New Horizons in Quality Control of Enzyme Pharmaceuticals: Combining Dynamic Light Scattering, Fourier-Transform Infrared Spectroscopy, and Radiothermal Emission Analysis

Comments 1: The first third of the Introduction (Section 1.1) is essentially a general review of hyaluronic acid that is only loosely connected to the core topic—quality control of hyaluronidase pharmaceuticals. This part should be significantly shortened; only the most relevant aspects (molecular-weight-dependent functions and their relation to fibrosis, oncology, and osteoarthritis) need to be retained.

Response 1: Thank You for Your comment. We have revised section 1.1 and removed some of the unnecessary information on hyaluronic acid, namely deleted paragraphs: “In the context of biodistribution, hyaluronan is a widespread component of the ex-tracellular matrix of various types of connective tissue. The main localizations of HA are as follows: tendons, cartilage tissue, and skin. Of these, skin, or more precisely the con-nective tissue of the dermis, accounts for over 50% of the total pool of hyaluronan in the body. Furthermore, significant concentrations of HA have been identified in the synovial fluid of joints and a number of other biological media [3]”;

“Hyaluronan deficiency has also been identified as a contributing factor in the de-velopment of other diseases. For example, in type 2 diabetes mellitus and atherosclerosis, reduced HA content in the vascular wall leads to endothelial dysfunction, increased vascular permeability, and accelerated formation of atherosclerotic plaques [33,34]. In the field of dermatology, hyaluronan deficiency has been clinically linked to accelerated skin ageing and reduced skin hydration. The clinical signs of this condition include a loss of elasticity and morphological changes in the dermis [35,36].”

“The pathological conditions described above were caused by an enzymatic degra-dation of hyaluronan. However, an alternative degradation pathway has been observed. For instance, HA is susceptible to degradation by hydroxyl radicals, peroxynitrite, and hypochlorite anions, which can be formed in vivo, and their concentration increases during inflammation [37]. Oxidative degradation of hyaluronan leads to a disruption of the protective function of this substance, with the result that the binding of active forms of oxygen and nitrogen formed during inflammatory pathologies is affected [38].”

“Sodium hyaluronate is a salt form of HA that has been shown to restore the viscoelastic properties of synovial fluid, improve joint function, and reduce the severity of inflammation [33,34]”.

Comments 2: The term “bovine hyaluronidase azoximer” has a strong non-native phrasing and should be uniformly replaced throughout the manuscript with the standard scientific nomenclature “azoximer-conjugated hyaluronidase” (or “azoximer–hyaluronidase”). Using the registered trade name “Longidaze®” for the modified product and “Lidaza®” for the conventional hyaluronidase products is strongly recommended, as these are widely recognised by the pharmaceutical community.

Response 2: Thank You very much for Your comment, we have changed the terms you suggestedthroughout the manuscript.

Comments 3: Section 2.1 provides only Russian registration numbers. The commercial/trade names, manufacturers, batch numbers, and expiry dates of the tested products must be explicitly stated to ensure traceability and reproducibility.

Response 3: Thanks a lot for Your comment, we added information about the manufacturer and the country of origin (See lines – 197, 200 and 203). However, since this work is scientific, the team of co-authors requests that international nonproprietary names be retained in the article, as they are unique and recognized by WHO for certain active substances. We indicate in the comments batch numbers for “Lidaza®” from LLC “Samson-Med” – 90923 (exp. date till 09.25) , for “Longidaze®” from LLC “NPO Petrovax” – 451121 (exp. date till 11.23), for “Lidaza®” from JCS “NPO Microgen” – P140322 (exp. date till 02.25). Research began back in the 21st year, starting with the study of azoximer conjugated hyaluronidase preparations, so batch numbers begin since 2021.

Comments 4: Shelf-life correlation using RTE was performed only for the azoximer-conjugated product (Longidaze®/BovHYAL). No expired samples of conventional hyaluronidase (Lidaza®) were examined, resulting in an obvious imbalance. This limitation should either be addressed experimentally or clearly acknowledged and justified in the text.

Response 4: Thank You very much for Your comment! We have added a paragraph explaining why we chose the Longidaze® preparation to study of changes in quality characteristics without opening the primary packaging. Lines 404-412.

Comments 5: The conclusions are severely overstated. Phrases such as “The study resolved the issue …” and claims that the proposed methods are “dependable and sensitive approaches” constitute overstatement. The work merely explores and preliminarily evaluates these techniques; it is far from resolving a recognised analytical challenge in the field. All such absolute claims must be toned down to appropriate wording (e.g., “suggests”, “offers a promising supplementary tool”, “preliminary results indicate …”).

Response 5: Thank You very much for Your comment! We rewrote the sentence in Abstract. See lines 25-26.

Comments 1: Figure 3. The disaccharide fragment produced by hyaluronidase has the structure D-glucuronic acid linked to N-acetyl D glucosamine. The brackets in Figure 3 are in the wrong place as they do not show the disaccharide produced by hyaluronidase cleavage.

Response 1: Thank you for your comment. This figure shows a molecule of hyaluronic acid in the form of a structural chemical formula. Hyaluronic acid consists of repeating disaccharide units. Therefore, these brackets do not indicate in any way the hyaluronic acid fragments cleaved by hyaluronidase. Cleavage is shown using scissors, which are aimed at the beta 1,4 bond, through which hyaluronic acid is cleaved by hyaluronidase.

Comments 2: Figure 3. There are extra lines in the sugar links, suggesting the presence of a CH2 group where there should be none. Please modify the drawing to remove lines that suggest the sugars are linked through OCH₂

Response 2: Thank You for Your comment. If we understood your question correctly, then we are referring to the relationship between the two carbohydrates (the oxygen bridge). Given that N-acetyl D glucosamine and D-glucuronic are linked by a beta 1,4 bond, there can certainly be no question of the presence of an additional CH2 bond. The figure shows a classic representation of the visualization of beta and alpha bonds for carbohydrates. To confirm that such an image is relevant, I am attaching accompanying images from other articles and textbooks. Of course, the view without bending the oxygen bridge can also be used to visualize the chemical structure of a molecule. However, bearing in mind the effect of hyaluronidase specifically on the beta 1,4 bond of hyaluronic acid during its cleavage. We believe that it would be quite appropriate to show this in the following way.

DOI: 10.1016/B978-0-444-64081-9.00009-7

DOI: 10.2174/187221108786241651

Comments 3: Page 5 line 181. Please rewrite the phrase to read “to study the enzyme activity of the protein”

Response 3: Thank You for Your comment, the articles provided by the references describe studies on the IR analysis of the effect of hyaluronidase on proteins. Therefore, the proposal was rewritten as follows: “A number of articles also mention the use of IR spectroscopy to study the effect of enzyme on proteins”. Line 181.

Comments 4: Three hyaluronidases were characterized. One was bovine hyaluronidase, but the species of the other two is not stated. Are they human hyaluronidases?  If they are recombinant hyaluronidases, were they expressed in E.coli?

Response 4: Thank You for Your question! All three hyaluronidases chosen for research in this article are commercially available pharmaceuticals. According to the information provided by the manufacturer, all three enzymes are derived from the testes of bovine animals. We have not synthesized or obtained any enzymes during the experimental part of the work. See lines 132-133.