Int J Immunopathol Pharmacol. 2004 Jan-Apr;17(1):33-8.
Growth hormone antibodies formation in patients treated with recombinant human growth hormone.
Here's another study that references the one heavyiron listed:
proteomesci.com/content/3/1/1
Mass spectrometrical analysis of recombinant human growth hormone (Genotropin®) reveals amino acid substitutions in 2% of the expressed protein
Abstract
Background
The structural integrity of recombinant proteins is of critical importance to their application as clinical treatments. Recombinant growth hormone preparations have been examined by several methodologies. In this study recombinant human growth hormone (rhGH; Genotropin®), expressed in E. coli K12, was structurally analyzed by two-dimensional gel electrophoresis and MALDI-TOF-TOF, LC-MS and LC-MS/ MS sequencing of the resolved peptides.
Results
Electrospray LC-MS analysis revealed one major protein with an average molecular mass of 22126.8 Da and some additional minor components. Electrospray LC-MS/MS evaluation of the enzymatically digested Genotropin® sample resulted in the identification of amino acid substitutions at the residues M14, M125, and M170; di-methylation of K70 (or exchange to arginine); deamidation of N149, and N152, and oxidation of M140, M125 and M170. Peak area comparison of the modified and parental peptides indicates that these changes were present in ~2% of the recombinant preparation.
Conclusion
Modifications of the recombinant human growth hormone may lead to structural or conformational changes, modification of antigenicity and development of antibody formation in treated subjects. Amino acid exchanges may be caused by differences between human and E. coli codon usage and/or unknown copy editing mechanisms. While deamidation and oxidation can be assigned to processing events, the mechanism for possible di-methylation of K70 remains unclear.
And here's the information about how Genotropin may actually reverse the antibody issue:
labeling.pfizer.com/ShowLabeling.aspx?id=577
Anti-hGH Antibodies
As with all therapeutic proteins, there is potential for immunogenicity. The detection of antibody formation is highly dependent on the sensitivity and specificity of the assay. Additionally, the observed incidence of antibody (including neutralizing antibody) positivity in an assay may be influenced by several factors including assay methodology, sample handling, timing of sample collection, concomitant medications, and underlying disease. For these reasons, comparison of the incidence of antibodies to GENOTROPIN with the incidence of antibodies to other products may be misleading. In the case of growth hormone, antibodies with binding capacities lower than 2 mg/mL have not been associated with growth attenuation. In a very small number of patients treated with somatropin, when binding capacity was greater than 2 mg/mL, interference with the growth response was observed.
In 419 pediatric patients evaluated in clinical studies with GENOTROPIN lyophilized powder, 244 had been treated previously with GENOTROPIN or other growth hormone preparations and 175 had received no previous growth hormone therapy. Antibodies to growth hormone (anti-hGH antibodies) were present in six previously treated patients at baseline. Three of the six became negative for anti-hGH antibodies during 6 to 12 months of treatment with GENOTROPIN. Of the remaining 413 patients, eight (1.9%) developed detectable anti-hGH antibodies during treatment with GENOTROPIN; none had an antibody binding capacity > 2 mg/L. There was no evidence that the growth response to GENOTROPIN was affected in these antibody-positive patients.
I want to add the paragraph from that article I just posted that cites the article heavyiron posted. It potentially appears the article he posted may have been based upon rHGH that had structural integrity issues, and that ultimately led to the antibody response in the patients it cited. See here:
Background
The structural integrity of recombinant products generated by prokaryotic and eukaryotic organisms is a major concern. Modifications such as amino acid sequence substitution/mutations of recombinant proteins may lead to pharmacological inactivation, autoimmune phenomena [1-3] and adverse effects [4,5].
References
1: Ahangari G, Ostadali MR, Rabani A, Rashidian J, Sanati MH, Zarindast MR: Growth hormone antibodies formation in patients treated with recombinant human growth hormone.
Int J Immunopathol Pharmacol 2004, 17:33-38.
2: Takano K, Shizume K: Current clinical trials with authentic recombinant human growth hormone in Japan.
Acta paediatr Scand Suppl 1986, 93-97.
3: Massa G, Vanderschueren-Lodeweyckx M, Buillon R: Five-year follow-up of growth hormone antibodies in growth hormone deficient children treated with recombinant human growth hormone.
Clin Endocrinol (Oxf) 1993, 38:137-142.
4: Cutfield WS, Jackson WE, Jefferies C, Robinson EM, Breier BH, Richards GE, Hofman PL: Reduced insulin sensitivity during growth hormone therapy for short children born small for gestational age.
J Pediatr 2003, 142:113-116.
5: Sugimoto S, Hanaki K, Kawashima Y, Kinoshita T, Nagaishi J, Hayashi A, Kanzaki S: Aplastic anemia during growth hormone (GH) treatment in a girl with idiopathic GH-deficiency.
