], ANC [Cycles 1 and 4]) and PK profile (Cycles 1 and 4) of single and multiple 6-mg doses of PF-06881894 concomitant with chemotherapy and after definitive breast surgery were assessed. RESULTS: Twenty-five patients (mean age 59 years) were enrolled (Cycle 0, n = 12; Cycles 1-4, n = 13). In Cycle 0, PD responses and PK values were lower with 3-mg versus 6-mg PF-06881894. In Cycles 1 and 4, mean DSN was 0.667 days after single or multiple 6-mg doses of PF-06881894, respectively. In Cycle 4 versus Cycle 1, PD responses were more robust; PK values (mean area under the curve, maximum concentration) were lower; and clearance values were higher. The safety profile of PF-06881894 was similar to that for reference pegfilgrastim. CONCLUSION: PF-06881894 as a single 3- or 6-mg dose prior to definitive surgery, or multiple 6-mg/cycle doses postoperatively, with/without myelosuppressive chemotherapy, was consistent with the clinical pharmacology and safety profile of reference pegfilgrastim. TRIAL REGISTRATION: October 2017. ClinicalTrials.gov Identifier: NCT02650193. EudraCT Number: 2015-002057-35.

Biosimilar Perceptions Among Healthcare Professionals and Commercial Medical Benefit Policy Analysis in the United States.

BACKGROUND: Biosimilars represent a significant cost savings opportunity for the entire healthcare system. Despite efforts from the United States Food and Drug Administration, adoption has not been as successful as originally hoped. Perceived barriers to adoption of biosimilars have been described previously, but more knowledge is needed. Further, increased understanding is needed surrounding commercial payer preferences of biosimilars. METHODS: A survey to assess perceived barriers to biosimilar adoption was dispersed to healthcare leaders who work in health-systems, physician practices, and the pharmaceutical industry. Policies from the top 15 commercial payers, by covered lives, were reviewed to collect information surrounding coverage and preferred products to assess if perceptions from healthcare leaders align with payer policies. RESULTS: The largest number of responses (n = 76) came from health-systems (n = 56), followed by pharmaceutical manufacturers (n = 12), and physician practices (n = 8). Responses from each cohort aligned very closely with the composite results of the group. Responses surrounding safety and efficacy were high amongst all groups, while rebate increases to payers for reference products were of highest concern for adoption. United Healthcare had the most policies preferring biosimilars (6/7, 86%). Filgrastim-sndz (Zarxio), had the most preferred statuses for a biosimilar (10/15, 67%). The infliximab reference product had the most preferred statuses for a reference product (9/15, 60%). CONCLUSIONS: Findings from this study outline the greatest perceived barriers to adoption of biosimilars from a variety of different stakeholders. Rebates from reference product manufacturers to payers was the main deterrent for biosimilar use.

Physicochemical stability study of MYL-1401O, a biosimilar of trastuzumab, following a transient temperature excursion.

INTRODUCTION: The extended stability of the trastuzumab biosimilar Ogivri™ (MYL-1401O; trastuzumab-dkst) was studied under different storage conditions, including following reconstitution of the lyophilized powder (21 mg/mL) but undiluted and stored in vials at 4°C; after dilution at two concentrations (0.8 and 2.4 mg/mL) in polyolefin bags and stored at 4°C; and following a three-day thermal excursion to 25°C. METHODS: Several methods were utilized to assess the physical and chemical stability of the drug under different storage conditions. RESULTS: At all storage conditions tested, there was no change in the tertiary structure of MYL-1401O as assessed by second-derivative ultraviolet and fluorescence-derived spectral analysis, and no evidence of oligomer formation or fragmentation was observed as assessed by gel exclusion chromatography and dynamic light scattering, confirmed by assessment of quinary structures using size-exclusion chromatography. Ion-exchange chromatography showed no significant changes in the distribution of ionic variants, particularly deamidations. Thermal denaturation curves indicated no destabilization of the three-dimensional structure after 90 days at 4°C or after thermal excursion for 72 h at 25°C. CONCLUSION: The trastuzumab biosimilar MYL-1401O maintained its physical and chemical stability for at least 90 days at 4°C or after thermal excursion to 25°C, supporting the safe use of MYL-1401O in several real-world settings, including advanced preparation for administration or when a break in the cold cycle occurs.

