Neste trabalho foram desenvolvidas condições específicas de RP-HPLC para análise de preparações recombinantes e naturais de hLH, de hCG, e de suas subunidades. O hLH e o hCG heterodimérico e suas subunidades e migraram com tempos de retenção (tR) significativamente diferentes, na seguinte ordem de hidrofobicidade crescente: -hCG < -hLH < hCG < hLH < -hCG < -hLH. Nestas condições, onze preparações foram estudadas: o Padrão Internacional recombinante hLH-WHO 96/602, uma preparação comercial recombinante, duas preparações hipofisárias altamente purificadas de hLH, uma preparação recombinante e duas preparações urinárias de hCG e quatro produtos urinários heterogêneos, contendo hLH + hFSH. Todas as preparações de hLH mostraram um tempo de retenção similar para o pico principal (tR = 38,35 ± 0,42 min; DPR = 1,1 %; n = 4 preparações), enquanto o pico principal do hCG migrou cerca de 4% mais rápido, quando comparado a este valor médio. Picos de hLH, hFSH e hCG foram também identificados nas preparações urinárias heterogêneas. O método foi validado para as sete preparações homogêneas, sendo a exatidão, precisão e sensibilidade calculadas com base na curva dose-resposta, altamente linear (r=0,99998; p<0,0001; n=20). A quantificação de diferentes gonadotrofinas nas preparações heterogêneas foi também realizada, embora com claras limitações de exatidão. / Specific RP-HPLC conditions for the analysis of recombinant and native hLH and hCG preparations and of their subunits were set up. Heterodimeric hLH and hCG and their - and - subunits all migrated with significantly different retention times (tR) in the following order of increasing hydrophobicity: -hCG < -hLH < hCG < hLH < -hCG < -hLH. With basis on these conditions, a total of eleven preparations were studied: the International Standard of recombinant hLH-WHO 96/602, a commercial recombinant and two highly purified pituitary hLH, a recombinant and two urinary hCG preparations and four heterogeneous urinary products containing hLH + hFSH. All hLH preparations showed very similar retention times for the main peak (tR = 38.35 ± 0.42 min; RSD = 1.1 %; n = 4 preparations), while the hCG main peak ran about 4 % faster when compared to this average value. Human LH, hFSH and hCG peaks could also be identified in the heterogeneous urinary preparations. Quantitative analysis could be validated for the seven homogeneous preparations and accuracy, precision and sensitivity were calculated on the basis of a highly linear dose-response curve (r=0.99998; p<0.0001; n=20). Quantification of the differents gonadotropins in the heterogeneous urinary preparations was also carried out, though with clear accuracy limitations.

Neste trabalho foram desenvolvidas condições específicas de RP-HPLC para análise de preparações recombinantes e naturais de hLH, de hCG, e de suas subunidades. O hLH e o hCG heterodimérico e suas subunidades e migraram com tempos de retenção (tR) significativamente diferentes, na seguinte ordem de hidrofobicidade crescente: -hCG < -hLH < hCG < hLH < -hCG < -hLH. Nestas condições, onze preparações foram estudadas: o Padrão Internacional recombinante hLH-WHO 96/602, uma preparação comercial recombinante, duas preparações hipofisárias altamente purificadas de hLH, uma preparação recombinante e duas preparações urinárias de hCG e quatro produtos urinários heterogêneos, contendo hLH + hFSH. Todas as preparações de hLH mostraram um tempo de retenção similar para o pico principal (tR = 38,35 ± 0,42 min; DPR = 1,1 %; n = 4 preparações), enquanto o pico principal do hCG migrou cerca de 4% mais rápido, quando comparado a este valor médio. Picos de hLH, hFSH e hCG foram também identificados nas preparações urinárias heterogêneas. O método foi validado para as sete preparações homogêneas, sendo a exatidão, precisão e sensibilidade calculadas com base na curva dose-resposta, altamente linear (r=0,99998; p<0,0001; n=20). A quantificação de diferentes gonadotrofinas nas preparações heterogêneas foi também realizada, embora com claras limitações de exatidão. / Specific RP-HPLC conditions for the analysis of recombinant and native hLH and hCG preparations and of their subunits were set up. Heterodimeric hLH and hCG and their - and - subunits all migrated with significantly different retention times (tR) in the following order of increasing hydrophobicity: -hCG < -hLH < hCG < hLH < -hCG < -hLH. With basis on these conditions, a total of eleven preparations were studied: the International Standard of recombinant hLH-WHO 96/602, a commercial recombinant and two highly purified pituitary hLH, a recombinant and two urinary hCG preparations and four heterogeneous urinary products containing hLH + hFSH. All hLH preparations showed very similar retention times for the main peak (tR = 38.35 ± 0.42 min; RSD = 1.1 %; n = 4 preparations), while the hCG main peak ran about 4 % faster when compared to this average value. Human LH, hFSH and hCG peaks could also be identified in the heterogeneous urinary preparations. Quantitative analysis could be validated for the seven homogeneous preparations and accuracy, precision and sensitivity were calculated on the basis of a highly linear dose-response curve (r=0.99998; p<0.0001; n=20). Quantification of the differents gonadotropins in the heterogeneous urinary preparations was also carried out, though with clear accuracy limitations.

