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Consideraes gerais 2.1.  As informaes contidas neste guia aplicam-se a mtodos bioanalticos, tais como cromatografia gasosa (CG), cromatografia lquida ... , utilizados na determinao quantitativa de frmacos e/ou metablitos em matrizes biolgicas, ... Tambm se aplica a outras tcnicas analticas, tais como mtodos microbiolgicos e imunolgicos, ou para outras matrizes biolgicas, embora, nestes casos, pode-se observar um alto grau de variabilidade. BG&G&GDGG ̙33___PPT10i. +D=' = @B +" (  ^  H ?Rectangle 3N?  0 2. Consideraes gerais 2.4. Para os estudos de biodisponibilidade relativa/bioequivalncia deve-se utilizar padro interno, sempre que mtodos cromatogrficos forem utilizados. Deve-se justificar a impossibilidade de sua utilizao. oGGG3G?G ̙33___PPT10i. +D=' = @B +"  (    H?Rectangle 3N  0 3.1. Especificidade 3.1.1. ...  Cada amostra branco deve ser testada utilizando o procedimento e as condies cromatogrficas propostas ... 3.1.2. ...  interferncia significativa no tempo de reteno do frmaco, metablito ou padro interno ... 3.1.4. ...  deve ser injetado separadamente para determinar os tempos de reteno individuais . 3.1.5. ...  A resposta de picos interferentes no tempo de reteno do frmaco deve ser inferior a 20% ... Fk`keGG:GGEGG"GGGGRG ̙33___PPT10i. +D=' = @B +" 0(    H ?Rectangle 3N  0 %m3.2. Curva de calibrao/linearidade 3.2.2. Para a determinao da curva de calibrao, deve-se analisar amostras extradas da matriz apropriada, no mnimo 6 (seis) concentraes diferentes. Procedimentos alternativos devem ser justificados, como na obteno de uma correlao no-linear, em que um maior nmero de concentraes de padres sero necessrios. hG+GMG]GGn ̙33___PPT10i. +D=' = @B +" @ (   |  H -?Rectangle 3N  0 3.5. Limite inferior de quantificao (LIQ) 3.5.1. Estabelecido por meio da anlise de matriz biolgica contendo concentraes decrescentes do frmaco at o menor nvel quantificvel com preciso e exatido aceitveis. 3.5.2. e 3.5.3. Razo de 5:1 entre o sinal e o rudo ,5sGhG@G ̙33___PPT10i. +D=' = @B +"    P$n (  $6  $ H@B?Rectangle 3,9 0   Because of these significant differences between small and macromolecule analytes, different technologies, such as LC-MS for small molecules and LBAs for macromolecules, are often employed to determine drug levels for PK assessments.  As a result of small molecule analysis moving to the LCMS platform, LBAs are now almost exclusively used to measure macromolecules. (Viswanathan et al. 2007)  Ligand-binding assays or immunoassays are nowadays especially used for macromolecules. In these assays, quantification is based on macromolecular interactions between the macromolecule and antibody. (EMEA 2009) $PPPPPP%PGGGGGGnGaGGTG>U ̙33___PPT10i. +D=' = @B +" `((  (8 ( H?Rectangle 2 `  0 :Cristal City (Viswanathan et al. 2007)0;!G G;  ( H?Rectangle 3'g 0 nVALIDAO DE LBAS PARA MACROMOLCULAS Preciso e exatido com CQs em matrizes equivalentes s previstas no estudo Preparao da matriz devido a substancias endgenas Avaliao de 5 ou mais concentraes cobrindo a faixa de quantificao do mtodo: 1  LIQ 2  baixa (3 X LIQ) 3  mdia (mdia geomtrica) 4  alta (75% LSQ) 5  LSQ &N'G0GG\ ̙33___PPT10i. +D=' = @B +" p,(  ,8 , H?Rectangle 2 `  0 :Cristal City (Viswanathan et al. 2007)0;!G G; , H?Rectangle 3'g 0 0VALIDAO DE LBAS PARA MACROMOLCULAS Para LBAs a variabilidade inter-corridas normalmente contribui mais para a variabilidade geral do que a variabilidade itra-corrida. recomendada ao menos 2 determinaes independentes de cada amostra de validao em cada corrida por no mnimo 6 corridas. &5GGG1 ̙33___PPT10i. +D=' = @B +"% ,$0 (  08 0 H`?Rectangle 2 `  0 :Cristal City (Viswanathan et al. 2007)0;!G G; 0 Hp?Rectangle 3'g 0 dVALIDAO DE LBAS PARA MACROMOLCULAS Para aceitao do mtodo a impreciso e a exatido inter-corrida devem ser at 20% (25% para LIQ e LSQ) recomendado que a soma da preciso mais a exatido seja menor que 30% (40% para LIQ e LSQ) Todas as corridas devem ser includas nos clculos finais, a no ser em casos de erros bvios e documentados. b&5eGe ̙33___PPT10i. +D=' = @B +" 4(  48 4 H q?Rectangle 2 `  0 :Cristal City (Viswanathan et al. 2007)0;!G G;E 4 Hs?Rectangle 3'g 0 CRITRIOS DE ACEITAO DE CORRIDAS ANALTICAS 75% dos CCs dentro de 20% de exatido (25% para LIQ e LSQ) CQB, CQM e CQA no mnimo em duplicata (5% do total de amostras desconhecidas) Aprovao mnima de 4 em 6 CQs dentro de 20% de exatido e ao menos 50% de cada concentrao X0GE  ̙33___PPT10i. +D=' = @B +"| {8c(  84 8 Hu?Rectangle 2 `  0 $DRAFT EMEA 2009 B%GG G% 8 H`v?Rectangle 3<9, 0 S Especificidade e Seletividade Especificidade do anticorpo e seletividade do mtodo so pontos crticos do ensaio. No deve haver reaes cruzadas do anticorpo com outros compostos de estrutura semelhante ao analito. A especificidade validada utilizando matriz de 10 fontes. A presena de anticorpos endgenos deve ser considerada. Devido interferncia de compostos endgenos a resposta do branco pode ser maior do que 20% da resposta do LIQ, o que aceitvel desde que no comprometa a exatido do mtodo. !PTPPfPP<PP9PPPPPG GGGG ̙33___PPT10i. +D=' = @B +"S ZR<:(  <4 < Hx?Rectangle 2 `  0 $DRAFT EMEA 2009 B%GG G% < H`y?Rectangle 3<9, 0 *" Curva de calibrao importante que a matriz das amostras e a matriz padro sejam equivalentes. Caso no sejam, deve ser demonstrado que a relao dose- resposta no afetada pela matriz. Quando padres de calibrao  ncora forem utilizados, seus valores devem ser muito bem documentados. A exatido dos padres de calibrao deve estar dentro de + ou  20% (25 para LIQ e LSQ). PPPPPPPPGGsGGG ̙33___PPT10i. +D=' = @B +" @(  @4 @ H{?Rectangle 2 `<  0 $DRAFT EMEA 2009 B%GG G% @ H`|?Rectangle 3<9, 0 z Controles de Qualidade Ao menos 3 concentraes de CQ devem ser includas em cada corrida analtica. A preciso e a exatido inter e intra-corrida deve estar dentro de 20%. O erro total deve ser menor do que 30% (40% LIQ e LSQ) Os resultados de todas as corridas devem ser includos para validao, exceto nos casos em que os erros so bvios e documentados. PPP7PPPPPGGXGGG{ ̙33___PPT10i. +D=' = @B +" D(  D4 D H`?Rectangle 2 `  0 $DRAFT EMEA 2009 B%GG G%c D H }?Rectangle 3<9, 0  Consideraes gerais sobre a validao recomendado que o tamanho da corrida de validao seja comparvel ao das corridas com amostras desconhecidas. Deve ser demonstrado que as caractersticas do analito no so afetadas pelos mtodos de preparao de amostras, aditivos (ex. anticoagulantes) e estabilidade durante todo o processo. A avaliao no deve apenas considerar propriedades fiisico-qumicas mas tambm a integridade biolgica. Cuidado com mudanas no mtodo aps a validao! (Diluentes, reagentes, pH, etc.) Os resultados de todas as corridas devem ser includos para validao, exceto nos casos em que os erros so bvios e documentados. +P*PPiPPRPPPPPG )GoGG G ̙33___PPT10i. +D=' = @B +" H(  H4 H H?Rectangle 2 `  0 $DRAFT EMEA 2009 B%GG G%M H H?Rectangle 3<9, 0  Kits Comerciais Kits comerciais podem ter sido desenvolvidos com propsitos diferentes de sua aplicao em estudos de bioequivalncia. Necessitam ser revalidados. Os princpios de validao listados acima se aplicam. PPPPGGGGG ̙33___PPT10i. +D=' = @B +" L(  L L H?Rectangle 2 `  0 r"Implicaes prticas para a ANVISA##G# L H?Rectangle 3<9, 0  Considerar as peculiaridades dos mtodos no-cromatogrficos na reviso da RE 899/2003. Para molculas passveis de anlise por mtodos cromatogrficos, recomendado que estes sejam a primeira escolha. Quanto uma anlise for possvel por mtodos cromatogrficos, mas a empresa quiser utilizar imunoensaios, os mesmos parmetros de preciso e exatido devem ser exigidos, acrescidos dos demais cuidados peculiares tcnica. Para drogas cujo ensaio cromatogrfico no for indicado, podem ser aceitos 20/25% como parmetros de preciso e exatido, acrescidos dos demais cuidados peculiares tcnica. Kits comerciais devem ser revalidados para estudos de biodisponibilidade relativa. PPXPPsPPPPPPTPPPGGGG ̙33___PPT10i. +D=' = @B +"*  1 ) P (  P P H ?Rectangle 2 `  0 [ Referncias  G  P H?Rectangle 3  0 > C. T. Viswanathan, Surendra Bansal , Brian Booth , Anthony J. DeStefano , Mark J. Rose , Jeffrey Sailstad , Vinod P. Shah , Jerome P. Skelly , Patrick G. Swann , and Russell Weiner. Workshop/Conference Report  Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays. The AAPS Journal. 2007; 9 (1) Article 4 John W. A. Findlay and Robert F. Dillard. Appropriate Calibration Curve Fitting in Ligand Binding Assays. The AAPS Journal. 2007; 9 (2) Article 29. EMEA. GUIDELINE ON VALIDATION OF BIOANALYTICAL METHODS. (DRAFT) 2009. Resoluo - RE n 899, de 29 de maio de 2003. 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    6Apresentao na tela (4:3)anvisaD Times New RomanArialVerdanaEstrutura padroKViso regulatria no Brasil para mtodos bioanalticos no-cromatogrficosSumrio*Mtodos No-Cromatogrficos e RE 899/2003Slide 4Slide 5Slide 6Slide 7Slide 8;Cristal City (Viswanathan et al. 2007);Cristal City (Viswanathan et al. 2007);Cristal City (Viswanathan et al. 2007);Cristal City (Viswanathan et al. 2007)%DRAFT EMEA 2009 %DRAFT EMEA 2009 %DRAFT EMEA 2009 %DRAFT EMEA 2009 %DRAFT EMEA 2009 #Implicaes prticas para a ANVISA Referncias*Obrigado! bioequivalencia@anvisa.gov.br5Comparao entre macromolculas e molculas pequenas!Ligand Binding Assay - DefinioImunoensaio - DefinioImunoensaio - CaractersticasImunoensaio - CaractersticasImunoensaio - Caractersticas Fontes usadasTemaTtulos de slides  8@ _PID_HLINKSA http://www.aaps.org/http://www.aaps.org/_HMorelliMorelli  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      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