Antineoplastic drugs prevent or inhibit neoplasm maturation and proliferation, destroying cancer cells through their movement within the body 1. Cancer is caused by uncontrolled cell proliferation, invasion, and metastasis, primarily due to the clonal expansion of a single neoplastic cell 2, 3. Parenteral dosage forms differ from other forms as they are directly injected into the human body 4. Anticancer drugs are typically administered orally and parenterally, with decreased shelf life associated with parenteral dosage forms 5. These forms include solutions, liposomes, emulsions, suspensions, nano-systems, microspheres, and powders for injections, which can be reconstituted as solutions 6. Antineoplastic agents are crucial in cancer therapies and other diseases. Lyophilization, or freeze-drying, is a process where water is sublimed from a composition, allowing unstable pharmaceutical and biological agents to be stored in liquid form. However, most agents require additional ingredients to protect the active ingredient during lyophilization, and reconstituting a lyophilized antineoplastic agent into an aqueous solution can be challenging. Anti neoplastic agents often require additional ingredients to protect the active ingredient during lyophilization 7, 8. Hence there is a need for lyophilization to improve the stability and shelf life of the drug candidate. The research aims to improve the stability and shelf life of a drug candidate through lyophilization. Key objectives include conducting pre-formulation studies, formulating an injectable dosage form, lyophilizing it, developing and optimizing the lyophilization cycle, evaluating the lyophilized product, performing accelerated stability studies, and documenting the results. The study focuses on formulating a generic Bleomycin product in lyophilized injection form and optimizing its preparative lyo cycle.

Bleomycin Sulphate is an antibiotic used for treating various cancers, including squamous cell carcinoma, Hodgkin's disease, testicular teratoma, malignant effusions, metastatic melanoma, thyroid, lung, and bladder carcinomas. It is administered via intramuscular, intravenous, subcutaneous, or intraarterial routes and is available in lyophilized injectable dosage form.

Studies showed that the potency of the drug candidate gradually decreases as the time increases. The present study found that the drug was unstable in solution form as the potency of the solution showed a significant change. Hence bleomycin aqueous injection was subjected for lyophilization in order to extend the shelf life needed for a marketable product. The potency of the antibiotic was found to be 17.626 mg (Table 1, Table 2, ).

Table 3

Long term storage condition at 5 ± 3 °C
Storage interval	Description	pH (Limit 4.5-6.0)	Assay (90 to 120%)	Reconstitution time (NMT 30sec)	Light transmittance (650nm) (NLT 97%)	% Moisture Content (NMT6.0%)	Contents of Bleomycins (in %)
							Bleomycin A2 (55 to 70%)	Bleomycin B2 (25 to 32%)	Bleomycin A2 & B2 (NTL 90%)	Demethyl bleomycin A2 (NMT1.05%)	Others (NMT 0.6%)
1 Month	A white to off- white powder	5.24	103.6	12 sec	99.66	1.78	66.4	28.6	95.2	0.89	0.25
2 Month		5.24	104.6	12 sec	98.89	1.76	67.7	28.9	94.2	0.88	0.29
3 Month		5.26	104.8	12 sec	98.06	1.83	68.5	29.3	94.3	0.86	0.24
Short term storage condition at 25 ± 2 °C and 60 ± 5% RH (Accelerated condition)
Storage interval	Description	pH [Limit 4.5-6.0]	Assay (90 to 120%)	Reconstitution time (NMT 30sec)	Light transmittance (650nm, NLT 97%)	% Moisture Content (NMT6.0%)	Contents of Bleomycins (%)
							Bleomycin A2 (55 to 70%)	Bleomycin B2 (25 to 32%)	Bleomycin A2 & B2 (NTL 90%)	Demethyl bleomycin	Others (NMT)
1 Month	A white to off- white powder	5.26	103.6	13 sec	99.16	1.84	66.4	27.6	94.2	0.86	0.26
2 Month		5.27	102.6	14 sec	98.19	1.89	68.3	29.9	92.1	0.79	0.25
3 Month		5.27	102.8	14 sec	99.06	1.84	67.5	29.1	92.3	0.89	0.29

The Bleomycin sulphate was tested for preformulation, and its IR spectrum identified it as a standard drug. The antibiotic microbial assay method revealed a 103.7% concentration of Bleomycin Sulphate, with 68.62% and 29.4% contents for Bleomycin A2 and Bleomycin B2, respectively. The Bleomycin Sulphate showed a 1.31% drying loss and a melting point of 275 °C, with a pH of 5.14 ± 0.03, and was used to create a parenteral dosage form using lyophilization technique.

