In-use Stability of Semaglutide Injection 15mg/3mL (5mg/mL)

In-use Storage Conditions, Study Design, Study Outcome and Labelling Recommendations

M.E. Kannan, Debjani Singh, Yogesh Wagh, Dinesh Patel, Bhavesh Patel,

Devendra Badgujar, Tushar Nahata*

Pharmaceutical Technology Center, Zydus Lifesciences Limited, Ahmedabad, Gujarat, India.

*Corresponding Author E-mail: tusharnahata@zyduslife.com

ABSTRACT:

Multidose parenteral products administered using reusable injection devices require a scientifically justified in‑use period due to repeated breaches of container closure integrity during use. Innovative Semaglutide injection 15 mg/3 mL (5 mg/mL) is a preserved multidose formulation supplied in a reusable pen system. An in‑use stability study was conducted to understand the in‑use storage stability. Prefilled cartridges were assembled into the reusable pen device were subjected to worst‑case simulated use involving up to 32 needle punctures and stored for up to 8 months at 2–8 °C and 56 days at 15–30 °C. Samples were evaluated at predefined intervals for physicochemical quality attributes, preservative content, particulate matter, and antimicrobial effectiveness in accordance with applicable compendial requirements and predefined acceptance criteria. All tested parameters remained within specifications throughout the study, with no clinically relevant trends observed. The generated data confirmed the in-use storage stability of semaglutide multidose formulation.

KEYWORDS: In-use stability, semaglutide injection, multidose parenteral product, antimicrobial effectiveness testing, physicochemical stability, preserved drug product, reusable pen injector.

INTRODUCTION:

Semaglutide is a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist widely used for the management of type 2 diabetes mellitus and obesity. The growing use of self-administered injectable therapies has led to the development of multidose pen delivery systems that improve convenience and adherence. However, repeated breaching of the primary container closure system during routine use introduces potential physicochemical and microbiological risks.

Regulatory expectations require that multidose parenteral products demonstrate stability throughout the proposed in use period, ensuring that product quality, safety, and efficacy are maintained after first use. In use stability studies therefore play a critical role in defining maximum permissible in-use periods and labelling instructions, particularly for preserved formulations.

This manuscript describes a simulated in-use stability evaluation of semaglutide injection 15mg/3mL (5 mg/mL) supplied in a reusable pen device. The study was designed to support a proposed in-use period under both refrigerated and controlled room temperature storage conditions, aligned with patient use scenarios.

MATERIALS AND METHODS:

Product Description

Semaglutide injection is a clear, colorless, sterile solution containing 5mg/mL Semaglutide and Phenol (0.45% w/v) as an antimicrobial preservative. The drug product is filled into 3mL Type I glass cartridges (Batch No. XXX027) and supplied for use with a reusable pen device and disposable 32G × 4mm pen needles.

In-use Study Design:

The study was conducted using final market presentation components, including cartridges, reusable pen devices, and compatible pen needles. The in-use simulation was designed to represent a worst-case clinical scenario.

Key study conditions were as follows:

· Refrigerated storage: 2–8 °C for up to 8 months (initial, 4 months, and 8 months)

· Controlled room temperature storage: 15–30 °C for up to 56 days (initial and 56 days)

· Simulated use: Each cartridge was punctured up to 32 times to simulate repeated weekly dosing over the maximum anticipated treatment duration

· All puncturing and sampling were performed under normal environmental conditions using new sterile pen needles for each withdrawal.

Figure 1 summarizes the in‑use study design and key evaluation aspects.

Sampling and Handling:

On Day 1, cartridges assembled into pen devices were initially punctured, and samples were withdrawn for baseline analysis. Remaining pens were capped and stored under the assigned temperature conditions. At each predefined time point, samples were withdrawn using new needles and subjected to analytical evaluation.

Physicochemical Testing:

Comprehensive analytical characterization of the drug product was performed to assess critical quality attributes relevant to identity, strength, purity, and overall suitability for parenteral administration. All testing was conducted using validated, stability indicating analytical methods that were qualified in accordance with internal procedures and applicable regulatory and pharmacopeial requirements. The selected test panel was designed to support product quality assessment across formulation performance, container–closure compatibility, and particulate control.

General appearance was evaluated by visual inspection under controlled illumination conditions to confirm the absence of visible particulate matter and to qualitatively assess clarity and colour. Visible particle inspection was conducted in accordance with pharmacopeial guidance for injectable products. Quantitative clarity assessment was performed by measuring percent transmittance at 650 nm using a calibrated UV–visible spectrophotometer. Colour was monitored by absorbance measurement at 420 nm to detect potential formulation discoloration associated with degradation pathways or excipient interactions.

