Stable SARS‐CoV‐2 antibody levels and functionality in serum and COVID‐19 convalescent plasma after long‐term storage

Abstract

Background and Objectives

The coronavirus disease 2019 (COVID‐19) pandemic necessitated various therapeutic approaches, including convalescent plasma (CP) administration. The administration timing of COVID‐19 CP (CCP), antibody specificity and quantity were identified as crucial factors for therapeutic success. Currently, antibody durability and storage time are still under debate. The aim of this study was to evaluate the stability and in vitro functionality of severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) antibodies in human plasma and serum after long‐term storage, to provide a framework for generally applicable rules regarding the long‐term storage of CCP.

Materials and Methods

Serum and plasma samples of CCP donations were investigated at the time of donation and after 2 and 3 years' storage at less than −30°C using (electro)chemiluminescence immunoassays and enzyme‐linked immunosorbent assays, with the plasma undergoing multiple freezing and thawing.

Results

Our data reveal robust levels of SARS‐CoV‐2 antibodies after long‐term storage. Furthermore, our findings also indicate that multiple freezing and thawing cycles do not affect the antibody levels or their neutralizing capability.

Conclusion

As antibody stability and in vitro functionality are maintained over extended periods, even after repeated freezing and thawing, our findings support long‐term storage of CCP, particularly benefiting vulnerable populations such as immunocompromised individuals. By now, donors have likely encountered various SARS‐CoV‐2 variants and vaccine‐acquired antibodies. This antibody mix present in CCP is suggested to protect even against new variants. Our data indicate that current regulations for the storage of CCP can be extended and that CCPs could be used for therapeutic purposes after long‐term storage without significant loss of antibody quantity.

Highlights.

• Severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) anti‐nucleocapsid (anti‐N) and anti‐spike (anti‐S) antibody levels and their in vitro functionality remain stable in serum and plasma after long‐term storage.

• Multiple freeze–thaw cycles do not affect SARS‐CoV‐2 antibody levels or their neutralizing capability.

• Current regulations for the storage of coronavirus disease 2019 convalescent plasma (CCP) can be extended and CCP could be used for therapeutic purposes after long‐term storage without any significant loss of antibody quantity.

INTRODUCTION

Coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2), resulted in global health challenges, necessitating the exploration of various therapeutic approaches. One approach is convalescent plasma (CP) administration, in which plasma containing specific SARS‐CoV‐2 antibodies is transfused from recovered to infected individuals, generating passive immunity and thus enhancing the recipient's immune response. CP therapy is based on serotherapy, which uses animal sera to transfer immunity against bacterial toxins to animals and humans [1, 2]. The first reported transfusion of human CP was conducted in 1906, where plasma of a man recovered from measles was transfused to a child suffering from a severe form of infection, resulting in rapid and significant improvement of symptoms [3]. Since then, CP has been used to treat numerous infectious diseases with varying degrees of success. During the 1918 influenza pandemic, CP showed the potential to reduce mortality rates. Later, CP was applied during different viral outbreaks, such as ebolavirus, chikungunya virus and SARS, with varying therapeutic success [4, 5]. The application of CP for treating COVID‐19 has been extensively studied and yielded deviating results. Studies conducted early in the pandemic had reported no significant benefit of COVID‐19 CP (CCP) regarding the mortality rate or improvement of clinical outcomes in hospitalized COVID‐19 patients [6, 7]. However, recent studies have suggested that CCP could be beneficial for immunocompromised patients [8, 9, 10] if administered early after symptom onset. In this setting, CCP was associated with reduced mortality and improved recovery rates of hospitalized individuals [11, 12, 13]. However, the efficacy of CCP also depends on sufficient titres of specific neutralizing antibodies [11, 12, 13]. Another factor that may affect efficacy is its long‐term storage. Some data indicate that freezing and thawing might reduce the activity of pathogen‐directed antibodies in human samples [14, 15]. However, others did not observe such an effect on antibodies directed against human pathogens [16, 17]. Nevertheless, there are uncertainties regarding antibody stability and storage periods. Currently, authorities worldwide suggest different storage conditions and shelf life for CCP. The U.S. Food and Drug Administration recommends expiry of fresh frozen plasma 1 year after collection and that of CCP 6 months after collection when stored at or below −18°C [18]. The European Medicines Agency (EMA) and the European Directorate for the Quality of Medicines and HealthCare (EDQM) have not published any recommendations on CCP storage conditions. European blood banks usually follow the EDQM guidelines for the storage of frozen plasma (depending on product specificities, up to 3 months at less than −18°C and up to 36 months at less than −25°C) [19, 20, 21, 22] and follow national recommendations. Furthermore, guideline from the International Society of Blood Transfusion on the use of CCP recommends a storage period of 12 months at below −18°C [23]. The aim of this study was to evaluate the stability and in vitro functionality of SARS‐CoV‐2 antibodies after long‐term storage and thus contribute to a decision‐making basis for general rules regarding the long‐term storage of CCP.

