title: Investigation of a Degradant in a Biologics Formulation Buffer Containing L-Histidine.
abstract: OBJECTIVE
  An unknown UV 280 nm absorbing peak was observed by SEC for protein stability samples formulated in L-histidine during a stress stability study. Understanding the source would enhance the confidence in the SEC results. We identified the unknown peak, studied the cause, and evaluated ways to eliminate it.
  
  
  METHODS
  The unknown peak was fractionated by preparative size exclusion chromatography separations, and subsequently analyzed by Hydrophilic Interaction Chromatography (HILIC) coupled with Time-of-Flight (TOF) high resolution mass spectrometry. The possible degradation was also studied with the presence of different excipients, including metal cations, chelating agents, and amino acids.
  
  
  RESULTS
  The unknown peak was identified to be trans-urocanic acid, a degradant of histidine, based on evidences from HILIC retention time, UV profile, accurate mass measurement, trans-cis isomerization, and pI measurement. The degradation from histidine to urocanic acids was not affected by the presence of Fe(2+), but slightly activated by Mn(2+). The chelating agents, EDTA and DTPA, counteracted the Mn(2+) effects. This degradation was evidenced to be caused by contamination. Adding alanine or cysteine as an excipient was found to reduce this degradation by 97 and 98%, respectively.
  
  
  CONCLUSIONS
  L-histidine formulation buffer can be contaminated to induce histidine degradation to trans-urocanic acid, which shows a large UV 280 nm absorbing peak at the total permeation volume under SEC conditions. Amino acids alanine and cysteine effectively inhibit this histidine degradation.
authors: Bioanalytical and Discovery Analytical Sciences, Research & Development, Bristol-Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey, 08543, USA, chunlei.wang@bms.com.
authors: Chunlei
authors: C
authors: Wang
authors: Aaron
authors: A
authors: Yamniuk
authors: Jun
authors: J
authors: Dai
authors: Sike
authors: S
authors: Chen
authors: Paul
authors: P
authors: Stetsko
authors: Noah
authors: N
authors: Ditto
authors: Yingru
authors: Y
authors: Zhang
mesh_terms: D001688:Biological Products / Q000008:administration & dosage* / Q000737:chemistry*; D002021:Buffers; D002614:Chelating Agents / Q000737:chemistry; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D004340:Drug Contamination; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D014560:Urocanic Acid / Q000737:chemistry*
chemical_list: D001688:Biological Products; D002021:Buffers; D002614:Chelating Agents; D005079:Excipients; D011506:Proteins; D006639:Histidine; D014560:Urocanic Acid