The concentration of semaglutide was determined using a validated reverse phase high performance liquid chromatography (HPLC) method capable of selectively resolving the active pharmaceutical ingredient from formulation excipients and potential degradants. Method validation demonstrated suitability with respect to specificity, linearity, precision, accuracy, and robustness over the intended analytical range. Phenol preservative content was quantified using a separate validated HPLC method to confirm preservative levels consistent with antimicrobial effectiveness requirements throughout the study.

Impurity profiling, including related substances and degradation products, was performed using a validated ultra high-performance liquid chromatography (UHPLC) method. This method provided enhanced resolution and sensitivity for the detection and quantification of known and unknown impurities, thereby enabling meaningful assessment of chemical stability. Aggregate content was evaluated using SEC-HPLC technique appropriate for detecting high molecular weight species, supporting assessment of protein integrity and aggregation propensity under the tested conditions.

Figure 1. In-use study design details

Physicochemical attributes were further characterized by measurement of pH and osmolality. pH measurements were performed using a calibrated pH meter under controlled temperature conditions to ensure consistency with formulation design intent. Osmolality was determined using a validated osmometric method to confirm suitability for subcutaneous administration and to monitor potential changes associated with degradation or excipient instability.

Sub-visible particulate matter was quantified using a light obscuration method in alignment with pharmacopeial requirements for injectable products. Particle counts were reported for particles ≥10μm and ≥25μm, providing assurance of particulate control related to formulation composition, manufacturing processes, and container–closure interactions.

Acceptance criteria for all quality attributes were based on approved product specifications and relevant pharmacopoeial standards, including compendial requirements applicable to sterile parenteral drug products. Collectively, these analytical evaluations provided a comprehensive assessment of drug product quality and supported the suitability of the formulation and manufacturing process for its intended use.

Microbiological Testing

The testing protocol employed compendial challenge organism’s representative of Gram‑negative bacteria, Gram‑positive bacteria, yeast, and Mold, thereby providing a comprehensive assessment of antimicrobial activity across a broad spectrum of potential contaminants. The organisms used included Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus brasiliensis, each prepared and qualified in accordance with compendial guidance.

Drug product samples were inoculated with standardized microbial suspensions to achieve the required initial challenge levels and were subsequently stored under the defined test conditions. Microbial recovery was evaluated at predetermined intervals of 7, 14, and 28 days following inoculation. Log reduction values were calculated by comparison of recovered microbial counts against initial inoculum levels at each time point.

The preservative system’s effectiveness was determined by comparing observed microbial log reductions to the acceptance criteria specified in the applicable pharmacopeial standards for parenteral preparations. Compliance with these criteria at both the initial and end‑of‑in‑use testing points demonstrated the continued adequacy of preservative performance throughout the labelled in‑use period under the studied storage conditions.

Acceptance Criteria as per United States Pharmacopeia (USP <51>):

Bacteria:

A reduction of not less than 1.0 log from the initial calculated count at 7 days, a reduction of not less than 3.0 log from the initial count at 14 days, and no increase in the bacterial count at 28 days compared to the 14‑day count.

Yeast and molds:

There should be no increase from the initial calculated count at 7, 14, and 28 days.

Acceptance Criteria as per Indian Pharmacopoeia (IP 2.2.2)

Bacteria:

The viable bacterial count should be reduced by not less than 1.0 log (or the remaining count should not exceed 10% of the initial concentration) at 7 days, and by not less than 3.0 log (or the remaining count should not exceed 0.1% of the initial concentration) at 14 days. There should be no increase in the bacterial count at 28 days relative to the 14‑day count.

Yeast and molds:

There should be no increase in the yeast or mold counts at 7, 14, and 28 days compared to the initial count.

RESULTS AND DISCUSSION:

Physicochemical Stability

Across all tested conditions, semaglutide injection remained clear and colorless, with no visible particles observed. Semaglutide assay values remained within 2% of label claim at all time points. Phenol content remained unchanged during studied period, supporting continued preservative efficacy during in-use storage.

Total aggregates and related substances remained low throughout the study, with no significant trend toward increased degradation under either refrigerated or room temperature conditions. pH and osmolality remained stable and within predefined limits. Sub-visible and visible particulate matter complied with USP <788>, USP <790> and IP 2.5.9 requirements.

