Abstract
Background
In late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus emerged in China and quickly spread into a worldwide pandemic. Prior to the development of specific drug therapies or a vaccine, more immediately available treatments were sought including convalescent plasma. A potential improvement from convalescent plasma could be the preparation of anti‐SARS‐CoV‐2 hyperimmune globulin (hIVIG).
Study Design and Methods
Convalescent plasma was collected from an existing network of plasma donation centers. A caprylate/chromatography purification process was used to manufacture hIVIG. Initial batches of hIVIG were manufactured in a versatile, small‐scale facility designed and built to rapidly address emerging infectious diseases.
Results
Processing convalescent plasma into hIVIG resulted in a highly purified immunoglobulin G (IgG) product with more concentrated neutralizing antibody activity. hIVIG will allow for the administration of greater antibody activity per unit of volume with decreased potential for several adverse events associated with plasma administration. IgG concentration and IgG specific to SARS‐CoV‐2 were increased over 10‐fold from convalescent plasma to the final product. Normalized enzyme‐linked immunosorbent assay activity (per mg/ml IgG) was maintained throughout the process. Protein content in these final product batches was 100% IgG, consisting of 98% monomer and dimer forms. Potentially hazardous proteins (IgM, IgA, and anti‐A, anti‐B, and anti‐D) were reduced to minimal levels.
Conclusions
Multiple batches of anti‐SARS‐CoV‐2 hIVIG that met regulatory requirements were manufactured from human convalescent plasma. The first clinical study in which the hIVIG will be evaluated will be Inpatient Treatment with Anti‐Coronavirus Immunoglobulin (ITAC) [NCT04546581].
Keywords: convalescent plasma, COVID‐19, COVID‐19 convalescent hyperimmune, hIVIG, immune globulin
1. INTRODUCTION
The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) emerged in late 2019 from the Wuhan region of China, 1 and by early 2020, the resulting infection, termed coronavirus disease 2019 (COVID‐19), had reached pandemic status according to the World Health Organization. 2 The novelty of this pathogen prompted an urgent search for therapeutic agents with efficacy against SARS‐CoV‐2. Convalescent plasma was quickly identified and investigated as a potential treatment for COVID‐19. 3
Convalescent plasma has a long history of treatment of infectious diseases extending from the Spanish flu pandemic 4 to more recent outbreaks of severe acute respiratory syndrome (SARS) 5 and Middle East respiratory syndrome. 6 There are, however, some disadvantages of convalescent plasma, including that the nature, titer, and neutralizing power of the antibodies therein can vary greatly from one donor to another. 7
Many of these disadvantages can be overcome through the purification and concentration of the specific antibodies into drug preparations. Human immunoglobulin products made from pooled plasma of convalescent or vaccinated donors have been used for many years in the treatment of various infections. Here, we report on collection of convalescent plasma and manufacture anti‐SARS‐CoV‐2 hyperimmune globulin (hIVIG) for use in clinical studies.
2. MATERIALS AND METHODS
2.1. Collection of convalescent plasma
Plasma was collected from COVID‐19 convalescent donors using a network of plasma donation centers geographically dispersed throughout the United States (Grifols network: Biomat USA Inc., Interstate Blood Bank Inc., and Talecris Plasma Resources, Inc.). All donations were collected by plasmapheresis and met the requirements for source plasma, which includes screening for a number of common viruses. 8 , 9 All donors were required to have laboratory evidence of COVID‐19 infection, either through nucleic acid amplification testing (NAT) or positive antigen test or by SARS‐CoV‐2 antibody test prior to enrollment. Symptomatic donors must have complete resolution of symptoms at least 14 days before donation if they were negative by follow‐up NAT or 28 days if they had no follow‐up test. Similarly, asymptomatic donors (positive by NAT or antigen tests) must wait 14 days after the initial test if they had a follow‐up negative NAT but must wait 28 days after the initial test if they had no follow‐up test. Asymptomatic donors tested only by a SARS‐CoV‐2 antibody test were required to wait 7 days prior to donation but could donate immediately if they had a negative NAT. Donors may be male or female, but females who have been pregnant must test negative for human leukocyte antigen (HLA) class I and class II antibodies. 10 All donations were tested by NAT (Procleix SARS‐CoV‐2 Assay; Grifols Diagnostic Systems, Emeryville, CA) to confirm absence of SARS‐CoV‐2 infection and by serology to confirm a suitable level of anti‐SARS‐COV‐2 immunoglobulin G (IgG) (Architect SARS‐CoV‐2 IgG Assay; Abbott Ireland, Diagnostics Division, Finisklin Business Park, Sligo, Ireland, or Recombivirus Human Anti‐SARS‐CoV‐2 Virus Spike 1 IgG ELISA Kit; Alpha Diagnostic International, San Antonio, TX). Plasma donors may donate up to two times a week, with individual donations of up to 880 mL, depending on donor weight. 8 , 9
2.2. Manufacture of convalescent SARS‐CoV‐2 immune globulin, human
Prior to pooling, plasma pools were modeled to maintain distribution consistent with overall donor ABO blood type distribution. This maintained consistent batch‐to‐batch levels of anti‐A and anti‐B. Type O and Type B donors were limited to no more than two units from any single donor to decrease the likelihood of having high anti‐A titers in the final product.