Evaluation of Structural, Biological, and Functional Similarity of Biosimilar Granulocyte Colony Stimulating Factor to its Reference Product.

PURPOSE: Granulocyte colony stimulating factor (GCSF; also known as filgrastim) is a growth factor used to induce production of granulocytes. As the first locally developed and approved biosimilar medicine of Turkey, Fraven® being a biosimilar of filgrastim has been ab initio manufactured from cell to finished product at two different production facilities. Comprehensive structural, biological and functional characterization studies were performed to compare Fraven® from two different production sites and its reference product Neupogen® sourced from Turkey. METHODS: Primary and higher-order protein structures were analyzed by high performance liquid chromatography electrospray ionization-time of flight mass spectrometry, circular dichroism, and two-dimensional nuclear magnetic resonance spectroscopy. Isoelectric focusing, SDS-Page, size exclusion chromatography, and related proteins analyses were used to compare impurities. In order to assess functional similarity, surface plasmon resonance (SPR) was used. In vitro cell proliferation assay was also performed to show dose related drug response in NFS-60 cell line. RESULTS: Primary, secondary and tertiary structures of biosimilar Fraven® manufactured at both sites were found to be highly similar to the reference Neupogen®. Product related substances and impurities were also highly similar to the reference. Comparability of GCSF receptor binding affinities of each product was shown using the KD values of SPR analysis. In vitro cell proliferation similarity was also evaluated and proven by PLA. CONCLUSION: Based on the similarity assessment, despite being manufactured at two different production sites, biosimilar Fraven® is highly similar to the reference product Turkey originated Neupogen®.

HDX and In Silico Docking Reveal that Excipients Stabilize G-CSF via a Combination of Preferential Exclusion and Specific Hotspot Interactions.

Assuring the stability of therapeutic proteins is a major challenge in the biopharmaceutical industry, and a better molecular understanding of the mechanisms through which formulations influence their stability is an ongoing priority. While the preferential exclusion effects of excipients are well known, the additional presence and impact of specific protein-excipient interactions have proven to be more elusive to identify and characterize. We have taken a combined approach of in silico molecular docking and hydrogen deuterium exchange-mass spectrometry (HDX-MS) to characterize the interactions between granulocyte colony-stimulating factor (G-CSF), and some common excipients. These interactions were related to their influence on the thermal-melting temperatures (Tm) for the nonreversible unfolding of G-CSF in liquid formulations. The residue-level interaction sites predicted in silico correlated well with those identified experimentally and highlighted the potential impact of specific excipient interactions on the Tm of G-CSF.

The incidence and clinical features of PEGylated filgrastim-induced acute aortitis in patients with breast cancer.

Although PEGylated filgrastim-induced aortitis is very rare and unknown clinically, some cases were reported and increasing, especially in breast cancer patients. The present study investigated the prevalence, clinical features and treatment of aortitis induced by PEGylated filgrastim in patients with breast cancer. A total of 2068 consecutive patients who underwent neoadjuvant/adjuvant chemotherapy with PEGylated filgrastim for breast cancer were enrolled. From the medical record, clinical, laboratory, medication, and imaging evaluation findings were collected. PEGylated filgrastim-induced aortitis was established in 0.3% of the study population. Common clinical presentations included extremely high fever and chest/back pain with high levels of inflammatory markers without any signs of infection. Contrast-enhanced computed tomography scans revealed typical enhancing wall thickening and periaortic soft tissue infiltration at various levels of aorta. All patients improved rapidly after treatment with modest doses of prednisolone (0.5 mg/kg/day) without any complications. Clinicians should be aware of aortitis as a possible complication of granulocyte-colony stimulating factor therapy, especially PEGylated filgrastim, given the frequent misdiagnoses in neutropenic patients undergoing chemotherapy.

Pegfilgrastim (PEG-G-CSF) Induces Anti-polyethylene Glycol (PEG) IgM via a T Cell-Dependent Mechanism.