Table 1. Results of the physicochemical tests during proposed In-use study period (Batch No. XXX027)

Sr. No.	Tests	Specifications	Initial	2°C to 8°C		15°C to 30°C
				4 months	8 months	56 days
1	Description	A clear, colourless solution	A clear, colourless solution	A clear, colourless solution	A clear, colourless solution	A clear, colourless solution
2	Assay of Semaglutide (By HPLC)	90.0% to 110.0%	99.0	98.9	98.1	98.2
3	Assay of Phenol (By HPLC)	80.0% to 110.0%	95.1	95.4	95.5	95.0
4	% Total Aggregates (By HPLC)	NMT 1.0%	BQL	0.07	0.09	0.17
5	Osmolality	Between 230 mOsm/kg to 330 mOsm/kg	248	253	255	253
6	pH	Between 7.1 and 7.7	7.4	7.4	7.5	7.4
7	% Transmittance at 650 nm	NLT 95%	100	100	100	100
8	Absorbance at 420nm	NMT 0.5 Au	0.000	0.001	0.001	0.001
9	Related substances (By UHPLC)	Total Degradation products: NMT 5.0%	0.21	0.32	0.30	1.07
10	Particulate Matter (Sub-visible)	Particles greater than or equal to 10µm: NMT 6000 particles/container	Complies	NP	Complies	Complies
		Particles greater than or equal to 25µm: NMT 600 particles/container	Complies	NP	Complies	Complies
11	Particulate Matter (Visible)	The solution should be free from particles of foreign matter that can be observed on visual inspection of cartridge	Complies	NP	Complies	Complies

ND: Not Detected; BQL: Below Quantitation Limit (LOQ = 0.05%); NP: Not performed; NLT: Not less than; NMT: Not more than

Figure 2-5 below illustrate the evaluation of the different physicochemical parameters studied.

Figure 2. Semaglutide assay during in‑use stability under refrigerated and room temperature conditions.

Semaglutide potency (% of label claim) was evaluated at initial release and during in‑use stability storage at 2–8°C for up to 8 months and 56 days at 15–30°C. Potency remained within acceptable limits upon subsequent storage under in‑use conditions at 2-8°C and 15–30°C. Data indicate overall chemical stability of semaglutide under the evaluated in‑use conditions.

Figure 3. Semaglutide % total aggregates during in use stability,

% aggregates remained within acceptable limits during in‑use stability storage at 2–8°C for up to 8 months and 56 days at 15–30°C. Data indicate overall chemical stability of semaglutide under the evaluated in‑use conditions. At initial, BQL equivalent to 0.05% levels considered for graphical presentation

Figure 4. pH during In-use stability, there is no change observed in pH during in‑use stability storage at 2–8 °C for up to 8 months, and 56 days at 15–30 °C.

Figure 5. Total impurity levels during In-use stability, Total impurity levels remained within acceptable limits during in-use stability storage at 2–8°C for up to eight months and 56 days at 15–30°C.

Antimicrobial Effectiveness Testing

AET results demonstrated that the formulation met compendial acceptance criteria at initial, refrigerated (8 months), and room temperature (56 days) in-use conditions. For bacterial challenge organisms, required log reductions were achieved at 7 and 14 days with no subsequent increase at 28 days. No increase in yeast or mold counts was observed throughout the test period.

Figure 6 below illustrate the evaluation of the antimicrobial effectiveness study data.

Table 2. Results of the antimicrobial effectiveness performance during proposed In-use study period

Stage	Name of Organisms	Log Reduction
		7 Days	14 Days	28 Days
Initial	E. coli	4.6946	4.6946	4.6946
	S. aureus	4.5118	4.5118	4.5118
	P. aeruginosa	4.4983	4.4983	4.4983
	C. albicans	2.3703	4.4842	4.4842
	A. brasiliensis	3.9542	3.9542	3.9542
2°C - 8°C, 8 months	E. coli	4.7403	4.7403	4.7403
	S. aureus	4.7032	4.7032	4.7032
	P. aeruginosa	4.5378	4.5378	4.5378
	C. albicans	3.7741	4.7283	4.7283
	A. brasiliensis	4.3117	4.3117	4.3117
15°C - 30°C, 56 days	E. coli	4.5682	4.5682	4.5682
	S. aureus	4.3802	4.3802	4.3802
	P. aeruginosa	4.1613	4.1613	4.1613
	C. albicans	4.5502	4.5502	4.5502
	A. brasiliensis	4.1303	4.1303	4.1303
Acceptance Criteria	For Bacteria (E. coli, S. aureus and P. aeruginosa)	Not less than 1.0 log reduction from initial count	Not less than 3.0 log reduction from initial count	No increase from the 14 days count at 28 days
	For Yeast and Mold (C. albicans, A. brasiliensis)	No increase from the initial calculated count at 7, 14 and 28 days

