Development of Low Endotoxin Gelatin for Regenerative Medicine

Kanayama, Yoshiharu; Aoki, Chizuru; Sakai, Yasuo

doi:10.1248/bpb.30.237

Cited by 23 publications

(19 citation statements)

References 6 publications

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“…Materials All chemicals and proteins employed in this study were obtained from the following sources: recombinant human albumin expressed in rice (rHA, Cellastim ), gelatin from porcine skin (gel strength 300, type A) (Sigma-Aldrich Co., St. Louis, MO); L-Arg, L-arginine monohydrochloride (LArg·HCl), monosodium L-glutamate monohydrate (Na·L-Glu, MSG), and sodium chloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan); purified lower molecular weight porcine gelatin (RM-50, 40-60 kDa; Jellice Co., Sendai, Japan) 13) ; dextran FP40 (SERVA Electrophoresis GmbH, Heidelberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…5,8,[12][13][14][15] Physical states (e.g., crystallinity, crystal polymorph) of the components are important factors that determine chemical and conformational stability of proteins during the freeze-drying process and subsequent storage. [16][17][18] The stabilizing effects of disaccharides are attributed to protection of protein conformation by the substitution of surrounding water molecules and by holding protein molecules in lower molecular mobility glassstate amorphous solids.…”

mentioning

confidence: 99%

“…[5][6][7][8][9][10][11] Application of the amino acid excipients that protect proteins through particular mechanisms not achievable by saccharides (e.g., aggregation-reducing effect of L-arginine (L-Arg) in aqueous solution) would increase formulation strategies in lyophilization of marginally stable proteins. 5,8,[12][13][14][15] Physical states (e.g., crystallinity, crystal polymorph) of the components are important factors that determine chemical and conformational stability of proteins during the freeze-drying process and subsequent storage. [16][17][18] The stabilizing effects of disaccharides are attributed to protection of protein conformation by the substitution of surrounding water molecules and by holding protein molecules in lower molecular mobility glassstate amorphous solids.…”

mentioning

confidence: 99%

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Amorphous–Amorphous Phase Separation of Freeze-Concentrated Protein and Amino Acid Excipients for Lyophilized Formulations

Izutsu¹,

Yoshida²,

Shibata³

et al. 2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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The objective of this study was to elucidate the mixing state of proteins and amino acid excipients concentrated in the amorphous non-ice region of frozen solutions. Thermal analysis of frozen aqueous solutions was performed in heating scans before and after a heat treatment. Frozen aqueous solutions containing a protein (e.g., recombinant human albumin, gelatin) or a polysaccharide (dextran) and an amino acid excipient (e.g., L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) at varied mass ratios showed single or double T g ′ (glass transition temperature of maximally freeze-concentrated solutes). Some mixture frozen solutions rich in the polymers maintained the single T g ′ of the freeze-concentrated amorphous solute-mixture phase. In contrast, amino acid-rich mixture frozen solutions revealed two T g ′s that suggested transition of concentrated non-crystalline solute-mixture phase and excipient-dominant phase. Post-freeze heat treatment induced splitting of the T g ′ in some intermediate mass ratio mixture solutions. The mixing state of proteins and amino acids varied depending on their structure, salt types, mass ratio, composition of co-solutes (e.g., NaCl) and thermal history. Information on the varied mixing states should be valuable for the rational use of amino acid excipients in lyophilized protein pharmaceuticals.Key words amorphous; freeze drying; thermal analysis; protein formulation; stabilization; mixing Freeze-drying is a popular method to formulate therapeutic proteins that are insufficiently stable in aqueous solutions during storage.1) Several freeze-dried therapeutic protein formulations contain multiple excipients, including stabilizers, bulking agents, pH buffer agents, and tonicity modifiers besides active pharmaceutical ingredient (API).2-4) Sucrose and trehalose are popular stabilizers that protect proteins from dehydration-induced irreversible structural changes during the process and from chemical degradation during storage. Certain amino acids and their salts are potent stabilizers in the freeze-drying of proteins, frozen storage of liposomes, and spray drying of vaccines.5-11) Application of the amino acid excipients that protect proteins through particular mechanisms not achievable by saccharides (e.g., aggregation-reducing effect of L-arginine (L-Arg) in aqueous solution) would increase formulation strategies in lyophilization of marginally stable proteins. 5,8,[12][13][14][15] Physical states (e.g., crystallinity, crystal polymorph) of the components are important factors that determine chemical and conformational stability of proteins during the freeze-drying process and subsequent storage. [16][17][18] The stabilizing effects of disaccharides are attributed to protection of protein conformation by the substitution of surrounding water molecules and by holding protein molecules in lower molecular mobility glassstate amorphous solids. Crystallization of some sugar alcohols (e.g., mannitol) during the freezing segment of lyophilization deprives the...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Amorphous–Amorphous Phase Separation of Freeze-Concentrated Protein and Amino Acid Excipients for Lyophilized Formulations

Izutsu¹,

Yoshida²,

Shibata³

et al. 2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…Four types of gelatin were prepared from low endotoxin Regenerative-Medicine Gelatin (RM-100; Jellice Co., Ltd. Miyagi, Japan) 12) . RM-100 was dissolved in distilled water.…”

Section: Introductionmentioning

confidence: 99%

Bone Formation in Gelatin/Calcium Phosphate Paste in a Subperiosteal Pocket of Rat Calvaria

