Investigation of the Charge Variant Profile of Non-cleavable Conjugated Antibodies

Ayat Abbood¹, Hassan Hadi Ali², Samir Azzat Malik³, Dima AlDiab⁴, Nasser Thallaj⁵*

¹Department of Medicinal Chemistry and Quality Control, University of Tishreen, Lattaquia, Syria.

²University of Al Mashreq, College of Engineering Technology, Baghdad, Iraq.

³MSC Pharmaceutical Chemistry, College of Pharmacy, University of Mustaqbal, Babylon, Iraq.

⁴Department of Food and Anlytical Chemistry, University of Tishreen, Lattaquia, Syria.

⁵Department of Medicinal Chemistry and Quality Control, University of AL-Rachid, Damascus, Syria.

*Corresponding Author E-mail: profthallaj@gmail.com

ABSTRACT:

This research aimed to analyze the charge variant profiles of monoclonal antibodies (mAbs) conjugated to maytansine derivatives or tomaymycin compounds via a non-cleavable linker, utilizing the imaging capillary isoelectric focusing (icIEF) technique. Initially, the charge variant profiles for three mAbs were assessed, revealing both major and minor variants among the samples. Specifically, mAB1 and mAB2 displayed two distinct charge variants with isoelectric points (pI) of 9.00 and 8.95, respectively. In contrast, mAB3 showed a predominant charge variant with a pI of 8.50, along with two minor variants (pI values of 8.30 and 8.60). The conjugation of mAB1 involved a maytansine derivative using non-cleavable linkers, while mAB2 and mAB3 were linked to tomaymycin molecules. The resulting non-cleavable antibody conjugates exhibited greater heterogeneity and acidity than their unconjugated forms. The observed pI ranges were 7.4 to 8.9 for mAB1 conjugates (∆pI: 1.4), 8.2 to 8.9 for mAB2 (∆pI: 0.7), and 7.4 to 8.4 for mAB3 (∆pI: 1). The icIEF method proved effective for tracking the charge profiles of antibody-drug conjugates (ADCs), showing excellent repeatability for both intra-day and inter-day measurements for unconjugated mAbs and ADCs.

KEYWORDS: Monoclonal Antibodies, Conjugates, icIEF, Charge Variant Profile.

INTRODUCTION: 

Antibody-drug conjugates (ADCs) have demonstrated significant promise of targeted therapeutics, particularly effective in treating various diseases, notably cancer¹. The FDA's approval of ozogamicin in 2000 heralded the emergence of ADCs as viable antitumor agents for acute myeloid leukemia (AML)². Since that time, the field has rapidly evolved, with 14 ADCs currently approved for different cancer types and over 100 more in clinical trials³.

ADCs leverage the specificity of antibodies to selectively target antigens found on tumor cells, delivering potent cytotoxic agents designed to destroy cancer cells⁴. These cytotoxic drugs are chemically attached to monoclonal antibodies (mAbs) through linkers, which can be classified as either cleavable or non-cleavable^3-7. Cleavable linkers release the cytotoxic agent in response to specific conditions within the tumor microenvironment, such as enzymatic activity or pH variations^3-7. In contrast, non-cleavable linkers necessitate complete enzymatic degradation of the mAb to release the cytotoxic compound^3-7. The use of non-cleavable linkers can offer benefits like longer plasma half-lives and diminished off-target toxicity^3-7.

The conjugation of drug molecules to mAbs typically involves cysteine (Cys) and lysine (Lys) residues, resulting in a complex and heterogeneous mixture that includes various species with differing drug-to-antibody ratios (DAR) and conjugation sites⁴. Due to this complexity, thorough characterization of ADCs is crucial for ensuring batch consistency⁸. Charge variant characterization is an important technique in assessing the quality of ADCs⁹.