MATERIALS AND METHODS

Ethics Statement

The study was conducted according to the Declaration of Helsinki and its later amendments, and was approved by the Ethics Committee of the Federal State of Salzburg (1127/2023) and the Ethics Committee of the Medical University of Innsbruck (1293/2023). Donors signed written informed consent on the use of blood/plasma donation as a therapeutic application for COVID‐19 patients and on the use of leftover material for research purposes. Samples were processed pseudonymized to protect donor privacy.

Donor selection and plasma collection

Plasma and whole blood were collected according to the Association for the Advancement of Blood & Biotherapies' guide ‘Apheresis: Principles and Practice’ [24] and the EDQM guidelines [21]. Two centres were involved, following different approaches to produce CCP: the Department for Transfusion Medicine, Salzburg, and the Central Institute for Blood Transfusion and Immunology, Innsbruck. In Salzburg, CCP was collected by plasmapheresis between 1 June 2020 and 15 January 2022. All donors fulfilled legal requirements for blood donation and were fully recovered from COVID‐19 for at least 4 weeks, as confirmed by RT‐qPCR and SARS‐CoV‐2 anti‐nucleocapsid (anti‐N) antibody screening. Donor characteristics are described in Table 1. Each donation was screened for infectious disease parameters and quality parameters, as described previously [25]. Donors were admitted to CCP donation using a Trima Accel cell separator (Terumo BCT, Lakewood, Colorado, USA), provided the following conditions were met: negative screening for infectious disease parameters; results within the normal range for quality parameters; and a SARS‐CoV‐2 anti‐spike (anti‐S) immunoglobulin G ratio of ≥2.0. A maximum volume of 650 mL of plasma was collected. Plasma pathogen reduction was performed using the INTERCEPT blood system (Cerus Corporation Europe BV, Amersfoort, The Netherlands) according to manufacturer's instructions.

TABLE 1.

Coronavirus disease 2019 convalescent plasma donor characteristics (Salzburg).

		Total number (%)
	CCPs	156
Sex	Women	38 (24.4)
	Men	118 (75.6)
ABO blood group	A	78 (50)
	AB	19 (12.2)
	B	30 (19.2)
	O	29 (18.6)
Age	Mean age (SD) in years	38.9 (11.0)

Abbreviation: CCP, coronavirus disease 2019 convalescent plasma.

In Innsbruck, CCP was produced from whole blood donations collected between 1 March and 31 July 2021. All donors fulfilled the legal requirements for blood donation; donor characteristics are summarized in Table 2. Pathogen inactivation was done according to the manufacturer's instructions using the Macotronic‐B2 and Theraflex‐MB sets (Macopharma). Each donation was examined for infectious disease parameters and SARS‐CoV‐2 antibodies using Alinity i instruments with corresponding assays (Abbott Ireland, Sligo, Ireland), as described previously [26, 27]. Plasma was used for CCP production provided negative results were found for infectious disease parameters and a SARS‐CoV‐2 anti‐S IgG titre of ≥120 binding antibody units per millilitre (BAU/mL), which is suggested by the EMA for plasma with intended therapeutic application [20].

TABLE 2.

Coronavirus disease 2019 convalescent plasma donor characteristics (Innsbruck).

