Neutralization of Rubella Vaccine Virus and Immunodeficiency-Related Vaccine-Derived Rubella Viruses by Intravenous Immunoglobulins.

The association between granulomas and vaccine-derived rubella virus (VDRV) in people with primary immune deficiencies (PID) has raised concerns about the ability of immunoglobulin (IG) preparations to neutralize VDRVs. We investigated the capacity of IG to neutralize rubella vaccine virus and four VDRV strains. As expected, the rubella vaccine virus itself was potently neutralized by IG preparations; however, the VDRV isolates from patients after intra-host evolution, 2-6 times less so. Diagnosis of immune deficiencies before possible live-virus vaccination is thus of critical importance, while IG replacement therapy can be expected to provide protection from rubella virus infection.

The occurrence of granulomas associated with vaccine derived rubella viruses (VDRV) in people with primary immune deficiencies (PID) challenges immunoglobulin (IG) preparations regarding their rubella neutralizing ability. This study confirmed potent rubella virus neutralization capacity of IG preparations and thus suggests protection of IG-treated PID patients against rubella. The study also highlights the importance of early diagnosis and timely given IG to prevent possible systemic spread of VDRV persisting locally in granulomas.

Clinical efficacy of SARS-CoV-2 Omicron-neutralizing antibodies in immunoglobulin preparations for the treatment of agammaglobulinemia in patients with primary antibody deficiency.

Immunocompromised individuals are at significantly elevated risk for severe courses of coronavirus disease 2019 (COVID-19). In addition to vaccination, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (nAbs) have been applied throughout the pandemic, with time of treatment onset and potency against the currently prevailing virus variant identified as relevant factors for medical benefit. Using data from the European Society for Immunodeficiencies (ESID) registry, the present study evaluated COVID-19 cases in three groups of patients with inborn errors of immunity (IEI; 981 agammaglobulinemia patients on immunoglobulin replacement therapy (IGRT); 8960 non-agammaglobulinemia patients on IGRT; 14 428 patients without IGRT), and the neutralizing capacity of 1100 immunoglobulin lots against SARS-CoV-2 ("Wuhan" and Omicron strains), throughout 3 years. From the first (2020/2021) to the second (2021/2022) cold season, i.e., during the virus drift to the more contagious Omicron variants, an increase in case numbers was recorded that was comparable (~2- to 3-fold) for all three study groups. During the same period, immunoglobulin lots showed a profound nAb increase against the archetypal SARS-CoV-2 strain, yet only low levels of Omicron nAbs. Notably, shortly before the third (2022/2023) cold season, Omicron-neutralizing capacity of released immunoglobulin lots had plateaued at high levels. From the second to the third cold season, COVID-19 cases dropped markedly. While a ~6-fold case reduction was recorded for the groups of non-agammaglobulinemia patients on IGRT and IEI patients not receiving IGRT, the decline was ~30-fold for the group of agammaglobulinemia patients on IGRT. These findings suggest a substantial COVID-19-protective effect of IGRT, at least for distinct groups of antibody-deficient patients.

Human Circovirus is not detected in plasma pools for fractionation.

BACKGROUND: Human Circovirus 1 and 2 were recently described in a French hepatitis case and in two Chinese drug users. Because of its small size and presumable high resistance to both inactivation and removal by nanofilters, such viruses-if determined to be even pathogenic-should be considered with respect to the safety of plasma derivatives. We, therefore, investigated the prevalence and titer of these viruses in plasma pools before fractionation. METHODS AND MATERIALS: We tested for the presence of Human Circovirus 1 and 2 by qPCR in 48 plasma pools derived from healthy donors from Europe, USA, and Japan, corresponding to more than 200,000 plasma donations. RESULTS: We did not detect the presence of Human Circovirus 1 and 2 in any of the plasma pools, with a limit of detection of 300-600 genome copies per mL of plasma. CONCLUSIONS: These results indicate that high levels of circovirus are not widely prevalent in such donations.

Detection of Minute virus of mice strains in different cell lines: Implications for adventitious agent testing.

Minute virus of mice (MMV) has contaminated biotechnological processes in the past and specific MMV testing is therefore recommended, if the production cell line is known to be permissive for this virus. Testing is widely done using cell-culture-based adventitious virus assays, yet MMV strains may differ in their in vitro cell tropism. Here, we investigated the growth characteristics of different MMV strains on A9 and 324K cells and identified significant differences in susceptibility of these widely used indicator cell lines to infection by different strains of MMV, which has implications for MMV detectability during routine testing of biotechnology process harvests. An MMV-specific polymerase chain reaction was evaluated as a more encompassing method and was shown as suitable replacement for cell culture-based detection of the different MMV strains, with the additional benefit that detection is more rapid and can be extended to other rodent parvoviruses that might contaminate biotechnological processes. Although no MMV contamination event of human-derived cell lines has happened in the past, biotechnological processes that are based on these also need to consider MMV-specific testing, as, for example, HEK293, a human-derived cell line commonly used in biopharmaceutical manufacturing, was shown as susceptible to productive MMV infection in the current work.