Charge variant profiles function as distinctive markers for evaluating the consistency and stability of ADC batches^9-10. Established methods such as ion exchange chromatography^10-13 and capillary isoelectric focusing (cIEF)^14-16 are commonly used to analyze these profiles by separating proteins based on their isoelectric point (pI). Imaged cIEF (icIEF) stands out for its reliability, sensitivity, resolution, and reproducibility in determining and quantifying protein pI. Additionally, icIEF facilitates real-time monitoring of charge variant separation, offering valuable insights into ADC stability and heterogeneity^8,17.

Maytansine derivatives and tomaymycin are often utilized as potent cytotoxic agents in ADC formulations. Maytansine derivatives work by inhibiting tubulin polymerization in cancer cells, thereby disrupting cell division¹⁸. Tomaymycin, an antibiotic derived from Streptomyces achromogenes, also demonstrates significant antitumor properties¹⁹.

This study aims to characterize the charge variant profiles of maytansine derivatives and tomaymycin conjugated to mAbs using a non-cleavable linker, employing the icIEF method developed in previous research²⁰.

MATERIALS AND METHODS:

Materials:

The ICE280 chemical test kit and ICE280 electrolyte solution kit were sourced from Convergent Bioscience. Methylcellulose was obtained in concentrations of 1% and 0.5%, along with pI markers of 6.61, 7.05, 8.18, and 9.5, also from Convergent Bioscience. Pharmalyte solutions with pH ranges of 3-10 and 8-10.5 were acquired from GE Healthcare. Furthermore, urea, sucrose, histidine, and phosphoric acid were purchased from Sigma.

mAbs and ADCs:

In this research, we examined three monoclonal antibodies: mAB1 and mAB2, which target EphA2, and mAB3, which targets CD19. Initially, we prepared unconjugated solutions of these antibodies at an approximate concentration of 10mg/mL in a phosphate buffer with a pH of 6.5. Following this step, mAB1 was conjugated to maytansinoid compounds, while mAB2 and mAB3 were linked to tomaymycin derivatives. The attachment of these cytotoxic agents to the monoclonal antibodies was performed using a non-cleavable linker. For the formulation of antibody-drug conjugates (ADCs), they were suspended in a buffer containing 10 mM histidine, 10% sucrose, and N-methyl-2-pyrrolidone (NMP) at a pH of 6.5, reaching an approximate concentration of 2mg/mL.

Sample preparation:

To prepare the samples of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), they were diluted to reach the desired final concentrations. The dilution was carried out in a solution containing 0.35% methylcellulose, a combination of 4% pharmalytes (with pH ranges of 3-10 and 8-10.5 in a 1:1 ratio), and 2M urea. pI markers with isoelectric points of 6.61, 8.81, 7.05, and 9.5 were also added to the mixture.

Following preparation, the samples were centrifuged at 6000rpm for 3 minutes to remove any particulate matter. The clarified samples were then transferred to glass autosampler vials, where a second centrifugation was performed to eliminate any remaining air bubbles. Finally, the prepared samples were loaded into the autosampler carousel for further analysis.

icIEF instrument:

In this research, icIEF method was performed using an iCE280 instrument paired with a PrinCE autosampler, both supplied by Convergent Bioscience. The analysis utilized a capillary column measuring 50mm in length, with an inner diameter (ID) of 100µm and an outer diameter (OD) of 200µm. This transparent capillary column was housed in a glass cartridge, and its inner surface was coated with a fluorocarbon material to reduce electroosmotic flow.

For the analysis of antibody-drug conjugates (ADCs) and monoclonal antibodies (mABs), a cathodic solution of 100mM NaOH and 0.1% methylcellulose was used, alongside an anodic solution containing 80mM H3PO4 and 0.1% methylcellulose. The protein focusing duration was set to either 7 (naked mABs) or 10 minutes (ADCs), during which a voltage of 3000V was applied. Detection of the focused proteins was carried out using a CCD camera set to a wavelength of 280nm.