Protein-based therapeutics are beginning to be widely used in various clinical settings. Conjugation of polyethylene glycol (PEGylation) to protein therapeutics improves their circulation half-lives in the body. However, we and other groups observed that the initial dose of some PEGylated protein-based therapeutics may induce anti-PEG antibodies (primarily immunoglobulin M (IgM)), resulting in the accelerated clearance of a second dose. The mechanism behind the induction of anti-PEG IgM by PEGylated protein-based therapeutics is still unclear. In this study, we found that Pegfilgrastim (PEG-G-CSF, the PEGylated form of the recombinant human granulocyte colony-stimulating factor) induced anti-PEG IgM in mice when administered via either intravenous or subcutaneous administration. However, the anti-PEG IgM induction is diminished both in athymic nude mice lacking T cells and in splenectomized mice. In addition, anti-PEG IgM production was significantly diminished in the cyclophosphamide-treated mice depleted of B-cells. These results indicate that anti-PEG IgM production by Pegfilgrastim occurs in spleen in a T cell-dependent manner, which differs from anti-PEG IgM induced by PEGylated liposomes. However, B cells, both marginal zone and follicular, are essential for anti-PEG IgM production in both PEGylated preparations.

Filgrastim loading in PLGA and SLN nanoparticulate system: a bioinformatics approach.

OBJECTIVE: In this research work, we hypothesized to predict the nanoparticulate system, best suited for targeted delivery of filgrastim. Significance: Targeted delivery of filgrastim to bone marrow is required to decrease the incidence of neutropenia/febrile neutropenia. This is achieved by nanoparticulate systems, duly designed by bioinformatics approach. METHOD: The targeted delivery of filgrastim in nanoparticulate system was achieved by molecular dynamics (MD) simulation studies. Two matrices comprising PLGA and SLN (tripalmitin, core component of SLN system) were modeled separately with proposed drug filgrastim. Energy minimization of all systems was done using the steepest descent method. PLGA and tripalmitin systems were equalized at 310 °C, at 1 bar pressure with Berendsen barostat for 200 ps using a v-rescale thermostat for 100 ps. Atomistic MD simulations of four model system and mass density of interacting systems were calculated. RESULTS: The mass density maps of each nanoparticle system, that is, PLGA and tripalmitin showed an increase in density toward the end of the simulation. The contact numbers attained equilibria with the average number of approx.. 1500 contacts in case of tripalmitin-filgrastim system. While PLGA-filgrastim system shows lesser contacts as compared to tripalmitin with average contacts of approx. 1000.The binding free energy was predicted to be -1104 kJ/mol in tripalmitin-filgrastim complex and -421 kJ/mol in PLGA-filgrastim system. CONCLUSION: Findings of study revealed that both nanoparticle systems assumed to be good model for drug-carrier systems. Though SLN systems were thought to be more appropriate than PLGA, still the in vivo findings could ascertain this hypothesis in futuristic work.

Simultaneous analysis of filgrastim and pegfilgrastim aggregates by size-exclusion chromatography.

A new size-exclusion high-performance liquid chromatographic (SE-HPLC) method for the simultaneous analysis of filgrastim and pegfilgrastim aggregates was developed and validated. A cross-linked agarose and dextran column was used at ambient temperature and an alkaline sodium phosphate buffer as mobile phase eliminated non-ideal interactions with the stationary phase. The robustness of the method was assessed by varying injection volumes, flow rates and sample vehicles. Other reliability assessments include calibration curve, intra and inter-day precision and accuracy, repeatability of retention times, application to real in-process production samples and column lifetime. The method exhibited linearity over the concentration of 0.02-4 mg/ml range for filgrastim and pegfilgrastim monomer with a correlation coefficient of greater than 0.999. The lower limit of quantification was 0.02 mg/ml and the limit of detection was 0.005 mg/ml. This SE-HPLC technique has been successfully used for several years and more than 10,000 samples.

Efficacy of Jackfruit365™ Green Jackfruit Flour Fortified Diet on Pegfilgrastim to Prevent Chemotherapy-Induced Leukopenia, Irrespective of Tumor Type or Drugs Used-A Retrospective Study.