The process involved four batches with varying cycle times, freezing, drying, ramp, and holding times while maintaining active pharmaceutical ingredient quantities. The first approach, lyo cycle 1, over 38.83 hours, produced a uniform, distinct, and intact cake without shrinkage or collapse. The process involved freezing for 3.6 hours, drying for 28.33 hours, and drying for 6.8 hours, with a vacuum gradually reduced from 250 to 150 mTorr. The resulting cake was a uniform, intact white to off-white cake with a pH of 5.14-5.19, a reconstitution time of 12 seconds, and a moisture content of 1.72-1.84%.

The product met the specified light transmittance and Bleomycin content limits. A second approach was attempted to reduce moisture content. Lyo cycle 2 was conducted for 42.8 hours, including 3.5 hours of freezing, with primary drying increased by 4 hours to 32.5 hours and secondary drying for 6.8 hours, maintaining the vacuum change protocol from cycle 1. The product was a uniform white to off-white cake with a pH range of 5.22-5.26 and a reconstitution time of 24 seconds. It had a light transmittance of 104.0% and moisture content of 3.2-3.56%. The light transmittance and Bleomycin content were within specified limits. Lyophilization was carried out for 38.82 hours in the third approach (lyo Cycle 3), with the same freezing rate and primary drying time as in the first cycle.

The secondary drying process increased the time by an hour to 7.16 hours, maintaining the same vacuum variation as in cycle 1, resulting in a white to off-white cake that appeared uniform, distinct, and intact without shrinkage or collapse. The sample had a pH range of 5.35-5.37, a reconstitution time of 25 seconds, a 102.0% assay value, and moisture content of 4.3-4.56%. Light transmittance and Bleomycin content were within specified limits. Lyophilization was conducted for 39.6 hours in lyo cycle 4, with annealing in freezing for 5.1 hours, primary drying for 28.5 hours, and secondary drying for 6 hours. The vacuum change protocol was maintained in lyo cycle 1.

The product had a collapsed cake, pH range of 5.30-5.34, and a reconstitution time of 25 seconds. It had a 103% assay value and moisture content of 5.4-5.95%. Despite its collapsed appearance, the light transmittance and Bleomycins content were within the specified limits. Lyo cycle 1 provided the best results, especially with low moisture content and reconstitution time, as the lyophilized product cake was uniform, distinct, intact, dry, and porous. Hence the protocol of the first lyo cycle was optimum and lab scale batch for 500 mL was taken and the product was loaded for stability studies. The accelerated stability studies were conducted according to ICH guidelines at two different temperature and relative humidity conditions: 5°C ± 3°C for long-term/real-time hold studies and 25°C ± 2°C/60% RH ± 5% for accelerated studies. The lyophilized products under stability studies were analyzed at time interval of first month, second month and third month. Products were analyzed for Parameters such as Description, pH, Assay, light transmittance, reconstitution time percentage moisture content and contents of Bleomycin.

The product's description remained unchanged after reconstituted with 10ml of WFI, forming a clear, colourless solution. The pH was checked, and no significant difference was found compared to the initial value, falling within the range of 5.24 - 5.28. Antibiotic microbial assay and contents of Bleomycin were analysed by HPLC and the difference was found to be within the limits. Percentage of water content was analysed and was found to be the same at 5 °C ± 3 °C, 25 °C ± 2 °C and 60 ± 5 % RH, conditions through a slight increase of 0.2 - 0.5 % was observed. Colour value and percentage light transmittance test were carried out by UV and the results were found to be within the limits. The results of stability study concluded that the product was stable at accelerated conditions.

Lyophilization technique was used to improve the cake characteristics of a drug in lyophilized form. Three different Lyo cycles were designed and investigated to optimize product characteristics. All cycles yielded the best results in parameters like Description, Assay, pH, Moisture Content, Reconstitution Time, and Bleomycin content. Cycle 1 showed better moisture content and was chosen for stability study. The lyophilization process involved a total cycle time of 38.83 hours, with a freezing time of 3.6 hours, primary drying time of 28.33 hours, and secondary drying time of 6.8 hours.