RESULTS AND DISCUSSION:

Charge variants represent a major source of heterogeneity in the production of therapeutic mABs and ADCs^3-7. This variability stems from modifications that occur during cell culture, purification, and formulation processes. Proper characterization of the charge variant profile is crucial for ensuring the purity of therapeutic mAbs and ADCs and maintaining consistency between batches. The main objective of this study was to analyze the charge variant profile of unconjugated mAbs.

Charge variant profile of unconjugated mAbs:

This study thoroughly characterized the charge variant profiles of three mAbs. The mAbs analyzed included mAB1 and mAB2, which are anti-CD19 antibodies targeting the CD19 cell surface antigen, and mAB3, an anti-EphA2 antibody specifically designed to bind to EphA2. The charge heterogeneity of these mAbs was assessed using the icIEF method, with the results illustrated in Figure 1. Notably, mAB1 and mAB2 exhibited greater charge heterogeneity and a more basic profile compared to mAB3.

Typically, the charge profile of a mAb includes major species along with minor ones that are either more acidic or more basic^21-22. Figure 2 further illustrates the percentage area occupied by charge variants for the three mAbs analyzed.

mAB1 showed two distinct charge variants with pI values of 9.00 (64%) and 8.95 (36%), resulting in a ΔpI of 0.1. Conversely, mAB2 presented three charge variants (ΔpI: 0.3), with the two primary peaks at pI values of 9.00 (60%) and 8.95 (30%). These observations are consistent with previous research on charge variant profiles in therapeutic mAbs. The presence of a more acidic variant in mAB1 and mAB2 (pI 8.95) compared to the major variant (pI 9.00) may be linked to the deamidation of one or two asparagine (Asn) residues, resulting in a shift in the pI value^11,23.

For mAB3, three charge variants were identified (ΔpI: 0.3), with a major variant at a pI of 8.5(85%) and two minor peaks at pI values of 8.3(13%) and 8.6(7%). The presence of a more acidic variant (pI 8.3) relative to the main species (pI 8.5) may also be attributed to deamidation, while the more basic variant (pI 8.6) could be influenced by the presence of a C-terminal lysine, as suggested by prior studies indicating that C-terminal lysine contributes to the formation of basic species^11-24.

The intra- and inter-day repeatability of the icIEF profiles for the mAbs demonstrated strong consistency in pI values (RSD% values below 0.26%) and in the percentage area occupied by charge variants (RSD% values below 8%). Detailed findings are summarized in Table 1.

Figure 1: Illustratation of the analysis conducted using icIEF to assess the charge variant profile of unconjugated mABs. The final concentration of the unconjugated mABs in the sample matrix was 0.2 mg/mL, and they were diluted in a solution of 0.35% methyl cellulose. Other conditions were mentioned in the materials and methods.

Figure 2. Representation of the area percentage of charge variants observed in the studied unconjugated antibodies.

Table 1. Statistical results of intra- day and inter-day repeatability of icIEF profile of mABs.

Intra- day repeatability (n=6)					Inter- day repeatability (n=6, 3 day)
Major charge species	pI		Area%		pI		Area%
	Mean	RSD%	Mean	RSD%	Mean	RSD%	Mean	RSD%
mAB1	8.95	0.2	34%	3	8.95	0.25	35%	4
	9.00	0.25	64%	3	9.00	0.20	65%	4
mAB2	8.95	0.15	30%	5	8.95	0.16	31%	6
	9.00	0.17	60%	5	9.00	0.18	60%	6
mAB3	8.5	0.2	85%	6	8.5	0.1	86%	7

Charge variant profile of non-cleavable ADCs:

The characterized monoclonal antibodies (mABs) were conjugated through the amino groups of lysine residues to maytansine derivatives for mAB1, and to tomaymycin for mAB2 and mAB3, using a cleavable linker (see Figure 3).

Figure 3. Showcases the structure of non-cleavable maytansinoid ADCs (DM4-NHAc-(PEG)4) in panel (A) and non-cleavable tomaymycin ADCs (tomaymycin-pyridine-(PEG)4) in panel (B).