		Total number (%)
	CCPs	147
Sex	Women	45 (30.6)
	Men	102 (69.4)
ABO blood group	A	81 (55.1)
	AB	7 (4.7)
	B	6 (4.1)
	O	53 (36.1)
Age	Mean age (SD) in years	43.5 (15.0)

Abbreviation: CCP, coronavirus disease 2019 convalescent plasma.

Plasma freezing, long‐term storage and thawing

CCPs from Salzburg were frozen after production using a blast freezer (KLF 16‐24; Cryo Life Science Technologies, Ebenthal, Austria, settings: 45 min, −40°C) and stored in a Tecto Plus 150 freezer room (Viessmann, Allendorf, Germany) at −40°C. CCP products were considered expired 2 years after production. Thawing after product expiry was performed using a rapid dry plasma thawing system (Barkey, Leopoldshoehe, Germany), which allowed controlled heating to 37°C. After the first thawing, the plasma sample was subjected to antibody measurements. Then the CCP was split into aliquots, which were re‐frozen and stored at −40°C. The aliquots were subjected to up to four freeze–thaw cycles. After every thawing, antibody screening was done. Additionally, after the first thawing, one aliquot of the CCP was not re‐frozen but instead stored for 7 days at 4°C followed by antibody measurements.

CCPs produced in Innsbruck were frozen using the blast freezer (KLF 24‐36; settings: 45 min, −30°C) and stored in a freezer room at –30°C (Groemer GmbH, Lochen am See, Austria), with expiry 2 years after production.

Anti‐SARS‐CoV‐2 serological screening

The Elecsys Anti‐SARS‐CoV‐2 total antibody electrochemiluminescence immunoassay (ECLIA, Roche Diagnostics, Basel, Switzerland) was applied using a cobas8000‐e801 device (Roche Diagnostics) to screen for SARS‐CoV‐2 anti‐N total antibodies according to the manufacturer's instructions in serum of CCP donors. Results were expressed as cut‐off indices (COIs). Furthermore, serum at the time of donation as well as serum and plasma after 2 years' storage were screened for SARS‐CoV‐2 anti‐S antibodies using the Euroimmun SARS‐CoV‐2 IgG ELISA (Euroimmun, Luebeck, Germany) on a Euroimmun Analyser I platform. Testing was performed according to manufacturer's instructions using serial dilutions of samples. Results were expressed as IgG ratio values. The in vitro functionality of SARS‐CoV‐2 antibodies was examined by the cPass™ SARS‐CoV‐2 neutralization antibody detection kit (surrogate virus neutralization test [sVNT], GenScript, Piscataway Township, New Jersey, USA) according to the manufacturer's instructions using an ETIMax3000 device (Diasorin, Saluggia, Italy). This enzyme‐linked immunosorbent assay (ELISA) detects whether antibodies present in the sample inhibit the interaction between a horseradish‐peroxidase‐conjugated recombinant viral receptor‐binding domain (RBD) protein and human angiotensin‐converting enzyme 2. Results are expressed as percent signal inhibition. Cell‐based pseudovirus neutralization and SARS‐CoV‐2 virus neutralization tests showed highly correlated results with this assay [28, 29]. As suggested by the manufacturer, signal inhibition rates of ≥30% were assumed to indicate the presence of neutralizing antibodies.

In Innsbruck, blood donations (serum and plasma) were tested for spike RBD IgG antibodies at the time of production and after 2 years of storage using the quantitative SARS‐CoV‐2 IgG II chemiluminescent microparticle immunoassay (Abbott) on the Alinity I system [26, 27]. Spike RBD IgG antibody values of ≥7.1 BAU/mL were considered seropositive.

Statistical analysis

For descriptive analysis, means, standard deviations and absolute and relative frequencies were calculated. The Pearson correlation coefficient was used to evaluate the strength and direction of the linear relationship between different times. The Wilcoxon signed‐rank test was used to investigate the decrease of values. For the analysis of the difference in values between donation and thawing, the median as well as the first and third quartiles of the differences were determined. The Bonferroni–Holm correction was applied to account for multiplicity. Metric values are visualized using box plots with error bars; categorical values are depicted by line plots. A one‐tailed significance level of α = 0.05 was applied. All analyses were performed using the R statistical software (version 4.3.2) [30].