Chemotherapy-Induced Leukopenia (CIL) is associated with increased mortality and economic burden on patients. This study was conducted to evaluate whether inclusion of green jackfruit flour in regular diet of those patients receiving chemotherapy, could prevent CIL. This was a retrospective study conducted among a group of patients undergoing chemotherapy for solid tumors at Renai Medicity Hospital, Palarivattom, Cochin, Kerala, India, since June 2018. The study group comprised of 50 consecutive subjects, who were supplemented with green jackfruit flour diet in their regular diet and further followed up prospectively. The control group was retrospective with 50 subjects prior to June 2018, with no diet supplements. Those who received less than three cycles were excluded from either arm. The mean age of the participants in study group and control group were 53.16 ± 11.06 and 56.96 ± 12.16 years respectively. In the study group, six patients out of 37, and 20 patients out of 50 in the control group, developed CIL. They received 38 and 105 vials of filgrastim respectively. After excluding those cycles in study group patients, where green jackfruit flour was not taken, the mean number of cycles in which CIL developed (p = 0.00) and number of vials of filgrastim taken per cycle (p = 0.00) were significantly different from control group and no patient in the study group developed CIL. Inclusion of green jackfruit flour as a dietary intervention prevents chemotherapy-induced leukopenia in patients undergoing chemotherapy along with pegfilgrastim.

Structural similarity, characterization of Poly Ethylene Glycol linkage and identification of product related variants in biosimilar pegfilgrastim.

The approval of biosimilars requires demonstration of biosimilarity, which rests on the base of thorough analytical characterization of the biosimilar product. In addition to demonstration of biosimilarity, the product related impurities need to be thoroughly characterized and controlled at minimal levels. Pegylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG (Poly Ethylene Glycol) heterogeneity, site of addition and number of attached pegylated moieties. A combination of several methods including circular dichroism, FTIR (Fourier-transform Infrared Spectroscopy), fluorescence spectroscopy, DSC (Differential Scanning Calorimetry), 1D and 2D NMR (Nuclear Magnetic Resonance), Edman degradation and peptide mapping by LC-MS (Liquid Chromatography Mass Spectrometry) were used for characterization of N-terminally pegylated filgrastim. Product related impurities such as oxidized, reduced, deamidated, dipegylated variants and monopegylated positional isomers have been characterized in detail using various HPLC (High Performance Liquid Chromatography) based methods and LC-MS techniques. The functional characterization in terms of receptor binding and cell proliferation assay was conducted for the similarity assessment and the potential impact of the product variants on the in vitro biological activity has also been assessed. In summary, this study presents, for the first time, a detailed structural and molecular level characterization of a biosimilar pegfilgrastim providing a strong base for the demonstration of overall biosimilarity of the product with the innovator product.

Quality Comparison of Biosimilar and Copy Filgrastim Products with the Innovator Product.

PURPOSE: Filgrastim, a recombinant human granulocyte-colony stimulating factor, is widely used to treat congenital and acquired neutropenia. Following patent expiration of the innovator filgrastim product, biosimilar filgrastim products have been approved in the EU and shown to be comparable with the innovator with respect to quality, safety and efficacy. In less regulated markets, copy filgrastim products are available but data about their quality are scarce. In the present study, we provide a head-to-head comparative study on the quality of biosimilar and copy filgrastim products. METHODS: Innovator filgrastim product, Neupogen®, two EU-licensed biosimilars, Zarzio® and Tevagrastim®, and two copy filgrastim products, Biocilin® and PDgrastim®, were subjected to peptide mapping, circular dichroism spectroscopy, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size-exclusion chromatography, reversed-phase ultra-performance liquid chromatography, endotoxin test, flow imaging microscopy and in vitro potency assay. RESULTS: Zarzio® and Tevagrastim® have comparable quality to Neupogen®, while Biocilin® showed a significantly lower and PDgrastim® a higher specific activity. Moreover, PDgrastim® showed a higher level of impurities and a lower thermo stability than the other products. CONCLUSIONS: Except for the deviating specific activities of the two copy filgrastim products, we found no substantial differences in product quality between the filgrastim products studied.

Characterisation of the site-specific monoPEGylated rhG-CSF analogue pegteograstim.

