RESUMEN
OBJECTIVE: We examined the charge heterogeneity and carbohydrate complexity of hCG in healthy pregnant Asian subjects and Asian women with gestational thyrotoxicosis to assess whether particular glycoforms of hCG are associated with the thyrotoxicosis of pregnancy. DESIGN: Blood was taken at 8-16 weeks of gestation from five pregnant Asian women with clinical symptoms of gestational thyrotoxicosis and biochemical indicators of hyperthyroidism and from six age-matched healthy pregnant women. hCG charge heterogeneity and carbohydrate complexity were assessed by fast protein liquid chromatography chromatofocusing and concanavalin A affinity chromatography, respectively. The degree of terminal sialylation of the different glycoforms was measured by ricin affinity chromatography, which detects exposed galactose residues following desialylation. RESULTS: Free T3, free T4 and hCG levels were elevated and TSH suppressed in the thyrotoxic subjects compared to controls (P < 0.007). Overall, the glycoform distribution of the hCG in the blood from the control and thyrotoxic groups was similar, with a median pI of 4.34 (median and range: 3.78-4.68) and pI 4.23 (3.98-4.38). Free T4 (P < 0.028) and free T3 (P < 0.03) levels were positively correlated to both the absolute hCG concentration and the percentage of hCG forms between pI 3.36-4.0. The distribution of simple (73-88.6%), intermediate (9.7-26.4%) and complex (0.1-7.3%) branching oligosaccharide forms was similar in both groups, as was the percent hCG which bound to ricin (< 3.2%). CONCLUSION: We conclude that excessive thyroid stimulation in the thyrotoxic patients is associated both with the absolute concentration of hCG and the relative proportion of acidic glycoforms between pI 3.36 and 4.0.
Asunto(s)
Gonadotropina Coriónica/sangre , Complicaciones del Embarazo/sangre , Tirotoxicosis/sangre , Adulto , Gonadotropina Coriónica/química , Cromatografía de Afinidad , Cromatografía Liquida , Femenino , Humanos , Concentración de Iones de Hidrógeno , Embarazo , Tirotropina/sangre , Tiroxina/sangre , Triyodotironina/sangreRESUMEN
The immunopotency and in-vitro biopotency of clinical batches of Gonal-F((R)) and Puregon((R)) (recombinant human follicle stimulating hormones) were compared and their carbohydrate chains investigated for charge heterogeneity and internal carbohydrate complexity. Immunopotency (IU/pmol) for both Gonal-F and Puregon was 0.35 +/- 0.01 and biopotency (ED(50), pmol/l) was similar, being 7.3 +/- 0.6 and 5.4 +/- 0.2 respectively. Charge distributions were essentially the same with no difference either in median isoelectric point (pI) (between 4.26 and 4.50), or in the bulk of material fractionated between pI 4 and 5 (66.0 +/- 1.8% Gonal-F and 72.0 +/- 1.8% Puregon). However, there were minor differences in charge at extremes of pI, Gonal-F being slightly more acidic: 18.2% Gonal-F versus 9.8% Puregon at pI 3.5-4.0 (P: = 0.03) and 6.7% Gonal-F versus 10.7% Puregon at pI 5.0-5.5 (P: = 0.03). Carbohydrate complexity was the same: 9.3 versus 10.9 (complex), 76.6 versus 78.6 (intermediate) and 14.1 versus 10.5% (simple). In summary, Gonal-F and Puregon have similar immunopotency, in-vitro biopotency and internal carbohydrate complexity, differing slightly in charge heterogeneity, Gonal-F having more acidic glycoforms. We conclude them to be intrinsically very similar, expecting no difference in clinical efficacy on the basis of respective structure.
Asunto(s)
Hormona Folículo Estimulante , Proteínas Recombinantes , Animales , Carbohidratos/química , Fenómenos Químicos , Química Física , Hormona Folículo Estimulante/química , Hormona Folículo Estimulante/inmunología , Hormona Folículo Estimulante/farmacología , Hormona Folículo Estimulante Humana , Humanos , Punto Isoeléctrico , Masculino , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacologíaRESUMEN
Glycoforms of recombinant human follicle stimulating hormone (rhFSH) (Org 32489, Puregon) were characterized using concanavalin A lectin affinity chromatography to reveal information about the internal carbohydrate complexity (extent of carbohydrate side-chain branching) of the preparations. The rhFSH glycoforms were measured by radioimmunoassay and a two-site immunoradiometric assay and compared with those in two urinary preparations (Metrodin and Metrodin-HP) used in assisted reproduction programmes and a urinary FSH international standard 70/45 (uFSH IS 70/45). Similar data were obtained with both assays; rhFSH had 6% complex internal carbohydrate structures compared with 22-27% for Metrodin, Metrodin-HP and uFSH. The proportion of simple carbohydrate structures was also different, with rhFSH having 18.5 compared with 4.5-9.3% for Metrodin, Metrodin-HP and uFSH. A linear relationship was observed between the percentage glycoforms with an isoelectric point (pl) < 4 and the log percentage simple forms (logarithmic regression; r = 0.93) indicating a direct relationship between carbohydrate complexity and charge heterogeneity. In summary, rhFSH contains fewer complex forms and an increased proportion of simple carbohydrate structures in comparison with Metrodin, Metrodin-HP and IS 70/45.