Wada

Amemiya

Hirota

et al. 2016

J. Hard Tissue Biology.

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In the present study, gelatin/calcium phosphate pastes with different degradation rates of gelatin were prepared and evaluated for the bone response after implantation into a subperiosteal pocket of rat calvaria. Four types of gelatin with different sizes and degrees of cross-linking were prepared: Sample A: smaller size and lower degree of cross-linking; Sample B: smaller size and higher degree of cross-linking; Sample C: larger size and lower degree of cross-linking; and Sample D: larger size and higher degree of cross-linking. A powder mixture of each gelatin with calcium phosphate was mixed with a liquid. Then, the mixed paste was placed into a polytetrafluoroethylene (PTFE) tube, which was implanted directly into a subperiosteal pocket of rat calvaria. At 8 weeks after implantation, each specimen was excised, and non-decalcified thin sections were prepared. After staining each section with toluidine blue, the bone response was observed histologically and histomorphometrically. New bone formation was observed from the calvaria surface, not from the periosteum for the four different gelatin/CaP paste samples. Significantly greater amounts of residual non-degraded gelatin was observed for Sample B/CaP and Sample D/CaP paste (p<0.05). It was revealed that Sample C/CaP paste with a lower degree of gelatin cross-linking produced greater amounts of new bone formation. The size of gelatin also influenced bone formation. Gelatin/CaP paste is advantageous because it can be applied to any shape of defect, and it is a promising material for bone regenerative treatment.

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“…RM gelatin was removed endotoxin using an ultra filtration membrane without heat sterilization at 250°C. 10) We created cisplatin-adsorbing RM gelatin sponge. The solubility of dried RM gelatin sponge can be controlled by heating for sterilization.…”

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confidence: 99%

Creation of Cisplatin-Adsorbing Regenerative-Medicine Gelatin Sponge and Its Cisplatin Release Pattern

Kanayama

Aoki

Kawai

et al. 2014

Biological & Pharmaceutical Bulletin

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The purpose of this study was to clarify the adsorption of cisplatin on regenerative-medicine (RM) gelatin sponge, and to verify the relationship between the cisplatin release pattern of cisplatin-adsorbed RM gelatin sponge and the dissolving time of RM gelatin sponge. We tested various RM gelatin sponges, one with a molecular weight of 50000 Daltons (RM-50 gelatin sponge) that is 100% saline soluble at 24 h, RM-50-120 (heated at 120°C) that is 54.3% saline soluble at 24 h, and RM-50-140 (heated at 140°C) that is 15.8% saline soluble at 24 h. We investigated the production of cisplatin-adsorbed RM gelatin sponge and measured free cisplatin released from cisplatin-adsorbed RM gelatin sponge. There was no significant difference in the weight of adsorbed cisplatin among the RM-50, RM-50-120, and RM-50-140. The results mean that cisplatin adsorbs onto RM gelatin sponge irrespective of heating temperature. The average adsorbed weight of cisplatin per gram of RM gelatin sponge was 29.3 mg, which was approximately five times more than that per g previously reported for Gelpart (non-soluble gelatin sponge, clinically available). Cisplatin release in the RM-50 gelatin was the most rapid at only 1 h after incubation; it was released gradually and increasingly in the RM-50-120 gelatin, and released slowly in the RM-50-140 gelatin for 24 h incubation. Cisplatin-adsorbed RM gelatin sponge released cisplatin proportional to the dissolving time of RM gelatin sponge, indicating that the cisplatin release time can be controlled by heating for sterilization of RM gelatin sponge. Key words regenerative-medicine (RM) gelatin; endotoxin; cisplatin; drug delivery system (DDS); transcatheter arterial chemoembolization (TACE)Gelatin has been extensively used by pharmaceutical manufactures as a pharmaceutical excipient (stabilizer, additive, etc.) Furthermore, it is used as a material for biomaterials. For example, it is used in the biomaterial sheet to facilitate regeneration of dermis to cover a skin deficit.1,2) The endotoxin in gelatin is lipopolysaccharide (LPS), which is the same component as the outer envelope of Gram-negative bacteria, and is commonly isolated from Gram-negative bacteria. The collapse of the cell wall due to autolysis and denaturation would cause much more LPS to be released, damaging various types of cells. [3][4][5] When LPS invades the body, it causes fever and can cause septic shock.6-8) Sterilization to 121°C is insufficient to eradicate endotoxic activity, this requires heating at 250°C for more than 60 min.9) It is preferable to use gelatin without bacteria or endotoxins when this biomaterial is to be introduced into the body. The gelatin that matches the standard for purified gelatin in Japanese Pharmacopoeia in this regard was developed in 2007, and is termed regenerative-medicine (RM) gelatin. RM gelatin was removed endotoxin using an ultra filtration membrane without heat sterilization at 250°C. 10)We created cisplatin-adsorbing RM gelatin sponge. The solubility of dried RM gelatin sponge can be contr...

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Development of Low Endotoxin Gelatin for Regenerative Medicine

Cited by 23 publications

References 6 publications

Amorphous–Amorphous Phase Separation of Freeze-Concentrated Protein and Amino Acid Excipients for Lyophilized Formulations

Amorphous–Amorphous Phase Separation of Freeze-Concentrated Protein and Amino Acid Excipients for Lyophilized Formulations

Bone Formation in Gelatin/Calcium Phosphate Paste in a Subperiosteal Pocket of Rat Calvaria

Creation of Cisplatin-Adsorbing Regenerative-Medicine Gelatin Sponge and Its Cisplatin Release Pattern

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