We describe the characterisation of a novel monoPEGylated recombinant human granulocyte colony-stimulating factor analogue, pegteograstim (Neulapeg), prepared by site-specific 20 kDa maleimide-PEG conjugation. An additional cysteine was inserted between Gly136 and Ala137 of filgrastim (methionyl human granulocyte colony-stimulating factor) for site-specific PEGylation, and Cys18 of filgrastim was replaced with Ser18 to prevent unwanted PEGylation. Pegteograstim was produced by Escherichia coli and purified by cation exchange chromatography, and its structural, physicochemical, biological and immunological properties were investigated. Male Sprague-Dawley rats were administered pegteograstim (100 µg/kg) and the pharmacokinetics and pharmacodynamics compared with those of filgrastim. The results of long-term stability testing of pegteograstim revealed no significant change in its quality attributes at 2-8 °C for 36 months. In addition, pegteograstim was stable under the accelerated conditions (25 ± 2 °C, RH of 60 ± 5%) for 6 months. The site-specific monoPEGylated pegteograstim is a highly pure, stable and novel drug for long-lasting treatment of chemotherapy-induced neutropenia.

A demonstration of analytical similarity comparing a proposed biosimilar pegfilgrastim and reference pegfilgrastim.

BACKGROUND: Recombinant human granulocyte-colony stimulating factor (G-CSF, filgrastim) is used primarily to reduce incidence and duration of severe neutropenia and its associated complications in cancer patients that have received a chemotherapy regimen. The pegylated form of filgrastim, "pegfilgrastim", is a long-acting form that requires only a once-per-cycle administration for the management of chemotherapy-induced neutropenia. Apobiologix, a division of ApoPharma USA, Inc., and Intas Pharmaceuticals Limited have co-developed a proposed pegfilgrastim biosimilar to US-licensed pegfilgrastim. METHODS: The analytical similarity of Apobiologix pegfilgrastim and US-licensed pegfilgrastim with respect to their physicochemical profile was established using a wide range of rigorous orthogonal analytical techniques. Biological function was compared using receptor binding analyses, in vitro proliferation assays, and in vivo hematopoietic progenitor mobilization. RESULTS: Apobiologix pegfilgrastim and the US-licensed pegfilgrastim reference product were found to be highly similar analytically with respect to molecular mass, primary, secondary and tertiary protein structures, purity, charge, and hydrophobicity. No differences in receptor binding affinity were observed, and all samples demonstrated similar in vitro and in vivo bioactivity. CONCLUSION: These studies provide robust evidence supporting the structural and functional similarity between Apobiologix pegfilgrastim and the US-licensed reference pegfilgrastim, and hence their biosimilarity.

Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay.

A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins.

Implantation of a Poly-L-Lactide GCSF-Functionalized Scaffold in a Model of Chronic Myocardial Infarction.

A previously developed poly-L-lactide scaffold releasing granulocyte colony-stimulating factor (PLLA/GCSF) was tested in a rabbit chronic model of myocardial infarction (MI) as a ventricular patch. Control groups were constituted by healthy, chronic MI and nonfunctionalized PLLA scaffold. PLLA-based electrospun scaffold efficiently integrated into a chronic infarcted myocardium. Functionalization of the biopolymer with GCSF led to increased fibroblast-like vimentin-positive cellular colonization and reduced inflammatory cell infiltration within the micrometric fiber mesh in comparison to nonfunctionalized scaffold; PLLA/GCSF polymer induced an angiogenetic process with a statistically significant increase in the number of neovessels compared to the nonfunctionalized scaffold; PLLA/GCSF implanted at the infarcted zone induced a reorganization of the ECM architecture leading to connective tissue deposition and scar remodeling. These findings were coupled with a reduction in end-systolic and end-diastolic volumes, indicating a preventive effect of the scaffold on ventricular dilation, and an improvement in cardiac performance.

Comparison of the Adsorption of Original and Biosimilar Preparations of Filgrastim on Infusion Sets and the Inhibition of Adsorption by Polysorbate 80.