Asunto(s)
Carbohidratos/análisis , Hormona Folículo Estimulante/química , Cromatografía de Afinidad , Concanavalina A , Hormona Folículo Estimulante/orina , Hormona Folículo Estimulante Humana , Glicosilación , Humanos , Radioinmunoensayo , Proteínas Recombinantes/química , Estándares de ReferenciaRESUMEN
The aim of this study was to partially characterize the glycoform composition of a recombinant human luteinizing hormone preparation (rhLH; Serono), an early version of the material (LHadi) which is currently being assessed for clinical application. Specifically, the charge (pl) and internal carbohydrate complexity of this rhLH was examined and compared with that of an alternative commercially available form of recombinant LH (Crystal Chem) and a pituitary International Reference Preparation (IRP). All preparations were separated by charge by chromatofocusing them on a pH gradient (7-4) using a 4 ml mono-P column is conjunction with a fast performance liquid chromatography system and by complexity of the oligosaccharide structures using concanavalin A (con-A) lectin affinity chromatography. LH in both the unfractionated and fractionated material was assessed by immunoradiometric assay (IRMA, I-LH) and by the in-vitro Leydig cell bioassay (B-LH). Both assays were calibrated against IRP 80/552. The in-vitro biopotency of the preparations was 18187 (Serono rhLH), 12063 (Crystal Chem rhLH) and 6658 (80/552) IU/mg; biological:immunological ratios were 1.14 (80/552), 1.90 (Crystal Chem rhLH) and 1.99 (Serono rhLH). However, similar qualitative data were obtained by both bioassay and immunoradiometric assay following fractionation, with the median pl of the bioactive LH in the preparations being 5.5 (24% > pH 6), 5.52 (18% > pH 6) and 4.97 (0% > pH 6) for the Serono, Crystal Chem and pituitary preparations respectively. Further all three contain < 1% of the complex carbohydrate structures and between 36-44% and 56-63% of the intermediate and simple forms of bioactive LH. In conclusion, the Serono recombinant LH preparation has a higher in-vitro bioactivity and is more basic than the other two preparations although the complexity of its carbohydrate moities appears to be similar.
Asunto(s)
Hormona Luteinizante , Animales , Células CHO , Cromatografía de Afinidad , Clonación Molecular , Concanavalina A , Cricetinae , Humanos , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Células Intersticiales del Testículo/efectos de los fármacos , Hormona Luteinizante/química , Hormona Luteinizante/genética , Hormona Luteinizante/inmunología , Hormona Luteinizante/aislamiento & purificación , Hormona Luteinizante/farmacología , Masculino , Ratones , Radioinmunoensayo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The within- and between-batch variation in the immunoreactive and in-vitro bioactive FSH content of Pergonal, Metrodin and Metrodin-HP was investigated. Three batches of Pergonal and Metrodin, consisting of three ampoules in each batch, and three batches of Metrodin-HP, consisting of between one and three ampoules per batch, were selected at random. The follicle stimulating hormone (FSH) content of Pergonal, Metrodin and Metrodin-HP was determined by radioimmunoassay (R-FSH) and the in-vitro rat Sertoli cell bioassay (B-FSH) using the international urinary standard 70/45. The variability in the FSH content of the preparations was evaluated within and between batches by analysis of variance. Within-batch variability of B-FSH was not observed in Pergonal or Metrodin-HP but was seen in two batches of Metrodin in which the potency varied by up to 2.4 fold (P = 0.03). The between-batch R-FSH potencies of Pergonal (P1-P3) varied, with P2 (59.8 +/- 0.6) and P3 (61.7 +/- 0.9) being higher than P1 (47.1 +/- 1.5 mean +/- SEM IU/ampoule, P < 0.01). A similar pattern of variability was observed for B-FSH. For Metrodin, each of the batch R-FSH potencies was dissimilar (P < 0.02), with estimates ranging from 34.9 +/- 1.2 to 64.3 +/- 1.8 IU/ampoule. Furthermore, the extensive within-batch B-FSH variation from two batches confounded any meaningful comparison of between-batch variability. For Metrodin-HP, there was no between-batch B-FSH variation (29.0 +/- 6.1 to 33.0 +/- 0.3 IU/ampoule) and the R-FSH content was also well controlled (41.2 +/- 0.5 to 46.9 +/- 1.1 IU/ampoule).(ABSTRACT TRUNCATED AT 250 WORDS)