PMID- 8899836
OWN - NLM
STAT- MEDLINE
DCOM- 19970331
LR  - 20210520
IS  - 0724-8741 (Print)
IS  - 0724-8741 (Linking)
VI  - 13
IP  - 10
DP  - 1996 Oct
TI  - Stable formulations of recombinant human growth hormone and interferon-gamma for 
      microencapsulation in biodegradable microspheres.
PG  - 1464-75
AB  - PURPOSE: The successful development of controlled release formulations for 
      proteins requires that the protein not be denatured during the manufacturing 
      process. The major objective was to develop formulations that stabilize two 
      recombinant human proteins, human growth hormone (rhGH) and interferon-gamma 
      (rhIFN-gamma), at high protein concentrations (> 100 mg/mL) in organic solvents 
      commonly used for microencapsulation, methylene chloride and ethyl acetate. 
      METHODS: Several excipients were screened to obtain the maximum solubility of 
      each protein. These formulations (aqueous, lyophilized, milled, spray dried, or 
      isoelectric precipitate) were then rapidly screened by emulsification in the 
      organic solvent followed by recovery into excess buffer. Additional screening was 
      performed with solid protein that was suspended in the organic solvent and then 
      recovered with excess buffer. The recovery of native protein was determined by 
      native size exclusion chromatography (SEC-HPLC) and circular dichroism (CD). The 
      selected formulations were encapsulated in polylactic-coglycolic acid (PLGA) 
      microspheres by either water-in-oil-in-water (W/O/W) or solid-in-oil-in-water 
      (S/O/W) methods. The initial protein released from the microspheres incubated at 
      physiological conditions was analyzed by SEC-HPLC, CD, and biological assays. 
      RESULTS: The stability of a given formulation in the rapid screening method 
      correlated well with stability during encapsulation in PLGA microspheres. 
      Formulations of rhGH containing Tween 20 or 80 resulted in lower recovery of 
      native protein, while trehalose and mannitol formulations (phosphate buffer, pH 
      8.0) yielded complete recovery of native rhGH. Other additives such as 
      carboxymethyl cellulose, gelatin, and dextran 70 were not effective stabilizers, 
      and polyethylene glycol provided some stabilization of rhGH. Trehalose/rhGH (1:4 
      mass ratio) and mannitol/rhGH (1:2 mass ratio) formulations (potassium phosphate 
      buffer, pH 8.0) were lyophilized, reconstituted to 200 and 400 mg/mL rhGH, 
      respectively, and then encapsulated in PLGA microspheres. The protein was 
      released from these microspheres in its native state. Lyophilized formulations of 
      rhGH yielded analogous results indicating the ability of trehalose and mannitol 
      to stabilize the protein. Small solid particles of rhGH generated by spray drying 
      (both air and freeze-drying) formulations containing Tween 20 or PEG were stable 
      in ethyl acetate, but not methylene chloride. Similar results were also obtained 
      with rhIFN-gamma (137 mg/mL in succinate buffer, pH 5.0), where both mannitol and 
      trehalose were observed to stabilize the protein during exposure to the organic 
      solvents resulting in the release of native rhIFN-gamma from PLGA microspheres. 
      CONCLUSIONS: The rapid screening method allowed the development of stable 
      concentrated protein solutions or solid protein formulations that could be 
      successfully encapsulated in PLGA microspheres. The excipients observed to 
      stabilize these proteins function by preferential hydration of the protein, and 
      in the dry state (e.g., trehalose) may stabilize the protein via water 
      substitution yielding a protective coating around the protein surface. Studies of 
      other proteins should provide further insight into this mechanism of protein 
      stabilization during encapsulation.
FAU - Cleland, J L
AU  - Cleland JL
AD  - Department of Pharmaceutical Research and Development, Genentech, Inc., South San 
      Francisco, California 94080, USA.
FAU - Jones, A J
AU  - Jones AJ
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Pharm Res
JT  - Pharmaceutical research
JID - 8406521
RN  - 0 (Polymers)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Solutions)
RN  - 1SIA8062RS (Polylactic Acid-Polyglycolic Acid Copolymer)
RN  - 26009-03-0 (Polyglycolic Acid)
RN  - 33X04XA5AT (Lactic Acid)
RN  - 82115-62-6 (Interferon-gamma)
RN  - 9002-72-6 (Growth Hormone)
SB  - IM
MH  - Chemical Precipitation
MH  - Chemistry, Pharmaceutical
MH  - Drug Compounding
MH  - Drug Stability
MH  - Growth Hormone/*chemistry
MH  - Humans
MH  - Interferon-gamma/*chemistry
MH  - Isoelectric Focusing
MH  - *Lactic Acid
MH  - Microspheres
MH  - Particle Size
MH  - *Polyglycolic Acid
MH  - Polylactic Acid-Polyglycolic Acid Copolymer
MH  - Polymers
MH  - Protein Conformation
MH  - Recombinant Proteins
MH  - Solutions
EDAT- 1996/10/01 00:00
MHDA- 1996/10/01 00:01
CRDT- 1996/10/01 00:00
PHST- 1996/10/01 00:00 [pubmed]
PHST- 1996/10/01 00:01 [medline]
PHST- 1996/10/01 00:00 [entrez]
AID - 10.1023/a:1016063109373 [doi]
PST - ppublish
SO  - Pharm Res. 1996 Oct;13(10):1464-75. doi: 10.1023/a:1016063109373.