The charge variant profiles of antibodies conjugated to non-cleavable maytansine and tomaymycin are shown in Figure 4. Our analysis indicated that these non-cleavable conjugated antibodies displayed increased levels of heterogeneity and acidity compared to their unconjugated counterparts.

Figure 4. Illustration of the analysis conducted using icIEF for the evaluation of (a) the non-cleavable maytansinoid mAB1 conjugate and (b, c) the non-cleavable tomaymycin mAB2 conjugate. The experiments were conducted under specific conditions, including a final concentration of 0.5 mg/mL in 0.35% methyl cellulose. Other conditions were mentioned in materials and methods.

Figure 5 provides a detailed analysis of the percentage area occupied by charge variants in the non-cleavable antibody-drug conjugates (ADCs). The pI ranges for the non-cleavable maytansinoid mAB1 conjugate were found to be between 7.4 and 8.9 (ΔpI: 1.4). In contrast, the non-cleavable tomaymycin mAB2 and mAB3 conjugates showed pI ranges of 8.2 to 8.9 (ΔpI: 0.7) and 7.4 to 8.4 (ΔpI: 1), respectively.

The observed increase in charge heterogeneity among the non-cleavable conjugates, as indicated by the broader ΔpI values, along with the lower pI values of the isoforms, can be attributed to the number of drugs linked to the free amino groups of lysine (Lys) residues in the antibodies. Specifically, the non-cleavable maytansinoid mAB1 conjugates had a greater number of charge variants (14) compared to the tomaymycin conjugates (8 for mAB2 and 9 for mAB3). This variation can be explained by the differing availability of lysine residues for conjugation in each mAb, with antibodies typically containing up to 80 lysine residues^25-30.

The charge variants in the ADCs originated from the different numbers of amine groups on the lysine residues that were conjugated to the linker-drug. This modification led to a reduction in pI values as the number of modified amino groups increased, contributing to a more acidic profile of the ADC species. Similar observations have been documented for mAb conjugates^{26, 41-50}. It's important to note that the percentages of charge isoforms corresponding to the unconjugated antibodies were relatively low, indicating a successful conjugation process for the mAbs.

Figure 5. % Area of major charge variant of non-cleavable conjugated antibody.

Table 2. Statistical results of intra- day and inter-day repeatability of icIEF profile of ADCs.

Intra- day repeatability (n=6)					Inter- day repeatability (n=6, 3 day)
Major charge species	pI		Area%		pI		Area%
	Mean	RSD%	Mean	RSD%	Mean	RSD%	Mean	RSD%
Maytansinoid  mAB1 conjugates	8.3	0.2	7%	3	8.3	0.25	7%	3
	8.4	0.25	11%%	3	8.4	0.20	10.5%	2
	8.5	0.1	15%	2	8.5	0.25	15.5%	5
	8.6	0.1	13%	5	8.6	0.1	13.1%	4
	8.7	0.2	11%	4	8.7	0.25	11.2%	3
	8.8	0.2	7%	3	8.8	0.1	6,8%	2
Tomaymycin mAB2 conjugates	8.5	0.2	5%	6	8.5	0.27	5%	6
	8.55	0.15	16%	2	8.55	0.17	16.1%	2
	8.6	0.17	20%	2	8.6	0.18	29.9%	2
	8.7	0.25	22%	3	8.7	0.29	21.95%	3
	8.8	0.1	17%	4	8.8	0.15	17.01%	4
Tomaymycin mAB3 conjugates	7.8	0.15	5%	5	7.8	0.15	5.1%	4
	7.9	0.17	11%	5	7.9	0.18	11.2%	3
	8.1	0.25	26%	5	8.1	0.28	25.6%	5
	8.3	0.1	17%	5	8.3	0.15	17%	4
	8.4	0.1	10%	3	8.4	0.16	10%	3

The icIEF (isoelectric focusing) profile of the antibody-drug conjugates (ADCs) demonstrated excellent repeatability, both within the same day and over multiple days, as evidenced by the low relative standard deviation (RSD%) values. The RSD% for the pI (isoelectric point) values was below 0.26%, reflecting high precision in measuring the pI of the charge variants. Furthermore, the RSD% for the area percentage occupied by charge variants was under 7%, indicating consistent and reliable quantification of the various charge species in the ADCs. Detailed results on repeatability are provided in Table 2.