The purpose of the study was to evaluate the adsorption of filgrastim on infusion sets (comprising infusion bag, line and filter) and to compare the adsorption of the original filgrastim preparation with biosimilar preparations using HPLC. The inhibitory effect of polysorbate 80 on this adsorption was also evaluated. Filgrastim was mixed with isotonic sodium chloride solution or 5% (w/v) glucose solution in the infusion fluid. Filgrastim adsorption on infusion sets was observed with all preparations and with both types of infusion solution. The adsorption ratio was about 30% in all circumstances. Filgrastim adsorption on all parts of the infusion set (bag, line and filter) was dramatically decreased by the addition of polysorbate 80 solution at concentrations at or over its critical micelle concentration (CMC). The filgrastim adsorption ratio was highest at a solution pH of 5.65, which is the isoelectric point (pI) of filgrastim. This study showed that the degree of filgrastim adsorption on infusion sets is similar for original and biosimilar preparations, but that the addition of polysorbate 80 to the infusion solution at concentrations at or above its CMC is effective in preventing filgrastim adsorption. The addition of a total-vitamin preparation with a polysorbate 80 concentration over its CMC may be an effective way of preventing filgrastim adsorption on infusion sets.

Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies.

Biosimilar drug products must have a demonstrated similarity with respect to the reference product's molecules in order to ensure both the effectiveness of the drug and the patients' safety. In this paper the fusion framework of a highly sensitive NMR fingerprinting approach for conformational changes and mathematically-based biosimilarity metrics is introduced. The final goal is to translate the complex spectral information into biosimilarity scores, which are then used to estimate the degree of similarity between the biosimilar and the reference product. The proposed method was successfully applied to a small protein, i.e., filgrastim (neutropenia treatment), which is the first biosimilar approved in the United States, and a relatively large protein, i.e., monoclonal antibody rituximab (lymphoma treatment). This innovative approach introduces a new level of sensitivity to structural changes that are induced by, e.g., a small pH shift or other changes in the protein formulation.

Identification of Low-Level Product-Related Variants in Filgrastim Products Presently Available in Highly Regulated Markets.

BACKGROUND: Filgrastim is a recombinant, non-glycosylated form of human granulocyte colony-stimulating factor, used to stimulate leukocyte proliferation in patients suffering from neutropenia. Since the expiration of patents associated with Amgen's filgrastim biopharmaceutical, Neupogen(®), in 2006, a number of filgrastim products have been marketed; however, a detailed characterization and comparison of variants associated with these products have not been publically reported. OBJECTIVE: The objective of this study was to identify and quantify product-related variants in filgrastim reference products and biosimilars thereof that are presently available in highly regulated markets. METHODS: In this study, we used intact and top-down mass spectrometry to identify and quantify product-related variants in filgrastim products. Mass spectrometry has become the method of choice for physicochemical characterization of biopharmaceuticals, allowing accurate and sensitive characterization of product-related variants. RESULTS: In addition to modifications ubiquitously present in biopharmaceuticals, such as methionine oxidation and asparagine/glutamine deamidation, we identified six different low-level, product-related variants present in some, but not all, of the tested products. Two variants, an acetylated filgrastim variant and a filgrastim variant containing an additional C-terminal tryptophan extension, are newly identified variants. CONCLUSION: This study demonstrates that filgrastim products already in widespread clinical use in highly regulated markets differ in low-level, product-related variants present at levels mostly below 1 % relative abundance. This study provides a comprehensive catalog of minor differences between filgrastim products and suggests that the filgrastim product-related variants described here are not clinically relevant when present at low abundance.

Rapid formulation assessment of filgrastim therapeutics by a thermal stress test.

The biosimilar versions of recombinant methionyl human granulocyte colony-stimulating factor (rh-Met-G-CSF, filgrastim) are now widely available. Because changes to the formulation often lead to subtle differences, there is a critical need to define techniques to test and insure the quality of these products. The present study was designed to compare formulation and thermal stress stability of filgrastim products. The formulation ingredients including acetate, polysorbate 80, and sorbitol were determined using state-of-the-art validated analytical methods. The formulation pH and osmolality were also measured. Moreover, the stability profiles of 8 filgrastim products using thermal stress at 57 °C for 4 h were assessed by size-exclusion high-performance liquid chromatography (SE-HPLC) and in vitro biological assay. The products had different stability profiles. More stable products were within the specification for formulation and less stable products were beyond the specification limits. Altogether, the results suggest that a short-time stress study at 57 °C and analysis of filgrastim by SE-HPLC could unveil formulation problems and is potentially useful for comparability studies.