RESULTS

Anti‐N and anti‐S antibody levels are stable after long‐term storage below −30°C

Serum samples from 87 CCPs were examined for SARS‐CoV‐2 total anti‐N and anti‐S antibodies at the time of donation and after 2 years of storage. As depicted in Figure 1a,b, serum levels of anti‐N and anti‐S antibodies did not significantly decrease during storage. We also examined serum samples of 69 CCPs that were stored for 3 years. Again, no significant reduction was observed for anti‐N (Figure 1c) or anti‐S antibodies (Figure 1d). Additionally, a high correlation (r > 0.9) was found for antibody levels between donation and long‐term storage. This finding was confirmed in terms of the actual anti‐S quantity for serum of CCPs produced from whole blood donations (n = 112) (Figure 2).

FIGURE 1.

Stable severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) anti‐nucleocapsid (anti‐N) and anti‐spike (anti‐S) antibody levels in serum after long‐term storage. (a) SARS‐CoV‐2 total anti‐N antibody levels (cut‐off indices [COI]) and (b) anti‐S IgG ratios in serum of donors at the time of donation and after 2 years' storage below −40°C (n = 87). (c) SARS‐CoV‐2 anti‐N antibody levels and (d) anti‐S IgG ratios in serum at the time of coronavirus disease 2019 convalescent plasma (CCP) donation and after 3 years' storage below −40°C (n = 69).

FIGURE 2.

Robust severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) anti‐spike (anti‐S) quantity in serum of whole blood donors after long‐term storage. Data shown are binding antibody units per millilitre (BAU/mL) × 1000 of anti‐S IgG in serum samples of whole blood donors (n = 112) at the time of donation and after 2 years' storage below −30°C.

Comparable SARS‐CoV‐2 antibody levels in serum and plasma after long‐term storage

Next, we examined the SARS‐CoV‐2 anti‐N and anti‐S levels in CCPs that were not required for therapeutic application prior to expiry (n = 16). Regarding anti‐N antibodies, we found no differences for serum samples, but there was a significant difference between serum and CCP samples after storage (p < 0.05, median of the differences: 6.1, interquartile range: 5.1–10.8) (Figure 3a). However, the correlation between serum and CCP levels after storage was high (r > 0.9). We found no significant differences for anti‐S IgG ratios over time or between samples (Figure 3b, r > 0.9). For plasma samples from CCPs produced from whole blood (n = 43), anti‐S quantities were determined at the time of production and after storage. We observed a decrease in anti‐S quantity after long‐term storage, particularly for plasma with initially high anti‐S levels. However, most samples of CCPs produced from whole blood revealed a high correlation of anti‐S IgG quantity between donation and after storage (r > 0.9), indicating stable anti‐S concentrations (Figure 4a). Furthermore, we found a significant reduction between the anti‐S levels in serum and the corresponding CCP (n = 8) after storage (p < 0.05, median of the differences 0.20 BAU/mL × 1000, interquartile range: 0.09–0.45) (Figure 4b). Again, correlations between sample types and times were high (r > 0.9).

FIGURE 3.

Comparable levels of anti‐nucleocapsid (anti‐N) and anti‐spike (anti‐S) antibodies in serum and coronavirus disease 2019 convalescent plasma (CCP). (a) Data shown are severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) total anti‐N antibody levels (cut‐off indices [COI]) and (b) anti‐S IgG ratios in serum at the time of donation, and for serum and corresponding CCP samples after 2 years' storage (n = 16). IgG ratios are grouped as indicated, with intensification of colour indicating higher ratios. *p < 0.05.

FIGURE 4.

Consistent quantity of anti‐spike (anti‐S) IgG in coronavirus disease 2019 convalescent plasmas (CCPs). (a) Severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) anti‐S quantity in CCP produced from whole blood at the time of donation and after 2 years of storage (n = 43) and of (b) serum measured at the time of donation, and serum and corresponding CCP samples measured after 2 years of storage (n = 8). Data given as binding antibody units per millilitre (BAU/mL) × 1000. *p < 0.05.

