**4. Discussion**

In European Union, the production and the quality of medical products for both human and animal use is strongly regulated and controlled from both national

health agencies and qualified bodies and from the European Medicines Agency. Companies producing medicines and medical devices must follow strict and detailed guidelines and secure that all operations are performed under governing GMP and GLP rules. These precautions shall secure the quality of the product and the safe use for the patients. Thennati et al. has described the method for the quality control of the recombinant product is ensured through analytical steps using SDS-gel separation and MALDI-ToF analysis of the final product [17]. Therefore, it was a great surprise to identify a number of proteins originating from the starting product (urine) in different batches of the final product.

A discussion on contaminant proteins in hCG formulations has already been published [4, 5, 7, 17–19], and several publications address the possibility of the presence of harmful substances in commercial formulation [13–15, 20–22] but no final decision was made and the urinary-derived hCG formulations are still widely used although it has been shown that recombinant hCG can be used with the same success rate.

Due to the lack of published data and reports on hCG formulations and the lack of information about these products, we have analyzed commercially available formulations that are routinely prescribed for patients undergoing IVF treatment.

The major active component, hCG, was identified in all analyzed samples and, in addition, human serum albumin (HSA), luteotropin subunit beta (LSHB), and glycoprotein hormones alpha chain (CGA). The presence of HSA in all samples can be explained by its secretion in urine and by the need of growing CHO cells, for recombinant hCG production, in culturing medium supplemented with HSA, which might contain a number of other proteins that were not removed when HSA purification was performed. The pathway and interaction analysis, shown in **Figure 3**, explains the dependence of co-expression between the hCG, LHB, and CGA and explains why these proteins can also be identified in recombinant products. Obviously, the production of hCG also induces the expression of other proteins involved in ovarian steroidogenesis, hormone activity, and regulation of hormone levels, which are identified with high confidence with MS/MS analysis. However, in this case, the identification of LHB is a false positive one, and we cannot claim that this protein is really present in the recombinant sample. The reason is that hCG and LHB have a common amino acid sequence between amino acids on positons 22

#### **Figure 3.**

*Pathway describing the interdependence of hCG and LHB both of which were identified in recombinant products.*

**17**

**Figure 4.**

*their abundancies in biological processes.*

*Background Proteins in Human Chorionic Gonadotropin Pharmaceutical Formulations…*

and 131 in the protein backbone. If other parts of the sequence cannot be identified, we cannot tell the proteins apart. However, in this case, the identification score and the identification of other amino acids in the hCG sequence show an unique

As for the contaminants in urinary-derived products, uromodulin, which is also the major urinary protein, was a major hit following the major component. However, other proteins, such as alpha-1-microglobulin or apolipoprotein D, and prostaglandin were identified with high confidence. The contaminants in urinaryderived hCG formulations resemble urinary proteins identified in urine-only samples, which were described in a recently published study on stress-induced

Keratin was one of the major alien contaminants, and its presence suggests a contamination during the sample production and the origin might be the insufficient air-conditioning or the contamination of the packaging units, which might be traced back to the use of latex-made gloves, dust containing skin particles, etc. We can exclude the contamination in our lab since all sample preparation steps have been performed using the laminar flow and using nitrile gloves. The blank samples

Additional distinction between the urinary and the recombinant products can be seen when looking at the GO analysis of the biological processes where these

Products originating from the production process employing recombinant method have significantly less contaminant proteins of human origin. To our best knowledge, until now, there are no reports describing contaminations for recombinant hCG. Upon analysis, the hCG was the major component identified with contaminants such as keratin and human serum albumin (HSA). These contaminants are easy to explain, we have given an explanation for keratin in previous section, and HSA is most probably a contaminant from the culturing medium of CHO that

*GO term analysis of proteins identified in both urinary and recombinant formulations showing differences in* 

*DOI: http://dx.doi.org/10.5772/intechopen.82652*

 **4.1 Contaminant proteins in urinary-derived samples**

show no identifications of keratin or of other contaminations.

proteins are involved, as shown in **Figure 4**.

 **4.2 Contaminant proteins in recombinant samples**

was not completely removed upon hCG extraction.

identification.

urinary incontinence [23].

and 131 in the protein backbone. If other parts of the sequence cannot be identified, we cannot tell the proteins apart. However, in this case, the identification score and the identification of other amino acids in the hCG sequence show an unique identification.
