Preparation of Lyophilized Reference Standard Anti-SARS-CoV-2 Antibody
Lyophilized Reference Standard Anti-SARS-CoV-2 Antibody
Keywords:
Reference standard SARS-CoV-2 antibody, PRNT, LyophilizationAbstract
High-quality vaccines that meet specific standards are the primary measures during the COVID-19 pandemic. Information on vaccine quality control and testing for anti-SARS-CoV-2 neutralizing antibodies against different variants is beneficial for developing vaccination policies and models to be used. The World Health Organization has established international antibody standards, which are still limited. This study aimed to develop the reference standard anti-SARS-CoV-2 antibody for Plaque Reduction Neutralization Test (PRNT), the gold standard for detecting neutralizing antibody against each SARS-CoV-2 variant, as for quality control of SARS-CoV-2 vaccines and for testing neutralizing antibody levels after vaccination. Serum antibodies in the form of lyophilized sera were tested comparatively to the lyophilized anti-SARS-CoV-2 monoclonal antibody (mAb). The serum antibody and mAb formulations were prepared, and freeze-drying conditions were optimized. After the quality of the freeze-dried serum antibodies was evaluated, the homogeneity of the mAbs did not meet the acceptance criteria, while that of the lyophilized pooled serum antibody was found to be physically and biologically acceptable as a reference standard. The serum antibody samples were tested against the Delta, Omicron BA.2, and Omicron BA.5 variants in terms of the PRNT50 titer (95% confidence interval) as 512.97 (473–556), 288.14 (265–313), and 161.49 (124–184), respectively. Post-production potency tests of actual utilization were conducted for stability assessment, and the results were within the acceptance criteria (geometric mean ±3 SD). Therefore, the pooled serum antibody is suitable as the reference standard antibody against SARS-CoV-2 for vaccine quality control and for evaluating post-vaccination immune response during the COVID-19 pandemic.
References
Rey L, May JC. Freeze-drying/lyophilization of pharmaceutical and biological products. 2nd ed. New York: Marcel Dekker; 2004.
Naddafi K, Moosavi GH, Mesdaghinia AR. Evaluation of lyophilization effects on operational parameters and characteristics of activated sludge. Iranian J Env Health Sci Eng 2004; 1(1): 20-5.
Matejtschuk P, Perrie Y, Gurney R. Freeze-drying microscopy-unravelling the complexities of freeze-drying pharmaceuticals with advanced microscopy techniques. Drug Dev Deliv. [online]. 2022; [cited 2024 Oct 25]; [5 screens]. Available from: URL: https://drug-dev.com/freezedrying-microscopy-unravelling-the-complexities-of-freeze-drying-pharmaceuticalswith-advanced-microscopy-techniques-2.
Barresi AA, Ghio S, Fissore D, Pisano R. Freeze-drying of pharmaceutical excipients close to collapse temperature: influence of the process conditions on process time and product quality. Dry Technol 2009; 27(6): 805-16.
Haeusera C, Goldbach P, Huwyler J, Friess W, Allmendinger A. Impact of dextran on thermal properties, product quality attributes, and monoclonal antibody stability in freeze-dried formulations. Eur J Pharm Biopharm 2020; 147: 45-56.
Cheng SS, Mok CK, Li JK, Ng SS, Lam BH, Jeevan T, et al. Plaque-neutralizing antibody to BA.2.12.1, BA.4 and BA.5 in individuals with three doses of BioNTech or CoronaVac vaccines, natural infection and breakthrough infection. J Clin Virol 2022; 156: 105273. (7 pages).
Bewley KR, Coombes NS, Gagnon L, McInroy L, Baker N, Shaik I, et al. Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays. Nat Protoc 2021; 16: 3114-40.
WHO International Standard. First WHO international standard for anti-SARS-CoV-2 immunoglobulin (human) NIBSC code: 20/136. Instructions for use. [online]. 2020; [cited 2024 Oct 25]; [2 screens]. Available from: URL: https://nibsc.org/documents/ifu/20-136.pdf.
WHO/BS/2020.2403. Establishment of the WHO international standard and reference panel for anti-SARS-CoV-2 antibody. [online]. 2020; [cited 2024 Oct 25]; [60 screens]. Available from: URL: https://www.who.int/publications/m/item/WHO-BS-2020.2403.
The United States Pharmacopeia. General chapters <921> water determination. In: USP-NF. Rockville, MD: United States Pharmacopeia; 2022. page 815-819.
ISO 13528:2022. Statistical methods for use in proficiency testing by interlaboratory comparison. 3rd ed. Geneva, Switzerland: International Organization for Standardization; 2022.
ISO 33405:2024. Reference materials-approaches for characterization and assessment of homogeneity and stability. Switzerland: International Organization for Standardization; 2024.
Kullabut K, Yusho A, Sapsutthipas S, Chumpol S, Ritthitham K, Phumiamorn S. Method validation of the plaque assay for SARS-CoV-2 Titration. Thai Food Drug J 2021; 28(3): 9-16.
Srisutthisamphan K, Saenboonrueng J, Wanitchang A, Viriyakitkosol R, Jongkaewwattana A. Cross-neutralization of SARSCoV-2-specific antibodies in convalescent and immunized human sera against the bat and pangolin coronaviruses. Viruses 2022; 14: 1793. (13 pages).
Okba NMA, Müller MA, Li W, Wang C, Geurts van Kessel CH, Corman VM, et al. Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients. Emerg Infect Dis 2020; 26(7): 1478-88.
Jamtsho R. Stability of lyophilized human serum for use as quality control material in bhutan. Indian J Clin Biochem 2013; 28(4): 418-21.
Kida H, Yamasaki Y, Feril LB Jr, Endo H, Itaka K, Tachibana K. Efficient mRNA delivery with lyophilized human serum albumin-based nanobubbles. Nanomaterials (Basel) 2023; 13(7): 1283. (18 pages).
Kamath S, Lip GY. Fibrinogen: biochemistry, epidemiology and determinants. QJM: An International Journal of Medicine. 2003; 96(10): 711-29.
Santa Maria F, Huang YS, Vanlandingham DL, Bringmann P. Inactivation of SARS-CoV-2 in all blood components using Amotosalen/ultraviolet a light and Amustaline/glutathione pathogen reduction technologies. Pathogens 2022; 11(5): 521. (13 pages).
Zäh M, Brandenbusch C, Groël S, Winter G, Sadowski G Water activity as an indicator for antibody storage stability in lyophilized formulations. Mol Pharm 2025; 22(2): 918-26.
WHO/BS/2022.2427. Establishment of the 2nd WHO international standard for anti-SARSCoV-2 immunoglobulin and reference panel for antibodies to SARS-CoV-2 variants of concern. [online]. 2022; [cited 2024 Oct 25]; [67 screens]. Available from: URL: https://www.who.int/publications/m/item/who-bs-2022.2427.
World Health Organization. Annex 2: WHO manual for the preparation of reference materials for use as secondary standards in antibody testing. In: WHO expert committee on biological standardization: seventy-fifth report. WHO Technical Report Series No. 1043. Geneva: World Health Organization; 2022. p. 41-129.
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