Com a intenção de estabelecer métodos físico-químicos como uma alternativa ao bioensaio in vivo para determinação de atividade biológica, o conteúdo de hFSH, hTSH e hLH de diferentes preparações, nativas e recombinantes, foi determinado por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) e comparado ao dado obtido pelo clássico bioensaio in vivo em camundongos ou ratos (BA). Para estes hormônios foi encontrada uma relação linear entre os dois métodos: hFSH BAUI = 0,9925 RP-HPLCUI - 1,3165, r = 0,9371, p < 0,001, n = 24; hTSH BA&mu;g = 0,9790 RP-HPLC&mu;g - 0,052, r = 0,8725 , p < 0,001, n = 14; hLH BAUI = 0,8771 RP-HPLCUI + 12,41; r = 0,9786, p < 0,01, n = 5. Para outras nove preparações de hFSH e onze preparações de hTSH foi determinada a diferença média (d) entre a bioatividade predita pela RP-HPLC através destas equações e da média das bioatividades obtidas com os dois métodos. Para o hLH não foi possível determinar esta diferença em virtude das poucas amostras disponíveis. No caso do hFSH, d ± DP = -2,11 ± 3,49 % sendo a precisão de 1,16% e no caso do hTSH, d ± DP = -2,01 ± 5,56 % com precisão de 1,68%. Amostras parcialmente alteradas apresentaram diferentes graus de atividade de hFSH, hTSH e hLH que puderam ser preditas por RP-HPLC com uma aceitável concordância com os bioensaios in vivo. Estes resultados demonstraram que o emprego de um ensaio físico-químico sem o uso de animais, tal como a RP-HPLC, é uma alternativa viável ao uso do bioensaio in vivo para a determinação da potência de hFSH e hTSH, reduzindo assim o número de animais em geral utilizados para assegurar a qualidade e eficácia de um produto farmacêutico. / With the intention of setting up physico-chemical methods as an alternative to in vivo bioassay for determining biological activity, the hFSH, hTSH and hLH content of native and recombinant preparations was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and compared with the data obtained by the classical mouse or rat in vivo bioassays (BA). A linear relationship between the two methods was found for these hormones: hFSH BAIU = 0.9925 RP-HPLCIU 1.3165, r = 0.9371, p < 0.001, n = 24; hTSH BA&mu;g = 0.9790 RP-HPLC&mu;g - 0.052, r = 0.8725, p < 0.001, n = 14; hLH BAIU = 0.8771 RP-HPLCIU + 12.41, r = 0.9786, p < 0.01, n = 5. For nine other hFSH and eleven hTSH preparations, the mean difference (d) between the bioactivity predicted from RP-HPLC data via these equations and the mean of the bioactivities obtained with the two methods was as follows. For hLH this difference could not be estimated due to lack of different samples. In the case of hFSH, d ± SD = -2.11 ± 3.49% with a precision of 1.16% and in the case of hTSH, d ± SD = -2.01 ± 5.56 %, with precision of 1.68%. Partly-degraded hFSH, hTSH and hLH samples presented different activity degrees that could be predicted by RP-HPLC, with an acceptable agreement with the in vivo bioassays. These results demonstrate that the employment of a non-animal physico-chemical assay, such as RP-HPLC, is a viable alternative to the use of an in vivo bioassay for hFSH and hTSH potency determination, thus reducing the number of animals currently used for assuring quality and efficacy of a pharmaceutical product.