Figure 6. Represents the antimicrobial effectiveness testing (AET) results during in‑use stability after refrigerated storage (2–8 °C for up to 8 months), followed by room‑temperature storage (15–30 °C). Across all storage conditions and time points, the formulation demonstrated consistent and effective microbial log reductions. Bacterial organisms (E. coli, S. aureus, and P. aeruginosa) showed high and sustained log reductions (>4 log), while fungal organisms (C. albicans and A. brasiliensis) also met acceptance criteria, with no significant loss of preservative effectiveness observed after prolonged refrigerated storage or subsequent

All antimicrobial effectiveness test results complied with USP <51> and IP 2.2.2 acceptance criteria. The data confirm that the preservative system remained effective throughout the proposed in-use storage duration under both refrigerated and controlled room temperature conditions.

Raw Data :Antimicrobial Effectiveness Test (AET)

This section provides the complete raw Antimicrobial Effectiveness Test (AET) data generated during the in-use stability evaluation. The tables below summarize the observed log reductions for each challenge microorganism at 7, 14, and 28 days. The complete numerical datasets were used to calculate log reductions and confirm compliance with pharmacopeial acceptance criteria at all evaluated in-use conditions.

Table 3. Detailed raw data antimicrobial effectiveness performance during proposed In-use study period

Details	Name of Organisms and ATCC Strains			Control Vial				Log 10 value of Test Product							Log Reduction
				Initial Conc. Cfu/mL		Log 10 Value (X)		Zero Hours (A)		At 7 days (B)		At 14 days (C)		At 28 days (D)	Zero Hours (X-A)	At 7 days (X-B)	At 14 days (X-C)	At 28 days (X-D)
Initial Batch No. XXX027	E. coli (ATCC 8739)			4.95 X 10^5		5.6946		5.6946		1.0000		1.0000		1.0000	0.0000	4.6946	4.6946	4.6946
	S. aureus (ATCC 6538)			3.25 X 10^5		5.5118		5.5440		1.0000		1.0000		1.0000	-0.0322	4.5118	4.5118	4.5118
	P. aeruginosa (ATCC9027)			3.15 X 10^5		5.4983		1.0000		1.0000		1.0000		1.0000	4.4983	4.4983	4.4983	4.4983
	C. albicans (ATCC10231)			3.05 X 10^5		5.4842		5.5250		3.1139		1.0000		1.0000	-0.0408	2.3703	4.4842	4.4842
	A. brasiliensis (ATCC16404)			0.9 X 10^5		4.9542		4.9777		1.0000		1.0000		1.0000	-0.0235	3.9542	3.9542	3.9542
Remark: The sample complies the acceptance criteria
Details	Name of Organisms and ATCC Strains			Control Vial				Log 10 value of Test Product							Log Reduction
				Initial Conc. Cfu/mL		Log 10 Value (X)		Zero Hours (A)		At 7 days (B)		At 14 days (C)		At 28 days (D)	Zero Hours (X-A)	At 7 days (X-B)	At 14 days (X-C)	At 28 days (X-D)
2 -8°C, 8 Months Batch No. XXX027	E. coli (ATCC 8739)			5.5 X 10^5		5.7403		5.6232		1.0000		1.0000		1.0000	0.1171	4.7403	4.7403	4.7403
	S. aureus (ATCC 6538)			5.05 X 10^5		5.7032		5.6766		1.0000		1.0000		1.0000	0.0266	4.7032	4.7032	4.7032
	P. aeruginosa (ATCC9027)			3.45 X 10^5		5.5378		5.1613		1.0000		1.0000		1.0000	0.3765	4.5378	4.5378	4.5378
	C. albicans (ATCC10231)			5.35 X 10^5		5.7283		5.6283		1.9542		1.0000		1.0000	0.1000	3.7741	4.7283	4.7283
	A. brasiliensis (ATCC16404)			2.05 X 10^5		5.3117		5.1461		1.0000		1.0000		1.0000	0.1656	4.3117	4.3117	4.3117
Remark: The sample complies the acceptance criteria as per USP <51> and IP 2.2.2.
Details	Name of Organisms and ATCC Strains		Control Vial				Log 10 value of Test Product								Log Reduction
			Initial Conc. Cfu/mL		Log 10 Value (X)		Zero Hours (A)		At 7 days (B)		At 14 days (C)		At 28 days (D)		Zero Hours (X-A)	At 7 days (X-B)	At 14 days (X-C)	At 28 days (X-D)
15 -30°C 56 Days Batch No. XXX027	E. coli (ATCC 8739)		3.7 X 10^5		5.5682		5.5185		1.0000		1.0000		1.0000		0.0497	4.5682	4.5682	4.5682
	S. aureus (ATCC 6538)		2.4 X 10^5		5.3802		5.4149		1.0000		1.0000		1.0000		-0.0347	4.3802	4.3802	4.3802
	P. aeruginosa (ATCC9027)		1.45 X 10^5		5.1613		4.8162		1.0000		1.0000		1.0000		0.3451	4.1613	4.1613	4.1613
	C. albicans (ATCC10231)		3.55 X 10^5		5.5502		5.5185		1.0000		1.0000		1.0000		0.0317	4.5502	4.5502	4.5502
	A. brasiliensis (ATCC16404)		1.35 X 10^5		5.1303		4.8750		1.0000		1.0000		1.0000		0.2853	4.1303	4.1303	4.1303
Remark: The sample complies the acceptance criteria as per USP <51> and IP 2.2.2.
Acceptance Criteria		For Bacteria (E. coli, S. aureus and P. aeruginosa)							Not less than 1.0 log reduction from the initial calculated count at 7 days. Not less than 3.0 log reduction from the initial count at 14 days. No increase from the 14 days count at 28 days.
		For Yeast and Mod (C. albicans, A. brasiliensis)							No increase from the initial calculated count at 7, 14, and 28 days.

