Effect of Lyophilization on Stability of PEG-Protein Conjugate: A Case Study with Peginterferon alfa-2b

Chintan, Patel; Gayatri, Patel

doi:10.1590/s2175-97902023e201120

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Brazilian Journal of Pharmaceutical Sciences

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Article • Braz. J. Pharm. Sci. 59 • 2023 • https://doi.org/10.1590/s2175-97902023e201120 copy

Effect of Lyophilization on Stability of PEG-Protein Conjugate: A Case Study with Peginterferon alfa-2b

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

The purpose of the present study was to develop stable lyophilized formulation of peginterferon alfa-2b which is acquiescent to the short lyophilization process. The present study evaluates the effect of buffering components and cryoprotectant(s) on depegylation of the peginterferon alfa-2b in combination with lyophilization process. Finally, a short lyophilization process was identified which can produce a stable pharmaceutical form of peginterferon alfa-2b without any depegylation during long-term storage. Formulations were analyzed mainly for depegylation by HP-size exclusion chromatography and in-vitro antiviral activity. Residual moisture content in the lyophilized product was also used as a key indicating parameter to check its role with respect to depegylation upon storage under various temperature conditions. It was observed that the peginterferon alfa-2b when formulated in presence of cryoprotectant like sucrose requires longer lyophilization process of about 5 days, irrespective of the buffering components used, to reduce the level of residual moisture content and thereby to produce the stable formulation without depegylation. A stable formulation in presence of high concentration of lactose as a cryoprotectant was developed which can withstand stresses exerted to protein-polymer conjugate during lyophilization phases without any significant depegylation. A short lyophilization process of about 48 hours can be utilized for peginterferon alfa-2b when formulated in presence of lactose as a cryoprotectant through which a stable lyophilized formulation can be produced as against longer process required when sucrose is used a cryoprotectant, which is essential from commercial point of view as lyophilization is a costly process.

Keywords:
Depegylation; Lyophilization; Stability; Residual moisture content; Peginterferon alfa-2b

Ingredients	Quantity (mg per mL)
Ingredients	Formulation A	Formulation B
Peginterferon alfa-2b	0.160 mg	0.160 mg
Dibasic sodium phosphate	1.58 mg	-
Monobasic sodium phosphate	1.58 mg	-
Sodium Succinate	-	2.57 mg
Sucrose	115 mg	115 mg
Polysorbate 80	0.005 mg	0.005 mg
Water for Injection	1 mL	1 mL
pH	6.8	6.8

Ingredients	Quantity (mg per mL)
Ingredients	Formulation C	Formulation D	Formulation E	Formulation F
Peginterferon alfa-2b	0.178	0.178	0.178	0.178
Sodium Succinate	1.89	1.89	1.89	1.89
Lactose	18	100	57.1	57.1
Trehalose	-	-	31.4	22.8
Glycine	-	-	1.05	1.05
Polysorbate 80	0.1	0.1	0.1	0.1
Water for Injection	1 mL	1 mL	1 mL	1 mL
pH	6.8	6.8	6.8	6.8

Time intervals	Formulation A			Formulation B
Time intervals	Potency† (IU / mg)	% RMC	% Free IFN	Potency† (IU / mg)	% RMC	% Free IFN
Stressed storage: 40 °C ± 2 °C; 75 % RH ± 5 % RH
Initial	0.38 × 10⁸	0.05 %	3.29 %	0.47 × 10⁸	0.17 %	3.06 %
15 d	0.35 × 10⁸	1.33 %	4.24 %	0.47 × 10⁸	0.97 %	4.49 %
30 d	0.47 × 10⁸	1.60 %	5.68 %	0.72 × 10⁸	1.49 %	6.01 %
Accelerated storage: 25 °C ± 2 °C; 60 % RH ± 5 % RH
Initial	0.38 × 10⁸	0.05 %	3.29 %	0.47 × 10⁸	0.17 %	3.06 %
1 m	0.57 × 10⁸	1.16 %	4.16 %	0.44 × 10⁸	0.94 %	3.69 %
4 m	0.34 × 10⁸	0.95 %	4.47 %	0.49 × 10⁸	0.92 %	4.90 %
10 m	0.45 × 10⁸	1.63 %	6.29 %	0.41 × 10⁸	1.50 %	6.22 %
Real-time storage: between +2 °C and +8 °C
Initial	0.38 × 10⁸	0.05 %	3.29 %	0.47 × 10⁸	0.17 %	3.06 %
1 m	0.36 × 10⁸	0.10 %	3.33 %	0.44 × 10⁸	0.11 %	2.87 %
4 m	0.44 × 10⁸	0.14 %	3.77 %	0.48 × 10⁸	0.09 %	2.40 %
10 m	0.40 × 10⁸	0.91 %	3.18 %	0.37 × 10⁸	0.61 %	3.26 %