Com a intenção de estabelecer métodos físico-químicos como uma alternativa ao bioensaio in vivo para determinação de atividade biológica, o conteúdo de hFSH, hTSH e hLH de diferentes preparações, nativas e recombinantes, foi determinado por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) e comparado ao dado obtido pelo clássico bioensaio in vivo em camundongos ou ratos (BA). Para estes hormônios foi encontrada uma relação linear entre os dois métodos: hFSH BAUI = 0,9925 RP-HPLCUI - 1,3165, r = 0,9371, p < 0,001, n = 24; hTSH BA&mu;g = 0,9790 RP-HPLC&mu;g - 0,052, r = 0,8725 , p < 0,001, n = 14; hLH BAUI = 0,8771 RP-HPLCUI + 12,41; r = 0,9786, p < 0,01, n = 5. Para outras nove preparações de hFSH e onze preparações de hTSH foi determinada a diferença média (d) entre a bioatividade predita pela RP-HPLC através destas equações e da média das bioatividades obtidas com os dois métodos. Para o hLH não foi possível determinar esta diferença em virtude das poucas amostras disponíveis. No caso do hFSH, d ± DP = -2,11 ± 3,49 % sendo a precisão de 1,16% e no caso do hTSH, d ± DP = -2,01 ± 5,56 % com precisão de 1,68%. Amostras parcialmente alteradas apresentaram diferentes graus de atividade de hFSH, hTSH e hLH que puderam ser preditas por RP-HPLC com uma aceitável concordância com os bioensaios in vivo. Estes resultados demonstraram que o emprego de um ensaio físico-químico sem o uso de animais, tal como a RP-HPLC, é uma alternativa viável ao uso do bioensaio in vivo para a determinação da potência de hFSH e hTSH, reduzindo assim o número de animais em geral utilizados para assegurar a qualidade e eficácia de um produto farmacêutico. / With the intention of setting up physico-chemical methods as an alternative to in vivo bioassay for determining biological activity, the hFSH, hTSH and hLH content of native and recombinant preparations was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and compared with the data obtained by the classical mouse or rat in vivo bioassays (BA). A linear relationship between the two methods was found for these hormones: hFSH BAIU = 0.9925 RP-HPLCIU 1.3165, r = 0.9371, p < 0.001, n = 24; hTSH BA&mu;g = 0.9790 RP-HPLC&mu;g - 0.052, r = 0.8725, p < 0.001, n = 14; hLH BAIU = 0.8771 RP-HPLCIU + 12.41, r = 0.9786, p < 0.01, n = 5. For nine other hFSH and eleven hTSH preparations, the mean difference (d) between the bioactivity predicted from RP-HPLC data via these equations and the mean of the bioactivities obtained with the two methods was as follows. For hLH this difference could not be estimated due to lack of different samples. In the case of hFSH, d ± SD = -2.11 ± 3.49% with a precision of 1.16% and in the case of hTSH, d ± SD = -2.01 ± 5.56 %, with precision of 1.68%. Partly-degraded hFSH, hTSH and hLH samples presented different activity degrees that could be predicted by RP-HPLC, with an acceptable agreement with the in vivo bioassays. These results demonstrate that the employment of a non-animal physico-chemical assay, such as RP-HPLC, is a viable alternative to the use of an in vivo bioassay for hFSH and hTSH potency determination, thus reducing the number of animals currently used for assuring quality and efficacy of a pharmaceutical product.

No description available.