CONCLUSION:

We utilized the icIEF (isoelectric focusing) method to analyze the charge variant profiles of three monoclonal antibodies (mAbs) along with their corresponding non-cleavable conjugates. A comparison of the profiles among the unconjugated mAbs indicated that mAB3 had a more acidic and homogeneous profile, with its primary charge variant exhibiting a pI of 8.50. In contrast, mAB1 and mAB2 displayed two charge variants, with pIs of 9.00 and 8.95, respectively. During the conjugation process, mAB1 was linked to a maytansine derivative via a non-cleavable linker, while mAB2 and mAB3 were conjugated to tomaymycin molecules. The resulting non-cleavable conjugated antibodies showed greater heterogeneity and acidity compared to their unconjugated counterparts. These results underscore the utility of icIEF as an effective tool for monitoring the charge profiles of antibody-drug conjugates (ADCs). Additionally, the pI and area percentage values for both the unconjugated mAbs and ADC charge variants exhibited strong repeatability both within and across different days.

REFERENCES:

1. Dumontet C, Reichert JM, Senter PD, Lambert JM, Beck A. Antibody–drug conjugates come of age in oncology. Nat Rev Drug Discov. 2023; June; 22: 641–661. https://doi.org/10.1038/s41573-023-00709-2.

2. Gottardi M, Sperotto A, Ghelli Luserna Di Rorà A, Padella A, Cangini D, Giannini MB, et al. Gemtuzumab ozogamicin in acute myeloid leukemia: past, present and future. Minerva Med. 2020; Oct; 111(5): 395-410. doi: 10.23736/S0026-4806.20.07019-6.

3. Yang Y, Wang S, Ma P, Jiang Y, Cheng K, Yu Y, et al. Drug conjugate-based anticancer therapy - Current status and perspectives. Cancer Letters. 2023; Jan: 215969. https://doi.org/10.1016/j.canlet.2022.215969.

4. Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J. Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res. 2020; Jan; 18(1): 3-19. doi: 10.1158/1541-7786.MCR-19-0582.

5. Baah S, Laws M, Rahman KM. Antibody-Drug Conjugates-A Tutorial Review. Molecules. 2021; May; 26(10): 2943. doi: 10.3390/molecules26102943.

6. Su Z, Xiao D, Xie F, Liu L, Wang Y, Fan S, Zhou X, et al. Antibody–drug conjugates: Recent advances in linker chemistry. Acta Pharm Sin B. 2021; Dec; 11(12): 3889-3907. https://doi.org/10.1016/j.apsb.2021.03.042.

7. Salloum R, Baddour  F, Abbood A. A Questionnaire to Evaluate Undergraduate Students’ Consumption and Awareness of Non-Steroidal Anti-Inflammatory Drugs in Syria. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR). 2024; 4(4): June 1-6. DOI: 10.54105/ijapsr.C4041.04040624.

8. Zanboua R, Abbood A. Survey of Knowledge About the Interaction Between Food and Drugs Among the Syrian Population. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR), 2024; 4(4): June: 22-28. DOI: 10.54105/ijapsr.D4044.04040624.

9. Mahfouz H, Assaf A, Abbood A. Survey of Usage and Awareness of Ibuprofen Among the Syrian Population. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR).  2024; 4(5): August 23-28. DOI:  10.54105/ijapsr.E4048.04050824.