Protection of biomanufacturing processes from virus contamination through upstream virus filtration of cell culture media.

Cell-based manufacturing processes have occasionally been exposed to adventitious viruses, leading to manufacturing interruptions and unstable supply situations. The rapid progress of advanced therapy medicinal products needs innovative approaches to avoid any unwelcome reminder of the universal presence of viruses. Here, we investigated upstream virus filtration as a clearance step for any product too complex for downstream interventions. Culture media virus filtration was investigated with respect to virus clearance capacities under extreme conditions such as high process feed loading (up to ~19,000 L/m²), long duration (up to 34 days), and multiple process interruptions (up to 21 h). The small nonenveloped Minute virus of mice was used as relevant target virus, and as worse-case challenge for the investigated virus filters with a stipulated pore-size of about 20 nm. Certain filters-especially of the newer second generation-were capable of effective virus clearance despite the harsh regimen they were subjected to. The biochemical parameters for un-spiked control runs showed the filters to have no measurable impact on the composition of the culture media. Based on these findings, this technology seems to be quite feasible for large volume premanufacturing process culture media preparations.

On-column virus inactivation by solvent/detergent treatment for a recombinant biological product.

Each process step in the manufacture of biological products requires expensive resources and reduces total process productivity. Since downstream processing of biologicals is the main cost driver, process intensification is a persistent topic during the entire product life cycle. We present here one approach for the intensification of bioprocesses by applying on-column virus inactivation using solvent/detergent (S/D) treatment during ion-exchange chromatography. The established purification process of a recombinant protein was used as a model to compare key process parameters (i.e., product yield, specific activity, impurity clearance) of the novel approach to the standard process protocol. Additional wash and incubation steps with and without S/D-containing buffers were introduced to ensure sufficient contact time to effectively eliminate enveloped viruses and to significantly decrease the amount of S/D reagents. Comparison of key process parameters demonstrated equivalent process performance. To assess the viral clearance capacity of the novel approach, XMuLV was spiked as model virus to the chromatographic load and all resulting fractions were analyzed by TCID50 and RT-qPCR. Data indicates the inactivation capability of on-column virus inactivation even at 10% of the nominal S/D concentration, although the mechanism of viral clearance needs further investigation.

Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

Detergent-Mediated Virus Inactivation in Biotechnological Matrices: More than Just CMC.

For decades, the ability of detergents to solubilize biological membranes has been utilized in biotechnological manufacturing to disrupt the lipid envelope of potentially contaminating viruses and thus enhance the safety margins of plasma- and cell-derived drugs. This ability has been linked to detergent micelles, which are formed if the concentration of detergent molecules exceeds the critical micelle concentration (CMC). Traditionally, the CMC of detergents is determined in deionized water (ddH2O), i.e., a situation considerably different from the actual situation of biotechnological manufacturing. This study compared, for five distinct detergents, the CMC in ddH2O side-by-side with two biopharmaceutical process intermediates relevant to plasma-derived (Immunoglobulin) and cell-derived (monoclonal antibody) products, respectively. Depending on the matrix, the CMC of detergents changed by a factor of up to ~4-fold. Further, the CMC in biotechnological matrices did not correlate with antiviral potency, as Triton X-100 (TX-100) and similar detergents had comparatively higher CMCs than polysorbate-based detergents, which are known to be less potent in terms of virus inactivation. Finally, it was demonstrated that TX-100 and similar detergents also have virus-inactivating properties if applied below the CMC. Thus, the presence of detergent micelles might not be an absolute prerequisite for the disruption of virus envelopes.

Rapidly Increasing Severe Acute Respiratory Syndrome Coronavirus 2 Neutralization by Intravenous Immunoglobulins Produced From Plasma Collected During the 2020 Pandemic.