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

Neste trabalho é descrito um método prático e eficiente para dissociar, em subunidades &alpha; e &beta;, quantidades pequenas (da ordem de microgramas) dos hormônios foliculotrofina (hFSH), luteotrofina (hLH) e tireotrofina (hTSH) humana, nativos e recombinantes. A dissociação destes hormônios foi conseguida incubando-os, durante 16 horas, a 37ºC, com diferentes concentrações de ácido acético: 3M, 5M e 0,4M respectivamente para o hFSH, hLH e hTSH. Nestas condições, uma eficiência de dissociação acima de 98% foi obtida. Esta eficiência foi calculada com base nas determinações de massa dos heterodímeros e das subunidades, realizadas por MALDI-TOF-MS. Uma separação rápida e quantitativa das subunidades, com rendimentos da ordem de 80-90%, foi conseguida por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) em uma coluna C4. As subunidades foram caracterizadas quanto à pureza, hidrofobicidade, massa molecular e distribuição de carga por HPLC de exclusão molecular e fase reversa, SDS-PAGE e focalização isoelétrica. Quando analisadas quanto à hidrofobicidade, as subunidades mostraram-se aproximadamente iguais, enquanto as subunidades &beta; dos três heterodímeros apresentaram a seguinte escala de hidrofobicidade: &beta;-hFSH < &beta;-hTSH < &beta;-hLH. Com relação à massa molecular relativa (Mr), as subunidades &alpha; e &beta; do hFSH apresentaram as maiores Mr enquanto as subunidades do hLH as menores. A distribuição dos isômeros de carga das subunidades dos três hormônios ocorreu em uma região ácida, para o hFSH, em uma região básica, para o hLH e em uma região intermediária, para o hTSH. As subunidades &alpha; dos três hormônios, quando analisadas via SDS-PAGE, apresentaram praticamente a mesma mobilidade eletroforética, enquanto as subunidades &beta; apresentaram diferentes taxas de migração (mR), sendo mR &beta;-hFSH < mR &beta;-hTSH < mR &beta;-hLH. Diferenças relativas à massa molecular, hidrofobicidade, migração eletroforética e distribuição de carga foram encontradas entre as preparações recombinantes e hipofisárias dos três hormônios. O método descrito é suave, prático e flexível e pode ser adaptado à dissociação de outras glicoproteínas heterodiméricas recombinantes ou nativas. Permite não só estudos e caracterização direta de cada subunidade, como também detectar a presença de subunidades livres em preparações farmacêuticas, que são contaminantes indesejáveis, sendo, portanto, uma ferramenta extremamente útil para o controle de qualidade de produtos farmacêuticos. / In this work a practical and efficient method for the dissociation into &alpha;-and &beta;-subunits of small amounts (microgram range) of pituitaryderived and recombinant human follicle-stimulating hormone (hFSH), human luteotropin (hLH) and human thyrotropin (hTSH) is described. Dissociation was achieved by overnight treatment of the glycoproteins, at 37ºC, with acetic acid in different concentrations: 3M, 5M and 0,4M for hFSH, hLH and hTSH respectively. In these conditions, a dissociation efficiency of > 98% was attained. This efficiency was calculated on the basis of relative mass determinations of the heterodimers and subunits carried out via mass spectrometry (MALDI-TOF-MS). The &alpha;-and &beta;-subunits were rapidly and quantitatively separated by reversed-phase high-performance liquid chromatography (RP-HPLC) on a C4 column with yields of the order of 80-90%. The isolated subunits were characterized concerning their purity, hidrophobicity, molecular mass and charge distribution, via size exclusion and RP-HPLC, SDS-PAGE and isoelectric focusing. When analyzed with relation to the hydrophobicity, the &alpha;-subunits presented approximately the same hydrophobicity, while &beta;-subunits showed the following scale: &beta-hFSH < &beta;-hTSH < &beta;-hLH. Concerning molecular mass, &alpha;- and &beta;-subunits of hFSH were shown to have the highest while hLH subunits the lowest. Charge isomers of the subunits of the three glycohormones were predominantly distributed in an acidic region for hFSH, in a basic region for hLH, and in a wider pH range (acidic and basic) for hTSH. Similar migration rates (mR), analyzed via SDS-PAGE, were observed for the &alpha;-subunits of the three hormones. A greater variation was found for the &beta;-subunits: mR &beta;-hFSH < mR &beta;-hTSH < mR &beta;-hLH. Differences between recombinant and pituitary preparations of three hormones were observed with relation to molecular mass, hydrophobicity, electrophoretic migration and charge distribution. The described method is mild, practical and flexible and can be adapted to dissociate any recombinant or native heterodimeric glycoprotein, allowing studies and direct characterization of each subunit as well as the detection of free subunits that are undesired contaminants in pharmaceutical preparations, being also an extremely useful tool for the quality control of pharmaceutical products.

