title: Glycine crystallization in frozen and freeze-dried systems: effect of pH and buffer concentration.
abstract: OBJECTIVE
  (1) To determine the effect of solution pH before lyophilization, over the range of 1.5 to 10, on the salt and polymorphic forms of glycine crystallizing in frozen solutions and in lyophiles. (2) To quantify glycine crystallization during freezing and annealing as a function of solution pH before lyophilization. (3) To study the effect of phosphate buffer concentration on the extent of glycine crystallization before and after annealing.
  
  
  METHODS
  Glycine solutions (10% w/v), with initial pH ranging from 1.5 to 10, were cooled to -50 degrees C, and the crystallized glycine phases were identified using a laboratory X-ray source. Over the same pH range, glycine phases in lyophiles obtained from annealed solutions (0.25, 2 and 10% w/v glycine), were characterized by synchrotron X-ray diffractometry (SXRD). In the pH range of 3.0 to 5.9, the extent of glycine crystallization during annealing was monitored by SXRD. Additionally, the effect of phosphate buffer concentration (50 to 200 mM) on the extent of glycine crystallization during freezing, followed by annealing, was determined.
  
  
  RESULTS
  In frozen solutions, beta-glycine was detected when the initial solution pH was < or =4. In the lyophiles, in addition to beta- and gamma-glycine, glycine HCl, diglycine HCl, and sodium glycinate were also identified. In the pH range of 3.0 to 5.9, decreasing the pH reduced the extent of glycine crystallization in the frozen solution. When the initial pH was fixed at 7.4, and the buffer concentration was increased from 50 to 200 mM, the extent of glycine crystallization in frozen solutions decreased with an increase in buffer concentration.
  
  
  CONCLUSIONS
  Both solution pH and solute concentration before lyophilization influenced the salt and polymorphic forms of glycine crystallizing in frozen solutions and in lyophiles. The extent of glycine crystallization in frozen solutions was affected by the initial pH and buffer concentration of solutions. The high sensitivity of SXRD allowed simultaneous detection and quantification of multiple crystalline phases.
authors.affiliation: Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, USA. varshneydu@lilly.com
authors.forename: Dushyant B
authors.initials: DB
authors.lastname: Varshney
authors.forename: Satyendra
authors.initials: S
authors.lastname: Kumar
authors.forename: Evgenyi Y
authors.initials: EY
authors.lastname: Shalaev
authors.forename: Prakash
authors.initials: P
authors.lastname: Sundaramurthi
authors.forename: Shin-Woong
authors.initials: SW
authors.lastname: Kang
authors.forename: Larry A
authors.initials: LA
authors.lastname: Gatlin
authors.forename: Raj
authors.initials: R
authors.lastname: Suryanarayanan
mesh_terms: D002021:Buffers; D003460:Crystallization / Q000379:methods; D005612:Freeze Drying / Q000379:methods*; D005615:Freezing; D005998:Glycine / Q000032:analysis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010710:Phosphates / Q000737:chemistry; D015203:Reproducibility of Results; D017356:Synchrotrons / Q000295:instrumentation; D013678:Technology, Pharmaceutical / Q000379:methods; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D002021:Buffers; D010710:Phosphates; D005998:Glycine