Immunoglobulin lots (N = 176) released since March 2020 were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies, with first positive results for September 2020 lots (mean, 1.7 IU/mL; 46% of lots positive). From there, values steadily increased, in correlation with the cumulative coronavirus disease 2019 (COVID-19) incidence, to reach a mean of 31.2 IU/mL and 93% of lots positive by January 2021. Extrapolating the correlation, immunoglobulins could reach an anti-SARS-CoV-2 potency of approximately 345 IU/mL by July 2021. At that stage, prophylactic immunoglobulin treatment for primary/secondary immunodeficiency could contain similar doses of anti-SARS-CoV-2 as convalescent plasma that is used for treatment of COVID-19.

Omicron Severe Acute Respiratory Syndrome Coronavirus 2 Neutralization by Immunoglobulin Preparations Manufactured From Plasma Collected in the United States and Europe.

After >2 years of the coronavirus disease 2019 (COVID-19) pandemic, immunoglobulins (IGs) contain highly potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies, based on the large proportion of United States (US) plasma donors who have gone through COVID-19 or vaccination against the virus. Neutralization of Omicron SARS-CoV-2 by antibodies generated after non-Omicron infection or vaccination has been lower though, raising concerns about the potency of IG against this new virus variant. Also, as plasma collected in the US remains the main source of IG, the neutralization of SARS-CoV-2 for plasma collected elsewhere has been less well studied. Here, we confirm Omicron neutralization by US as well as European Union plasma-derived IG lots.

Orthopox viruses and the safety margins of solvent-detergent treated plasma-derived medicinal products.

BACKGROUND: The currently ongoing outbreak of monkeypox virus in many non-endemic countries around the world has also raised concerns about the safety of plasma-derived medicinal products. Based on what is known about the poxviridae, that is, that members are exceedingly large and carry a lipid envelope, effective removal and inactivation by plasma product manufacturing processes is expected. For the widely used solvent-detergent (S/D) treatments, however, poxviruses have been reported as potentially being a bit more resistant. STUDY DESIGN AND METHODS: Using a S/D mixture comprising tri-n-butyl-phosphate, polysorbate 80 and Triton X-100 (TX-100), inactivation of vaccinia virus (a model closely resembling monkeypox virus, both within the same genus, i.e., Orthopoxvirus) in a plasma-derived process intermediate was analyzed over 60 min. As use of Triton X-100 will, based on environmental concerns, be restricted, similar experiments were conducted with a physicochemically virtually identical alternative, Nereid. RESULTS: Fast inactivation of vaccinia virus to the assay detection limit, that is, reduction of infectivity by greater than 4 log10 within 10-20 min, was measured for the TX-100 S/D mixture. The alternative S/D mixture (Nereid instead of TX-100) was found fully equivalent. CONCLUSION: As for other lipid-enveloped viruses, treatment of process intermediates with S/D mixtures containing TX-100 or the closely related detergent Nereid are highly effective in inactivating poxviruses. Thus, the current spread of monkeypox virus does not compromise the viral safety margins of plasma-derived medicines.

Plasma procurement and plasma product safety in light of the COVID-19 pandemic from the perspective of the plasma industry.

This review, written from the perspective of the plasma industry, discusses plasma procurement and plasma product safety in light of the COVID-19 pandemic. The COVID-19 pandemic impacted the whole world and, therefore, not unexpectedly, the pharmaceutical industry too. In spite of this, the plasma protein industry has continued to provide life saving therapies to critically ill patients. Moreover, companies have collected COVID convalescent plasma (CP) to support development of investigational therapies, for example, hyperimmune globulins to potentially treat SARS-CoV-2 infection, and collaborated with those collecting COVID CP for direct transfusion, which has been made available under emergency use in the United States. For plasma that is fractionated to become a therapy, general knowledge of coronaviruses and numerous new studies on the structure and function of SARS-CoV-2 provide reassurance that existing industry precautions, including donor selection, as well as virus inactivation and removal steps during the manufacturing process are sufficient to maintain the high standards of virus safety of plasma products. The pandemic also revealed the vulnerability and inadequacy of the current plasma ecosystem. There is a need for more plasma to be collected around the world to meet the growing need for safe and efficacious plasma-derived therapies. This requires outdated regulatory and policy restrictions to be realigned with current scientific evidence. More countries around the world should be in a position to contribute to global supply of plasma so that patients with life-threatening conditions - and often no alternative therapeutic solutions - have better access to care.

Highly Potent SARS-CoV-2 Neutralization by Intravenous Immunoglobulins manufactured from Post-COVID-19 and COVID-19-Vaccinated Plasma Donations.