Neste trabalho é descrito um método prático e eficiente para dissociar, em subunidades &alpha; e &beta;, quantidades pequenas (da ordem de microgramas) dos hormônios foliculotrofina (hFSH), luteotrofina (hLH) e tireotrofina (hTSH) humana, nativos e recombinantes. A dissociação destes hormônios foi conseguida incubando-os, durante 16 horas, a 37ºC, com diferentes concentrações de ácido acético: 3M, 5M e 0,4M respectivamente para o hFSH, hLH e hTSH. Nestas condições, uma eficiência de dissociação acima de 98% foi obtida. Esta eficiência foi calculada com base nas determinações de massa dos heterodímeros e das subunidades, realizadas por MALDI-TOF-MS. Uma separação rápida e quantitativa das subunidades, com rendimentos da ordem de 80-90%, foi conseguida por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) em uma coluna C4. As subunidades foram caracterizadas quanto à pureza, hidrofobicidade, massa molecular e distribuição de carga por HPLC de exclusão molecular e fase reversa, SDS-PAGE e focalização isoelétrica. Quando analisadas quanto à hidrofobicidade, as subunidades mostraram-se aproximadamente iguais, enquanto as subunidades &beta; dos três heterodímeros apresentaram a seguinte escala de hidrofobicidade: &beta;-hFSH < &beta;-hTSH < &beta;-hLH. Com relação à massa molecular relativa (Mr), as subunidades &alpha; e &beta; do hFSH apresentaram as maiores Mr enquanto as subunidades do hLH as menores. A distribuição dos isômeros de carga das subunidades dos três hormônios ocorreu em uma região ácida, para o hFSH, em uma região básica, para o hLH e em uma região intermediária, para o hTSH. As subunidades &alpha; dos três hormônios, quando analisadas via SDS-PAGE, apresentaram praticamente a mesma mobilidade eletroforética, enquanto as subunidades &beta; apresentaram diferentes taxas de migração (mR), sendo mR &beta;-hFSH < mR &beta;-hTSH < mR &beta;-hLH. Diferenças relativas à massa molecular, hidrofobicidade, migração eletroforética e distribuição de carga foram encontradas entre as preparações recombinantes e hipofisárias dos três hormônios. O método descrito é suave, prático e flexível e pode ser adaptado à dissociação de outras glicoproteínas heterodiméricas recombinantes ou nativas. Permite não só estudos e caracterização direta de cada subunidade, como também detectar a presença de subunidades livres em preparações farmacêuticas, que são contaminantes indesejáveis, sendo, portanto, uma ferramenta extremamente útil para o controle de qualidade de produtos farmacêuticos. / In this work a practical and efficient method for the dissociation into &alpha;-and &beta;-subunits of small amounts (microgram range) of pituitaryderived and recombinant human follicle-stimulating hormone (hFSH), human luteotropin (hLH) and human thyrotropin (hTSH) is described. Dissociation was achieved by overnight treatment of the glycoproteins, at 37ºC, with acetic acid in different concentrations: 3M, 5M and 0,4M for hFSH, hLH and hTSH respectively. In these conditions, a dissociation efficiency of > 98% was attained. This efficiency was calculated on the basis of relative mass determinations of the heterodimers and subunits carried out via mass spectrometry (MALDI-TOF-MS). The &alpha;-and &beta;-subunits were rapidly and quantitatively separated by reversed-phase high-performance liquid chromatography (RP-HPLC) on a C4 column with yields of the order of 80-90%. The isolated subunits were characterized concerning their purity, hidrophobicity, molecular mass and charge distribution, via size exclusion and RP-HPLC, SDS-PAGE and isoelectric focusing. When analyzed with relation to the hydrophobicity, the &alpha;-subunits presented approximately the same hydrophobicity, while &beta;-subunits showed the following scale: &beta-hFSH < &beta;-hTSH < &beta;-hLH. Concerning molecular mass, &alpha;- and &beta;-subunits of hFSH were shown to have the highest while hLH subunits the lowest. Charge isomers of the subunits of the three glycohormones were predominantly distributed in an acidic region for hFSH, in a basic region for hLH, and in a wider pH range (acidic and basic) for hTSH. Similar migration rates (mR), analyzed via SDS-PAGE, were observed for the &alpha;-subunits of the three hormones. A greater variation was found for the &beta;-subunits: mR &beta;-hFSH < mR &beta;-hTSH < mR &beta;-hLH. Differences between recombinant and pituitary preparations of three hormones were observed with relation to molecular mass, hydrophobicity, electrophoretic migration and charge distribution. The described method is mild, practical and flexible and can be adapted to dissociate any recombinant or native heterodimeric glycoprotein, allowing studies and direct characterization of each subunit as well as the detection of free subunits that are undesired contaminants in pharmaceutical preparations, being also an extremely useful tool for the quality control of pharmaceutical products.