Endocr J 2003, 50:469-471.
Further reading:
Protropin (Somatrem, 192aa) literature stated:
As with all protein pharmaceuticals, a small percentage of patients may develop antibodies to the protein. Growth hormone antibody binding capacities below 2 mg/L have not been associated with growth attenuation. In some cases when binding capacity exceeds 2 mg/L, growth attenuation has been observed. In clinical studies and postmarketing experience of patients treated with Protropin® (somatrem for injection), approximately 0.4 percent of patients screened for antibody production developed antibodies with binding capacities > 2 mg/L at six months. Out of approximately 26,000 patients who have been treated with Protropin (somatrem) , 5 patients have had growth deceleration associated with binding capacities > 2 mg/L. If growth deceleration is observed that is not attributable to another cause, the patient should be tested for antibodies to growth hormone. Although no evidence exists to indicate that the methionine on the N-terminus of somatrem causes antibodies to growth hormone, the physician should consider transferring the patient to somatropin (rDNA origin) for injection, if a patient has antibody binding capacity > 2 mg/L, and has exhibited growth attenuation.
Humatrope (Somatropin, 191aa) literature stated:
As with all protein pharmaceuticals, a small percentage of patients may develop antibodies to the protein. During the first 6 months of Humatrope therapy in 314 naive patients, only 1.6% developed specific antibodies to Humatrope (binding capacity = to or >0.02 mg/L). None had antibody concentrations which exceeded 2 mg/L. Throughout 8 years of this same study, two patients (0.6%) had binding capacity >2 mg/L. Neither patient demonstrated a decrease in growth velocity at or near the time of increased antibody production. It has been reported that growth attenuation from pituitary***8211;derived growth hormone may occur when antibody concentrations are >1.5 mg/L.
And yet further readings in regards to HGH antibodies. The important aspects of each study are in red.
ncbi.nlm.nih.gov/pubmed/12434920
The use of an animal immunogenicity model in the development of Protropin somatrem (methionyl human growth hormone).
Abstract
The clinical development of methionyl human growth hormone, with particular emphasis on immunogenicity and the effects of antibody development, are summarized. In an animal model in rhesus monkeys, the immunogenicity of dinical preparations was reduced by the inclusion of additional purification steps in the manufacturing process. The immunogenic response in patients was also decreased by these improvements. No safety consequences related to antibody formation were observed and the occurrence of growth attenuation due to antibodies was found to be extremely low (<0.1%). The data suggest that the immunogenicity was not due to the N-terminal methionine or E. coli protein impurities: rather it was probably caused by small amounts of growth hormone with subtle structural alterations whose nature remains unknown.
ncbi.nlm.nih.gov/pubmed/3296632
Human growth hormone produced with recombinant DNA technology: development and production.
Abstract
The molecular basis of recombinant DNA technology is described, and the principles of genetically engineered proteins developed. The production of hGH by such methods utilizes a strain of Escherichia coli as host and a vector plasmid containing the appropriate information. Fermentation and purification of the hGH produced gives a preparation of high purity, containing only 1-2 ppm of E. coli polypeptide (ECP). This somatrem (Somatonorm) is identical to pituitary hGH except for an additional methionine residue at the N-terminal. Monoclonal antibodies fail to distinguish between pituitary hGH and somatrem. Preclinical studies of a variety of pharmacological and toxicological parameters indicate that the two hGH preparations have identical biological effects; no toxicological or mutagenic effects of somatrem have been detected.
ncbi.nlm.nih.gov/pubmed/2582244
Monoclonal antibodies to human growth hormone can distinguish between pituitary and genetically engineered forms.
Abstract
Monoclonal antibodies (MABs) prepared against human pituitary growth hormone (hGH) have been compared for their binding to pituitary-derived and genetically engineered methionyl growth hormone (met-hGH). The antibodies bind to four non-overlapping epitopes of which two are completely shared with human choronic somatomammotropin (hCS). The determinant defined by MAB NA27 was expressed on met-hGH to a lesser degree than on hGH of pituitary origin. However, another antibody, QA68, which binds to a determinant closely related to NA27, failed to discriminate between hGH and met-hGH. A further two MABs (EB1 and NA71) were similarly ineffective in distinguishing between the two forms of the hormone. The determinant recognized by antibody EB2 was equally represented on hGH and met-hGH when assessed by a liquid-phase radioimmunoassay: however, measurement of the binding in a solid-phase assay resulted in a two-four-fold lower binding to met-hGH. Bioactivity assessed by both an in vitro cell proliferation assay and an in vivo cartilage sulphation bioassay failed to distinguish between the two hormones. It is therefore concluded that the NH2-terminal methionine on bacterially derived growth hormone results in altered antigenicity of the hormone without any measurable effect on bioactivity.