10. Abbood A. Overview of Analytical Methods for Characterizing the Charge Heterogeneity of Antibody-Drug Conjugates. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR),  2024Volume-4 Issue-5 16-22. DOI:  10.54105/ijapsr.E4047.04050824

11. Beck A, Nowak C, Meshulam D, Reynolds K, Chen D, Pacardo DB, et al. Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants. Antibodies (Basel). 2022; Nov.20; 11(4): 73. doi: 10.3390/antib11040073.

12. Fekete P, Beck A, Veuthey JL, Guillarme D. Ion-exchange chromatography for the characterization of biopharmaceuticals. J Pharm Biomed Anal. 2015; Sep; 113: 43-55. https://doi.org/10.1016/j.jpba.2015.02.037.

13. Matsuda Y, Kliman M, Mendelsohn BA. Application of Native Ion Exchange Mass Spectrometry to Intact and Subunit Analysis of Site-Specific Antibody-Drug Conjugates Produced by AJICAP First Generation Technology. J Am Soc Mass Spectrom. 2020; Jul1. doi: 10.1021/jasms.0c00129.

14. Gahoual R, Beck A, Leize-Wagner E, François YN. Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products. J. Chromatogr. B Biomed. Appl. 2016; Oct; 1032: 61-78. https://doi.org/10.1016/j.jchromb.2016.05.028.

15. Chen T, Chen Y, Stella C, Medley CD, Gruenhagen JA, Zhang K. Imaged capillary isoelectric focusing (icIEF) tandem high resolution mass spectrometry for charged heterogeneity of protein drugs in biopharmaceutical discovery. J Pharm Biomed Anal. 2016; Oct; 1032: 39-50. https://doi.org/10.1016/j.jchromb.2016.07.023.

16. Abbood A, Herrenknecht C, Proczek G, Descroix S, Rodrigo J, Taverna M, Smadja C. Hexylacrylate-based mixed-mode monolith, a stationary phase for the nano-HPLC separation of structurally related enkephalins. Anal Bioanal Chem. 2011; Apr. 400(2): 459-68. doi: 10.1007/s00216-011-4762-4.

17. Wu J, McElroy W, Pawliszyn J, Heger CD. Imaged capillary isoelectric focusing: Applications in the pharmaceutical industry and recent innovations of the technology. TrAC Trends Analyt Chem. 2022; May; 150: 116567. https://doi.org/10.1016/j.trac.2022.116567.

18. Cao S, Dong YH, Wang DF, Liu ZP. Tubulin Maytansine Site Binding Ligands and their Applications as MTAs and ADCs for Cancer Therapy. Curr Med Chem. 2020; 27(27): 4567-4576. doi: 10.2174/0929867327666200316144610.

19. Abbood A. Optimization of the Imaged cIEF Method for Monitoring the Charge Heterogeneity of Antibody-Maytansine Conjugate. J Anal Methods Chem. 2023; 8150143. https://doi.org/10.1155/2023/8150143.

20. Tozuka Z, Yazawa H, Murata M, Takaya T. Studies on tomaymycin. III. Synthesis and antitumor activity of tomaymycin analogs. J Antibiot (Tokyo). 1983; Dec; 36(12): 1699-708. doi: 10.7164/antibiotics.36.1699.

21. Goyon A, Excoffier M, Janin-Bussat MC, Bobaly B, Fekete S, Guillarme D, et al.. Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies. J Chromatogr B. 2017; 1065: 119–128. doi: 10.1016/j.jchromb.2017.09.033.

22. Wagner-Rousset E, Fekete S, Morel-Chevillet L, Colas O, Corvaïa N, Cianférani S, et al. Development of a fast workflow to screen the charge variants of therapeutic antibodies. J. Chromatogr. A. 2017; May: 1498: 147–154. doi: 10.1016/j.chroma.2017.02.065.