Formulation	Time intervals	Potency† (IU / mg)	% RMC	% Free IFN	Fold depegylation
C	Initial	0.48 × 10⁸	1.4 %	0.63 %	3.14
C	After 3 m	0.43 × 10⁸	2.5 %	1.98 %	3.14
D	Initial	0.58 × 10⁸	0.25 %	0.84 %	No change
D	After 3 m	0.72 × 10⁸	ND	0.79 %	No change
E	Initial	0.72 × 10⁸	0.23 %	0.91 %	No change
E	After 3 m	0.51 × 10⁸	ND	0.92 %	No change
F	Initial	0.48× 10⁸	1.96 %	0.84 %	1.91
F	After 3 m	0.26 × 10⁸	2.43 %	1.61 %	1.91

Parameters	50 µg / 0.5 mL	80 µg / 0.5 mL	120 µg / 0.5 mL	180 µg / 0.5 mL
Appearance	Intact cake	Intact cake	Intact cake	Intact cake
pH*	6.87	6.8	6.87	6.85
% RMC	< 1 %	< 1 %	< 1 %	< 1 %
% Purity	94.96	95.0	94.42	94.11
% HMW	3.25	3.0	3.36	3.27
% Free interferon	1.79	2.0	2.22	2.62
Specific activity†	0.44 × 10⁸ IU / mg	0.50 × 10⁸ IU / mg	0.42 × 10⁸ IU / mg	0.50× 10⁸ IU / mg

Time intervals	Potency† (IU / mg)	% RMC	% Free IFN
Real-time storage: between +2 °C and +8 °C
Initial	0.51 × 10⁸	0.16 %	2.38 %
3 m	0.53 × 10⁸	0.40 %	2.27 %
6 m	0.37 × 10⁸	0.78 %	2.46 %
9 m	0.33 × 10⁸	1.21 %	2.59 %
12 m	0.51 × 10⁸	2.21 %	3.27 %
18 m	0.47 × 10⁸	0.91 %	3.30 %
24 m	0.54 × 10⁸	2.01 %	3.79 %
Accelerated storage: 25 °C ± 2 °C; 60 % RH ± 5 % RH
Initial	0.51 × 10⁸	0.16 %	2.38 %
3 m	0.67 × 10⁸	1.50 %	4.45 %
6 m	0.29 × 10⁸	2.05 %	6.85 %
9 m	0.29 × 10⁸	2.44 %	6.98 %
Stressed storage: 40 °C ± 2 °C; 75 % RH ± 5 % RH
Initial	0.51 × 10⁸	0.16 %	2.38 %
7 d	0.85 × 10⁸	0.93 %	3.88 %
14 d	0.53 × 10⁸	1.32 %	4.69 %
21 d	0.28 × 10⁸	1.73 %	5.19 %

	Real-Time storage stability (+2 °C to +8 °C):
Strength	Time intervals	Potency† (IU / mg)	% RMC	% Free IFN
50 µg / 0.5 mL	Initial	0.62 × 10⁸	0.63 %	1.21 %
	3 m	0.81 × 10⁸	0.84 %	1.65 %
	6 m	0.73 × 10⁸	0.84 %	1.67 %
	9 m	0.43 × 10⁸	0.92 %	1.42 %
	18 m	0.59 × 10⁸	0.73 %	2.25 %
150 µg / 0.5 mL	Initial	0.71 × 10⁸	0.31 %	0.89 %
	3 m	0.60 × 10⁸	0.44 %	1.73 %
	6 m	0.59 × 10⁸	0.52 %	1.95 %
	9 m	0.39 × 10⁸	0.67 %	2.01 %
	18 m	0.63 × 10⁸	0.48 %	2.41 %

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br

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[1] *Correspondence: G. Patel. Email: gayatripatel26@gmail.com.