Neben der Sicherheit für Lebensmittel stehen auch immer mehr die Lebensmittelverpackungen im Fokus der Öffentlichkeit. Der Übergang von Stoffen aus der Verpackung in das Lebensmittel ist unerwünscht und gesetzlich reglementiert. Um den Verbraucherschutz zu gewährleisten, müssen Grenzwerte und gesetzliche Anforderungen eingehalten werden. Der Übergang von rechtlich geregelten und nicht geregelten Substanzen muss überprüft werden, was eine analytische Herausforderung darstellt. Die Untersuchung der migrierenden stickstoffhaltigen Substanzen aus Doseninnenbeschichtungen mittels eines Screenings aller migrierenden nicht-flüchtigen stickstoffhaltigen Substanzen mit einer molaren Masse kleiner 1000 Da wurde durchgeführt. Die Anwendbarkeit eines Stickstoff-selektiven Detektors für das Screening von Coating-Extrakten, welche stickstoffhaltige Verbindungen enthalten konnte gezeigt werden. Gegenstand der Untersuchung waren Vernetzersubstanzen, Flüssiglacke sowie Migrate der fertigen Beschichtung. Stickstoffhaltige potenziell migrierende Substanzen wurden zunächst in den Ausgangsmaterialien der Beschichtung identifiziert, um diese dann im Migrat der Beschichtung zu quantifizieren. Es sollte gezeigt werden, ob Substanzen, welche als Ausgangsstoffe im Lack eingesetzt werden, oder entstehende Reaktionsprodukte in ein Lebensmittelsimulanz migrieren. Um die Relevanz der migrierenden stickstoffhaltigen Verbindungen im Hinblick auf weitere nicht stickstoffhaltige migrierende Verbindungen zu zeigen, wurde das Gesamtmigrat der zur Verfügung stehenden Coatings bestimmt. Es zeigte sich, dass der Anteil von NCS an den insgesamt migrierenden Verbindungen zwischen 0,2 und 6,3 % liegt. Der Fokus des zweiten Teils der vorliegenden Arbeit liegt auf Lebensmittelverpackungen aus Kunststoff. Zunächst wurde eine HPLC-Methode mit Hilfe des Verdampfungslichtstreudetektors zur Bestimmung der Gesamtmigration mit dem Simulanz Sonnenblumenöl etabliert werden. Das Ziel dieser Untersuchungen ist, den Einfluss von Temperatur, Zeit und Schichtdicke auf das Migrationsverhalten von Siegelschichten für den Hochtemperaturbereich (> 70 °C) mit fetthaltigen Lebensmitteln mit Hilfe von statistischer Versuchsplanung vorherzusagen. Mit Hilfe einer statistischen Software konnte eine Regressionsgleichung zur Berechnung der Gesamtmigration auf der Grundlage eines Box-Behnken-Versuchsplans erstellt werden. Dabei hatte die Temperatur den größten Einfluss auf die Gesamtmigration. Die Einflüsse von Zeit und Schichtdicke waren im untersuchten Bereich des hier gezeigten Modells linear und stiegen mit Erhöhung der Temperatur. Weiterhin konnte je 10 °C Temperaturerhöhung eine Verdopplung des ermittelten Gesamtmigrationswertes beobachtet werden. Die Bestimmung der Additive aus den Ersatzsimulanzien 95 % Ethanol und Iso-Octan von Verpackungen sollte ebenfalls gezeigt werden. Ein Screening-Gradient zur Bestimmung von 25 Additiven in den Ersatzsimulanzien wurde etabliert. Die Identifizierung der migrierenden Additive erfolgte mittels der Detektoren UVD (DAD), FLD, ELSD und CLND. Mit Hilfe der verschiedenen Detektionsarten ist es möglich, die strukturelle Vielfalt der eingesetzten Additive abzudecken. Eine Absicherung der Ergebnisse konnte zudem über MS-Detektion erfolgen. Mit Hilfe der Untersuchungen wurden die gesamtmigrierenden Substanzen aus Verbundfolien zu 50 % (95 % Ethanol-Migrat) bzw. 10 % (Isooctan-Migrat) aufgeklärt. Die Konzentration der quantifizierten Additive zeigte im Verhältnis gesehen annähernd gleiche Werte. Der Unterschied in den ermittelten Gesamtmigraten (95 % Ethanol: 1,2 mg/dm2, Iso-Octan: 5,6 mg/dm2) konnte demnach nicht über die migrierenden Additive erklärt werden. Als weitere migrierende Substanzen wurden Ethylen-Oligomere identifiziert. Die Quantifizierung dieser erfolgte erstmals mit Hilfe der 1H-NMR-Spektroskopie. Die nahezu vollständige