Repeated freezing and thawing does not alter antibody levels or in vitro functionality

We investigated the effect of multiple freezing and thawing on antibody levels and their in vitro functionality. Sixteen expired CCPs were thawed and split into five aliquots. One aliquot was stored at 4°C for 7 days. The other aliquots were subjected to further freeze–thaw cycles, and anti‐N and anti‐S screening and determination of in vitro functionality were carried out after every thawing step. In this way, CCPs underwent one, two, three, four or five freeze–thaw cycles, or one thawing process followed by a 1‐week storage at 4°C (Figure 5a). When comparing the levels of anti‐N antibodies from serum at the time of donation with the corresponding CCP after the first thawing process, we found no significant reduction. Anti‐N levels were not altered after multiple freezing and thawing and remained stable after thawing followed by a 1‐week storage at 4°C. We also found a high correlation between different times (r > 0.9) and no significant reduction, except when comparing the anti‐N levels of the donation and the first thawing event (p < 0.05, median of the differences: 5.9, interquartile range: 4–12.6) (Figure 5b). Similar observations were made for anti‐S, with no significant decrease and a high correlation between different samples (r > 0.9) (Figure 5c). Additionally, we examined the in vitro functionality of SARS‐CoV‐2 antibodies. All samples showed an inhibition rate of >30% after the first thawing step, with 14 of 16 samples revealing rates of >80%. We found no significant decrease in inhibition rate after multiple freezing and thawing or storage at 4°C (Figure 5d).

FIGURE 5.

Multiple freezing and thawing does not affect severe acute respiratory syndrome coronavirus type 2 (SARS‐CoV‐2) antibody levels or functionality. (a) After 2 years' storage below −40°C, coronavirus disease 2019 convalescent plasmas (CCPs) were thawed to 37°C and split into aliquots. One aliquot was stored 7 days at 4°C and the others were subjected to multiple freeze–thaw cycles (1–5 times). After every thawing and after 7 days at 4°C, CCPs were subjected to antibody screening. (b) SARS‐CoV‐2 anti‐nucleocapsid (anti‐N) levels, (c) SARS‐CoV‐2 anti‐spike (anti‐S) IgG ratios and (d) in vitro functionality of antibodies (given in percent inhibition). All assays were done for serum of corresponding CCPs at the time of donation and for plasma after 2 years' storage followed by multiple freeze–thaw cycles. *p < 0.05. COI, cut‐off indices.

DISCUSSION

Since the beginning of the pandemic, CCP has been used for therapeutic purposes in our clinics. However, CCP was sometimes not immediately available in the required quantities, and we therefore asked ourselves whether longer storage periods than currently applied would be possible. Our study provides insights into the stability and in vitro functionality of SARS‐CoV‐2 anti‐N and anti‐S antibodies in CCP and serum after 2 and 3 years' storage at below −30°C. Overall, no significant differences in the change of antibody levels were found. For the few reductions observed, the differences were minimal, suggesting the overall preserved integrity of anti‐N and anti‐S antibodies after storage. The reductions observed for CCP compared to the corresponding serum could be explained by a dilution effect, as citrate phosphate dextrose is not present in serum but is present in CCP. Furthermore, we investigated the effects of repeated freezing and thawing of plasma samples which were stored for 2 years at less than −40°C with regard to SARS‐CoV‐2 antibody levels and their in vitro functionality. We found no significant reduction in quantity or in vitro inhibition. Our data reflect the impact of long‐term storage and repeated freezing and thawing, confirming the preservation of antibody functionality. This resistance to repeated freeze–thaw cycles allows flexible storage and transportation, which is practical in remote or underserved areas where fresh donations may be limited.