From September 2020, some immunoglobulin lots from US plasma contained neutralizing antibodies against the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Paralleled by the increasing numbers of post-coronavirus disease 2019 (COVID-19) donors, immunoglobulin lot antibody positivity increased to 93% by January 2021, at a mean titer of approximately 30 IU/mL. The correlation predicted that anti-SARS-CoV-2 potency would reach 345 IU/mL by July 2021. In addition to post-COVID-19 donors, the rapidly increasing number of plasma donors vaccinated against COVID-19 resulted in a mean antibody titer of >600 IU/mL in July 2021 immunoglobulin lots, with SARS-CoV-2 antibody titers for several lots even higher than those of earlier produced hyperimmune globulin products.

Synthesis of "Nereid," a new phenol-free detergent to replace Triton X-100 in virus inactivation.

In the 1980s, virus inactivation steps were implemented into the manufacturing of biopharmaceuticals in response to earlier unforeseen virus transmissions. The most effective inactivation process for lipid-enveloped viruses is the treatment by a combination of detergents, often including Triton X-100 (TX-100). Based on recent environmental concerns, the use of TX-100 in Europe will be ultimately banned, which forces the pharmaceutical industry, among others, to switch to an environmentally friendly alternative detergent with fully equivalent virus inactivation performance such as TX-100. In this study, a structure-activity relationship study was conducted that ultimately led to the synthesis of several new detergents. One of them, named "Nereid," displayed inactivation activity fully equivalent to TX-100. The synthesis of this replacement candidate has been optimized to allow for the production of several kg of detergent at lab scale, to enable the required feasibility and comparison virus inactivation studies needed to support a potential future transition. The 3-step, chromatography-free synthesis process described herein uses inexpensive starting materials, has a robust and simple work-up, and allows production in a standard organic laboratory to deliver batches of several hundred grams with >99% purity.

Longitudinal analysis of SARS-CoV-2 antibodies in 8000 U.S. first-time convalescent plasma donations.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent individuals carry antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that, through a plasma donation, can be used as a potential therapeutic either in direct transfusion or for the manufacture of hyperimmune globulin (HIG). The success of such interventions depends on the antibody potency in such plasma donations, but little information on the collection of potent units is currently available. STUDY DESIGN AND METHODS: A total of 8749 plasma units, collected from April until September 2020 from first-time U.S. COVID-19 convalescent plasma donors, were characterized for SARS-CoV-2 immunoglobulin G (IgG) antibodies by Abbott chemiluminescent microparticle immunoassay (CMIA). The period between COVID-19 onset until donation and donor age, ethnicity, sex, and COVID-19 severity were evaluated against the obtained signal (index S/C). RESULTS: A marked decrease in mean index S/C was seen over the plasma collection period surveyed, which was significantly correlated to decreases in mean plasma donor age (p < .0001; R2 = .726) and percentage of donations obtained from COVID-19 convalescent patients who had been hospitalized (p = .001; R2 = .4426). The highest titer plasma units were obtained soon after convalescence from COVID-19 patients who required hospitalization, from advanced age donors, and from Black/African/Hispanic American versus White/Caucasian ethnicities, whereas there was no effect of donor sex on the values obtained with the Abbott CMIA. CONCLUSION: Since the onset of the pandemic, the average SARS-CoV-2 IgG values of first-time U.S. COVID-19 convalescent plasma donations have significantly dropped, mainly due to donations from progressively younger aged donors who tend to experience less severe COVID-19.

Characterization of 100 sequential SARS-CoV-2 convalescent plasma donations.

BACKGROUND: Transfusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent plasma is a promising treatment for severe coronavirus disease 2019 (COVID-19) cases, with success of the intervention based on neutralizing antibody content. Measurement by serologic correlates without biocontainment needs as well as an understanding of donor characteristics that may allow for targeting of more potent donors would greatly facilitate effective collection. STUDY DESIGN AND METHODS: One hundred convalescent plasma units were characterized for functionally active SARS-CoV-2 neutralizing antibodies, as well as for SARS-CoV-2 binding antibodies, with the intention to establish a correlation between the functionally more relevant neutralization assay and the more accessible enzyme-linked immunosorbent assay (ELISA). Donor demographics such as COVID-19 severity, age, and sex were correlated with antibody titers. RESULTS: A mean neutralization titer 50% of 230 (range, <8-1765) was seen for the 100 convalescent plasma units, with highly significant (P < .0001) yet quantitatively limited (R2 = 0.2830) correlation with results of the ELISA. Exclusion of units with particularly high titers (>500) from analysis improved correlation (R2 = 0.5386). A tendency of higher-titer plasma units from donors with increased disease severity, of advanced age, and of male sex was seen, yet the functional relevance of this difference is questionable. CONCLUSION: The ELISA-based correlation to neutralization titer enabled a threshold proposal that could be used to eliminate lower-titer units from the clinical supply for COVID-19 treatment. Disease severity may be associated with the development of higher titers of neutralizing antibodies, although larger case numbers will be needed for additional confirmation.

