Change炉o爎ead燝ENERAL燩RINCIPLE.PDFVIP

11/21/2016 35(1)?First?Interim?Revision?Announcement:?1054?BIOTECHNOLOGY-DERIVED?ARTICLES-ISOELECTRIC?FOCUSING 1054 ?BIOTECHNOLOGY-DERIVED?ARTICLES—ISOELECTRIC?FOCUSING This?chapter?provides?guidance?and?procedures?used?for?the?characterization?of?biotechnology-derived articles?by?isoelectric?focusing.?This?chapter?is?harmonized?with?the?corresponding?chapters?in?JP?and?EP. Other?characterization?tests,?also?harmonized,?are?shown?in?the?USP?general?information?chapters Biotechnology-Derived?Articles—Amino?Acid?Analysis? 1052 ?,?Biotechnology-Derived?Articles— Capillary?Electrophoresis? 1053 ?,?Biotechnology-Derived?Articles—Peptide?Mapping? 1055 ?, Biotechnology-Derived?Articles—Polyacrylamide?Gel?Electrophoresis? 1056 ?,?and?Biotechnology- Derived?Articles—Total?Protein?Assay? 1057 ?. Change?to?read: ?GENERAL?PRINCIPLE? 1 Isoelectric?focusing?(IEF)?is?a?method?of?electrophoresis?that?separates?proteins?according?to?their isoelectric?points.?Separation?is?carried?out?in?a?slab?of?polyacrylamide?or?agarose?gel?that?contains?a mixture?of?amphoteric?electrolytes?(ampholytes).?When?subjected?to?an?electrical?field,?the?ampholytes migrate?in?the?gel?to?create?a?pH?gradient.?In?some?cases,?gels?containing?an?immobilized?pH?gradient, prepared?by?incorporating?weak?acids?and?bases?to?specific?regions?of?the?gel?network?during?the preparation?of?the?gel,?are?used.?When?the?applied?proteins?reach?the?gel?fraction?that?has?a?pH?that?is the?same?as?their?isoelectric?point? ?(pI),? 1?their?charge?is?neutralized?and?migration?ceases.?Gradients can?be?made?over?various?ranges?of?pH,?according?to?the?mixture?of?ampholytes?chosen. Change?to?read: ?THEORETICAL?ASPECTS? 1 When?a?protein?is?at?the?position?of?its?isoelectri