Page 63 - ITPS-8-2

INNOSC Theranostics and
Pharmacological Sciences Preclinical study of GBpoietin biosimilar

Janssen (USA). Ethylenediaminetetraacetic acid (EDTA) plan and procedures were approved by the internal ethical
was purchased from Merck (USA). review board of Globe Biotech Limited, complying with
local ethical regulations. No treatment randomization and
2.2. Formulation of GBpoietin ® blinding methods were used in the study and sample sizes

The sample was formulated using formulation buffer were determined by the resource equation method.
(L-glycine, sodium chloride, sodium phosphate,
Polysorbate 80, water for injection; pH: 6.8 – 7.2) and 2.4. Single-dose toxicity assay
AKTA Flux S (GE Healthcare, USA). After sterile filtration A total of 36 Wister rats, including both male and female,
through a 0.22-micron PES Sartopore 2 filter (Sartorius were used for the study comprising four treatment groups,
Stedim, Germany), the pre-formulated bulk sample was a placebo group, and a control group – each group consists
transferred to quality control (QC) for testing as per of three male and three female rats. The four treatment
the specification. The sterile, pre-formulated bulk drug groups were assigned to either normal (500 IU/kg) or toxic
substance was then transferred to a fill-finish facility in a dose (1500 IU/kg) to assess the toxicological similarity
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2D single-use bag (Sartorius Stedim, France) for filling and of GBpoietin to Eprex . Treatment-1 and Treatment-2
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packaging while maintaining a temperature of 4 – 8°C. The were injected with normal and toxic doses of GBpoietin ,
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sterile 1 mL empty long syringes (Schott, Switzerland) were respectively, while Treatment-3 and Treatment-4 were
filled using automatic combo filling and closing machine injected with normal and toxic doses of Eprex , respectively.
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(Tofflon, China) at 1 mL volume for dose preparation. The placebo group was injected with 100 µL of formulation
The pre-filled syringes were packaged (blistered) using the buffer used for GBpoietin and Eprex . The control
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HM-AV Plus blister machine (Hoonga, The Republic of group was not given any injection, serving as a negative
Korea). After blistering completion, the drug product was control of the study. Two days before dosing, whole blood
stored at 4 – 8°C. Finally, the pre-filled syringes were tested (approximately 200 µL) was collected in 2% EDTA from
for QC as per the specification and transferred to the pre- each rat for the complete blood count (CBC) analysis.
clinical (animal) study center, maintaining a temperature Similarly, whole blood was also collected after the last
of 4 – 8°C, for the toxicology study. dosing at day 14 for the CBC analysis. Pharmacodynamic
(PD) endpoints including RBC count, white blood cell
2.3. Animal selection (WBC) count, hemoglobin (HGB) level, platelet (PLT)
Wistar rats (Rattus norvegicus) were used for the count, hematocrit (HCT) level, mean corpuscular volume
single- and repeated-dose safety and toxicity studies. (MCV), mean corpuscular hemoglobin (MCH), and
A total of 75 Wistar male and female rats (10 – 15 weeks mean corpuscular hemoglobin concentration (MCHC)
old) were selected and isolated 5 days before the dosing. were measured for all test subjects before injection and
After attentive monitoring and conditioning, 36 rats on the last day of the 14-day study using BK-6190-Vet
(18 males and 18 females) were subjected to single-dose auto hematology analyzer (Biobase, Germany). Similarly,
toxicity analysis and 24 rats (12 males and 12 females) alanine aminotransferase (ALT)/glutamic pyruvic
were subjected to repeated-dose toxicity analysis. The transaminase (GPT), aspartate aminotransferase (AST)/
temperature of the experimental animal room was 26 ± serum glutamic-oxaloacetic transaminase (sGOT), and
2°C and the relative humidity was 60 ± 5%. The room was blood urea nitrogen (BUN) assays were performed on
an HVAC-controlled ISO class 7 room with 70% fresh air blood serum using a semi-automatic chemistry analyzer
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intake and full exhaust. The rats were individually housed (Biobase, Germany) to assess the toxic effect of GBpoietin
in a polypropylene cage with proper water and feed and and Eprex on the liver and kidney. Body temperature and
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kept under 12 h of the day-night cycle. The target weight weight were also measured during the whole study period.
of male animals was 185 ± 20 g and female animals were A necropsy of representative subjects from each treatment,
175 ± 20 g. In the entire experiment, 20% of additional placebo, and control groups was done to check for
animals (males and females) were used as substitutes for abnormalities in the kidney, lung, liver, spleen, and heart.
the excluded animals. Healthy young adult animals were During the necropsy, external surfaces, all orifices, cranial
used. Females were nulliparous and non-pregnant. At the cavities, external surfaces of the brain and spinal cord,
beginning of the study, each animal was between 10 and thoracic, abdominal, and pelvic cavities, cervical tissues,
15 weeks old. The weight difference of animals used was and organs were also examined. Finally, histopathological
minimal, not exceeding ± 20% of the mean weight for each evaluation was carried out to find any pathological
sex. These animals were used to replace any individuals significance like degeneration or cellular necrosis in the rat
that were excluded during the study periods. The study internal organs such as the kidney, liver, lung, and spleen.

Volume 8 Issue 2 (2025) 57 doi: 10.36922/itps.5797