Aufklärung der Gesamtmigration einer Verbundfolie in den Ersatzsimulanzien konnte gezeigt werden. Die migrierenden Ethylen-Oligomere des Iso-Octan-Migrats wurden eingehender untersucht. Mit Hilfe von verschiedenen chromatographischen und spektroskopischen Methoden gelang eine Charakterisierung dieser im Migrat identifizierten Substanzen. / Besides the safety of food the focus on food packaging material increases in public. The migration of substances from the packaging into food is undesired and regulated by law. To ensure consumer protection legal limits and requirements have to be kept. The migration of regulated und not regulated substances has to be verified which means an analytical challenge. The determination of nitrogen containing substances (NCS) from food can coatings by screening of migrating, non-volatile substances with a molecular mass below 1000 Da from the coatings was carried out. The applicability of a nitrogen selective detector for the screening of coating extracts which contain nitrogen containing susbtances was shown. For the investigations crosslinking substances, liquid lacquers as well as migrates of the finished coatings have been available for determination. Nitrogen containing and potential migrating substances have been identified first in the raw marterial of the coating in order to quantify them in the migrates of the coating. It should be shown if substances from the raw materials or reaction products migrate into the food simulant. In order to show the relevance of the migrating nitrogen containing substances in respect to other non nitrogen containing compounds the overall migration of the available coatings was determined. It could be shown that the amount of NCS in the overall migrating substances was between 0.2 and 6.3 %. Focus of the second part of the work was on food packing made of plastic. First an HPLC-method with ELS detection for the determination of the overall migration in sunflower oil was developed. Purpose of this determination was to predict the influence of temperature, time and thickness of the layer on the migration behavior with fatty food of sealing layers in high temperature range (> 70 °C) by means of design of experiments. A statistical software computed a regression equation for the calculation the overall migration based on a Box-Behnken-Design. The highest influence could be shown for the temperature. The modell showed a duplication of the determined overall migration with 10 °C increase of temperature. The determination of plastic additives out of the 95 % ethanol and isooctane migrates of packaging material should also be conducted. An HPLC-screening method for the determination of 25 additives in the fat substitutes was established. The identification of the migrating additives was carried out with UV detection (DAD), FLD, ELSD and CLND. By means of the different detection systems it was possible to cover the structural diversity of the mainly used additives. To insure the results MS detection was used. By means of this investigations a clarification of the total migrating substances of a multilayer film was 50 % (95 % ethanol) and 10 % (isooctane), respectively. The concentration of the migrating substances on the scale of things is nearly identical. The difference in the overall migration (95 % ethanol: 1.2 mg/dm2, isooctane: 5.6 mg/dm2) can not be clarified by migration of additives. As other migrating substances ethylen oligomers can be identified. The quantification was carried out for the first time with 1H﷓NMR spectroscopy. An almost complete identification of migration substances of the overall migrate in food simulants can be shown. The migrating ethylen oligomers have been further investitgated. With the help of different chromatographic and spectroscopic methods a further characterisation of the migrating ethylen oligomers was successul.