23. Spanov B, Govorukhina N, van de Merbel NC, Bischoff R. Analytical tools for the characterization of deamidation in monoclonal antibodies. Journal of Chromatography Open. 2022; Nov; 2: 100025. https://doi.org/10.1016/j.jcoa.2021.100025.

24. Cai B, Pan H, Flynn GC. C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo. Biotechnol Bioeng. 2011; Feb; 108(2): 404-12. doi: 10.1002/bit.22933.

25. Dennler P, Fischer E, Schibli R. Antibody Conjugates: From Heterogeneous Populations to Defined Reagents. Antibodies. 2015; 4(3): 197-224. https://doi.org/10.3390/antib4030197.

26. Lin J, Lazar AC. Determination of charge heterogeneity and level of unconjugated antibody by imaged cIEF. Methods Mol. Biol. (Clifton, N.J.), 2013; 1045: 295–302. https://doi.org/10.1007/978-1-62703-541-5_19.

27. Isbera M, Abbood A, Ibrahim W. Weight and Content Uniformity of Warfarin Sodium Half Tablets. Research J. Pharm. and Tech. 2016; 9(3): 215-218. doi: 10.5958/0974-360X.2016.00039.1

28. Abbood A, Layka R. Weight and content uniformity Study of captopril half-tablets. Research J. Pharm. and Tech. 2017; 10(6): 1621-1626. doi: 10.5958/0974-360X.2017.00285.2.

29. Chbani D, Abbood A, Alkhayer M. Determination of Nitrite and Nitrate Ions levels in some types of processed meats marketed locally. Research J. Pharm. and Tech. 2018; 11(4): 1442-1447. doi: 10.5958/0974-360X.2018.00269.X.

30. Abbood A, Malek Z, Al-Homsh Y, Thallaj N. In vitro Study for Antibiotic resistance of bacteria causing Urinary Tract Infection from Syrian adults. Research J. Pharm. and Tech.  2022; 15(10): 4727-2. doi: 10.52711/0974-360X.2022.00794.

31. Abbood A, Malek Z, Thallaj N. Antibiotic resistance of urinary tract pathogens in Syrian children. Research J. Pharm. and Tech. 2022; 15(11): 4935-9. doi: 10.52711/0974-360X.2022.00829.

32. Deeb DN, Aldiab D, Sulaeman S. Effect of Storage Conditions on Bacteriological Growth in Infant Cereals. Research J. Pharm. and Tech. 2021; 14(2): 667-672. doi: 10.5958/0974-360X.2021.00119.0.

33. Alsubot S, Aldiab D. 5-hydroxymethylfurfural Levels in Coffee and Study of some effecting factors. Research J. Pharm. and Tech. 2019; 12(9): 4263-4268. doi: 10.5958/0974-360X.2019.00733.9.

34. Asaad RA. Hormone Receptor Status and its Relation to C-Reactive Protein and other Prognostic factors in Breast Cancer in Jableh- Syria. Research J. Pharm. and Tech. 2017; 10(9): 3003-3010. doi: 10.5958/0974-360X.2017.00532.7.

35. Asaad RA, Abdullah SS. Breast Cancer Subtypes (BCSs) Classification according to Hormone Receptor Status: Identification of Patients at High Risk in Jableh- Syria. Research J. Pharm. and Tech. 2018; 11(8): 3703-3710. doi: 10.5958/0974-360X.2018.00680.7.

36. Besherb S, Alallan L, Hassan Agha MA, AIshamas I, Thallaj N. Influence of soil salinity on the chemical composition of essential oil of Rosmarinus Officinalis in Syria, Research J. Pharm. and Tech. 2024; 17(4).

37. Machkour A, Thallaj NK, Benhamou L, Lachkar M, Mandon D. he Coordination Chemistry of FeCl3 and FeCl2 to Bis [2‐(2, 3‐dihydroxyphenyl)‐6‐pyridylmethyl](2‐pyridylmethyl) amine: Access to a Diiron (iii) Compound with an Unusual Pentagonal‐Bipyramidal/Square‐Pyramidal EnvironmentChemistry–A European Journal. 2006: 25; 12(25): 6660-6668. https://doi.org/10.1002/chem.200600276.