No SARS-CoV-2 Neutralization by Intravenous Immunoglobulins Produced From Plasma Collected Before the 2020 Pandemic.

The 2020 SARS-CoV-2 pandemic is caused by a zoonotic coronavirus transmitted to humans, similar to earlier events. Whether the other, seasonally circulating coronaviruses induce cross-reactive, potentially even cross-neutralizing, antibodies to the new species in humans is unclear. The question is particularly relevant for people with immune deficiencies, as their health depends on treatment with immunoglobulin preparations that need to contain neutralizing antibodies against the pathogens in their environment. Testing 54 intravenous immunoglobulin preparations, produced from plasma collected in Europe and the United States, confirmed highly potent neutralization of a seasonal coronavirus; however, no cross-neutralization of the new SARS-CoV-2 was seen.

Antibody-enhanced hepatitis E virus nanofiltration during the manufacture of human immunoglobulin.

BACKGROUND: Circulation of hepatitis E virus (HEV) in areas where plasma is sourced for the manufacture of plasma-derived medicinal products (PDMPs) has prompted verification of HEV clearance. HEV exists as quasi lipid-enveloped (LE) and non-lipid-enveloped (NLE) forms, which might be of relevance for HEV clearance from manufacturing processes of antibody-containing PDMPs with solvent/detergent (S/D) treatment upstream of further clearance steps. STUDY DESIGN AND METHODS: Presence of different HEV particles in stocks used in clearance studies was investigated, with nanofilters graded around the assumed HEV particle sizes and by gradient centrifugation. HEV removal by 35-nm nanofiltration was investigated in the presence or absence of HEV antibodies, in buffer as well as in immunoglobulin (IG) manufacturing process intermediates. RESULTS: HEV particles consistent with LE, NLE, and an "intermediate" (IM) phenotype, obtained after S/D treatment, were seen in different HEV stocks. In the absence of HEV antibodies, log reduction factors (LRFs) of 4.0 and 2.5 were obtained by 35-nm nanofiltration of LE and IM HEV, consistent with the larger and smaller sizes of these phenotypes. Addition of HEV antibodies enhanced IM HEV removal around 1000-fold (LRF, 5.6). Effective (LRF, >4.8 and >4.0) HEV removal was obtained for the nanofiltration processing step for IG intermediates with varying HEV antibody content. CONCLUSION: HEV spikes used in clearance studies should be carefully selected, as differences in physicochemical properties might affect HEV clearance. Antibody-mediated enhancement of HEV nanofiltration was demonstrated in IG process intermediates even at low HEV antibody concentration, illustrating the robustness of this manufacturing step.

Nanofiltration as a robust method contributing to viral safety of plasma-derived therapeutics: 20 years' experience of the plasma protein manufacturers.

BACKGROUND: Nanofiltration entails the filtering of protein solutions through membranes with pores of nanometric sizes that have the capability to effectively retain a wide range of viruses. STUDY DESIGN AND METHODS: Data were collected from 754 virus validation studies (individual data points) by Plasma Protein Therapeutics Association member companies and analyzed for the capacity of a range of nanofilters to remove viruses with different physicochemical properties and sizes. Different plasma product intermediates were spiked with viruses and filtered through nanofilters with different pore sizes using either tangential or dead-end mode under constant pressure or constant flow. Filtration was performed according to validated scaled-down laboratory conditions reflecting manufacturing processes. Effectiveness of viral removal was assessed using cell culture infectivity assays or polymerase chain reaction (PCR). RESULTS: The nanofiltration process demonstrated a high efficacy and robustness for virus removal. The main factors affecting nanofiltration efficacy are nanofilter pore size and virus size. The capacity of nanofilters to remove smaller, nonenveloped viruses was dependent on filter pore size and whether the nanofiltration process was integrated and designed with the intention to provide effective parvovirus retention. Volume filtered, operating pressure, and total protein concentration did not have a significant impact on the effectiveness of virus removal capacity within the investigated ranges. CONCLUSIONS: The largest and most diverse nanofiltration data collection to date substantiates the effectiveness and robustness of nanofiltration in virus removal under manufacturing conditions of different plasma-derived proteins. Nanofiltration can enhance product safety by providing very high removal capacity of viruses including small non-enveloped viruses.

Truly continuous low pH viral inactivation for biopharmaceutical process integration.

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities-which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.