CONCLUSION:

Multidose injectable products are inherently exposed to increased risk of contamination and degradation following first use. This study demonstrates that semaglutide injection 15 mg/3 mL maintains robust physicochemical integrity and antimicrobial protection under simulated worst case in-use conditions. The combination of repeated cartridge puncturing, long term refrigerated storage, and defined room temperature exposure represents a conservative approach to in-use stability assessment. The absence of meaningful changes in potency, degradation profile, or particulate burden supports the suitability of the formulation and delivery system for extended patient use. Importantly, antimicrobial effectiveness was preserved throughout the study, confirming the continued functionality of phenol as a preservative in the multidose presentation. These findings align with regulatory expectations for preserved parenteral products and support patient centric labeling flexibility. The in-use stability study of semaglutide injection 15 mg/3 mL (5 mg/mL) demonstrated that the product remains stable, safe, and effective when used in a reusable pen device for up to 8 months at 2–8 °C or for up to 56 days at 15–30 °C after first use. The results provide a strong scientific basis for the proposed in-use storage conditions and labeling recommendations.

ACKNOWLEDGEMENTS:

The authors acknowledge Zydus Lifesciences Limited for their support during the conduct of the study.

CONFLICTS OF INTEREST DECLARATION

All authors are employees of Zydus Lifesciences Limited. The study was conducted as part of the company’s product development activities. The authors declare that there are no conflicts of interest associated with this manuscript.

REFERENCES:

1. United States Pharmacopeia–National Formulary (USP–NF) Antimicrobial Effectiveness Testing ⟨51⟩; United States Pharmacopeial Convention: Rockville, MD, 2026.

2. Indian Pharmacopoeia Commission Effectiveness of Antimicrobial Preservatives (IP 2.2.2); Indian Pharmacopoeia Commission: Ghaziabad, India, 2026.

3. United States Pharmacopeia–National Formulary (USP–NF) Particulate Matter in Injections ⟨788⟩; United States Pharmacopeial Convention: Rockville, MD, 2026.

4. United States Pharmacopeia–National Formulary (USP–NF) Visible Particulate in Injections ⟨790⟩; United States Pharmacopeial Convention: Rockville, MD, 2026.

5. Indian Pharmacopoeia Commission Particulate Contamination (IP 2.5.9); Indian Pharmacopoeia Commission: Ghaziabad, India, 2026.

6. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) ICH Q1A(R2): Stability Testing of New Drug Substances and Products; ICH: Geneva, Switzerland, 2003.

Received on 05.01.2026      Revised on 21.03.2026

Accepted on 08.05.2026 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2229-2235.

DOI: 10.52711/0974-360X.2026.00321

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