38. Labban L, Thallaj N, Malek Z. The implications of E-cigarettes or" vaping" on the nutritional status. Journal of Medical Research and Health Sciences, 2019; 2(11): 784-787. https://doi.org/10.15520/jmrhs.v2i11.128.

39. Labban L, Thallaj N, labban A. Assessing the Level of Awareness and Knowledge of COVID 19 Pandemic among Syrians. Archives of Medicine, 2020; 12, 2:8, 1-5. DOI: 10.36648/1989-5216.12.2.309.

40. Thallaj NK, Przybilla J, Welter R, Mandon D. A ferrous center as reaction site for hydration of a nitrile group into a carboxamide in mild conditions. J. Am. Chem. Soc. 2008; 130: 2414-2415. https://doi.org/10.1021/ja710560g. 

41. Thallaj N. Microwave-Assisted Synthesis of Oxadiazole and Thiazolidine Derivatives. Indian Journal of Advanced Chemistry, 1, 3, 2022. 10-14. DOI:10.54105/ijac.d2015.102222.

42. Thallaj N. Quick Review of Chemistry Related to the [Fe]- Hydrogenases. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR) 2022; 2(4): 1-15. DOI:10.54105/ijapsr.C4016.062422.

43. Thallaj N.  A Short Review of Some Examples of the Binding of Fullerenes C60 to Transition Metal Complexes. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR) 2022. 2,6, 1-12. DOI: 10.54105/ijapsr.C4015.102622.

44. Thallaj N. Review of a Few Selected Examples of Intermolecular Dioxygenases Involving Molecular Oxygen and Non-Heme Iron Proteins. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR) 2023. 3, 2, 1-18. DOI:10.54105/ijapsr.C4011.023223.

45. Thallaj N. A Brief Overview of the General Characteristics and Reactivity Towards Dioxygen of the Ferrous Tris (2-Pyridylmethyl Amine) Series Complexes is Presented. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR) 2023. 3, 3, 1-18. DOI:10.54105/ijapsr.C4012.043323.

46. Thallaj N.  Detecting Antioxidant Behavior for Phenolic Content of Some Beauty Care Creams in Syrian Market. Indian Journal of Advanced Chemistry. 2024; 2(1): 10–14, Jan., doi: 10.54105/ijac.C2013.041322.

47. Thallaj NK, Mandon  D, White KA. The Design of Metal Chelates with a Biologically Related Redox‐Active Part: Conjugation of Riboflavin to Bis (2‐pyridylmethyl) amine Ligand and Preparation of a Ferric Complex Eur. J. of Inorg. Chem., 2007: 44–47.  https://doi.org/10.1002/ejic.200600789.

48. Wane A, Thallaj NK, Mandon D. The Reactivity of Molecular Dioxygen on a Series of Isostructural Dichloroferrous Complexes with Tripodal Tetraamine Ligands: General Access to µ-oxo Diferric Complexes, and Effect of α-Fluorination on the Kinetics of the Reaction. Chemistry A European Journal 14 (22), 6742-6753. https://doi.org/10.1002/chem.200701967

49. Abbood A, Thallaj N. Comparison between chromatofocusing and icIEF charge variant profiles of unconjugated monoclonal antibodies and their drug conjugates. Arab Journal of Pharmaceutical Sciences. 2023: 7; (1).

50. Thallaj N. Characterization of charge heterogeneity of antibody -drug conjugate by anion-exchange chromatofocusing. Tishreen University Journal-Medical Sciences Series. 2023; 44 (6):21-29.

Received on 22.05.2024      Revised on 12.08.2024

Accepted on 21.10.2024   Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):185-190.

DOI: 10.52711/0974-360X.2025.00028

© RJPT All right reserved

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.