Our findings support the usefulness of stockpiling CCP for extended periods, which is of particular interest for individuals with immunosuppression who are unable to build up sufficient antibody response and thus still remain impacted by SARS‐CoV‐2. Patients with a primary immunodeficiency, usually characterized by B‐cell defects and suppressed or non‐functional antibody production, showed rapid clinical improvement after CCP treatment [8, 10]. For individuals with a secondary immunodeficiency such as haematological malignancies and solid organ transplant, CCP treatment has been shown to result in enhanced viral clearance and a mortality benefit [8, 10, 31]. Vaccination is recommended for immunocompromised individuals; however, the rapid SARS‐CoV‐2 mutation rate leads to variants resistant to antibodies provided by available vaccines [32]. Furthermore, the need for immunosuppressant therapy can result in blocking the humoral response to the vaccine, resulting in a reduced protective effect [33, 34]. Therefore, immunocompromised individuals are frequently still in need of CCP treatment. In addition, CCP may also be beneficial for patients with post‐COVID‐19 syndrome, as described recently [35, 36]. Thus, stockpiling of CCPs ensures a ready supply of therapeutic plasma for these vulnerable populations. The rapid mutation rate of SARS‐CoV‐2 could be an issue regarding therapeutic efficacy. However, CCP is thought to confer hybrid immunity, as donors are likely to have been exposed to different virus variants and may have antibodies acquired through the vaccine, with this mixture of antibodies protecting against new SARS‐CoV‐2 variants [37, 38]. This could be an advantage in fighting new variants, as the production of vaccinations takes time whereas CCPs are more readily available.

For the present study, CCPs collected in 2020 contain endogenous antibodies only, as no vaccination was available at that time. As described previously, 77% of Austrian blood donors were vaccinated against SARS‐CoV‐2 at least once since their enrolment in 2021 [39]. Thus, plasma analysed for this study is likely to contain a mixture of natural and vaccine‐acquired antibodies. Furthermore, our study encompassed plasma covering 3 years of the COVID‐19 pandemic, including different circulating virus variants. As we did not observe significant differences at any time, our findings suggest that antibody levels remain stable in CCPs derived from different donors with different modes of antibody acquisition and likely exposure to various SARS‐CoV‐2 variants.

Our study has several strengths and limitations. It provides data regarding the stability of SARS‐CoV‐2 anti‐N and anti‐S antibodies in serum and plasma for up to 3 years post donation. Data included were obtained from two collection centres producing different plasma product types and applying different screening assays. Furthermore, our study assessed the impact of repeated freezing and thawing on antibody quantity and in vitro functionality, revealing stable antibody levels and neutralizing capacities. Limitations include the small sample size, especially for tests involving multiple freeze–thaw cycles, which may limit the statistical power. Although sVNTs are suggested as valid alternatives for conventional neutralization assays [28], this assay might yield false‐negative results for samples with low viral titre and because only neutralizing antibodies directed against RBD are measured. Moreover, mutations resulting in variants with diverging virulence, life cycle and virion composition may negatively affect these results. Our study involved plasma collected during different phases of the pandemic but did not account for potential donor variability in terms of immune response. This includes differences in antibody titres due to vaccination status, severity of infection and donor demographics such as age. Furthermore, our study focused on laboratory measurements without direct correlation to clinical outcomes. To address these limitations, future studies should stratify donors based on age, general health status and vaccination history to provide insights on how these factors may affect antibody durability. Further research should aim to correlate antibody stability and functionality with clinical outcomes, proving the effectiveness of later‐thawed samples. This will provide a more comprehensive understanding of the therapeutic value of stored plasma. In addition, our study focused on SARS‐CoV‐2 antibodies, so our results may not be applicable to antibodies against other pathogens.

In conclusion, our data show that CCPs subjected to long‐term storage retain the initially present SARS‐CoV‐2 anti‐N and anti‐S antibody levels and that antibody functionality may be preserved. Our results suggest an extension of the current regulatory proposed periods for CCP storage and support the usefulness of stockpiling plasma, thus ensuring a constant and immediate supply of effective therapeutic plasma for vulnerable populations, including individuals with haematological malignancies, solid organ transplantation or autoimmune diseases.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Laner‐Plamberger S, Siller A, Lauth W, Kern JM, Baskova L, Held N, et al. Stable SARS‐CoV‐2 antibody levels and functionality in serum and COVID‐19 convalescent plasma after long‐term storage. Vox Sang. 2025;120:784–792.

DATA AVAILABILITY STATEMENT

Data supporting the findings of this study are available within the article. Datasets are available from corresponding authors on reasonable request.