Pooled convalescent plasma was subjected to alcohol fractionation and caprylate/chromatography purification, resulting in highly purified IgG solutions formulated at 10% protein with glycine at a low pH. These production processes, including formulation, are identical to those for Gamunex immune globulin. 11 , 12 These processes include multiple steps validated for the removal and/or inactivation of viruses. 13 Impurity fractions such as albumin were not used for further manufacture of clinical product.
2.3. Characterization of hIVIG product
2.3.1. Routine batch testing
Routine batch quality control testing of hIVIG includes several measurements to characterize the product and ascertain that it is suitable for use. This characterization includes analyses for glycine, pH, protein concentration, osmolality, composition by electrophoresis, molecular weight profiling by size exclusion chromatography, and anticomplement activity. Analyses are also performed for sodium caprylate, residual IgA and IgM, prekallikrein activator, factor XIa, anti‐A, anti‐B, and anti‐D. These tests have recently been described for other immune globulin products manufactured with the caprylate/chromatography process. 12 In addition, compendial tests for sterility and pyrogenic substances are performed on all batches. 14
2.3.2. Anti‐SARS‐CoV‐2 ELISA
Anti‐SARS‐CoV‐2 IgG titers were determined using Human Anti‐SARS‐CoV‐2 Virus Spike 1 (S1) IgG assay (Alpha Diagnostic). hIVIG batches were tested using multiple serial dilutions and a curve constructed by plotting the log of the optical density as a function of the log of the dilution. The titer was defined as the reciprocal dilution at which this curve is equal to the low kit standard. Antibody levels are also expressed as a ratio to an in‐house control, which consists of a commercially available chimeric monoclonal SARS‐CoV‐2 S1 antibody (Sino Biologicals, Beijing, China) spiked into plasma from non‐COVID‐19 donors at levels intended to be similar to those found in the plasma of COVID‐19 donors.
2.3.3. SARS‐CoV‐2 neutralizing antibody assay
Multiple batches of hIVIG were tested for COVID‐19 antibodies using an immunofluorescence‐based neutralization assay performed at the National Institutes of Health Integrated Research Facility, Frederick, MD. 15 This assay quantifies the anti‐SARS‐CoV‐2 neutralization titer using a dilution series of test material tested for inhibition of infection of cultured Vero (CCL‐81) cells by SARS‐CoV‐2 (Washington isolate, CDC) at a multiplicity of infection of 0.5. Potency was assessed using a cell‐based immunosorbent assay to quantify infection by detecting the SARS‐CoV‐2 nucleoprotein using a specific antibody raised against the SARS‐CoV‐2 nucleoprotein (Sino Biological, Wayne, PA). The secondary antibody (Life Technologies, San Diego, CA) was conjugated to Alexa Fluor 594, and cells were quantified with a an Operetta high‐content imaging system (Perkin Elmer, Waltham, MA). A minimum of 16,000 cells are counted per sample dilution across four wells–two each in duplicate plates. Data are reported based on a four‐parameter regression curve (using a constrained fit) as a 50% neutralization titer (IC50).
3. RESULTS
The ABO blood typing results from the initial 500 plasma units used for manufacturing hIVIG batches are presented in Table 1. Results from two published studies are included as comparators.
TABLE 1.
ABO blood type distribution of convalescent plasma from test batches for this study compared with published values
To assess the recovery of the SARS‐CoV‐2–specific antibodies, results from testing with an IgG‐specific ELISA and a neutralizing antibody assay are presented in Table 2 for the first product batches produced and their corresponding plasma pools. The IgG‐specific ELISA titers increased more than 10‐fold when processing the pooled plasma into the final product. Neutralizing activity increased approximately threefold. The IgG concentration was also increased by more than 10‐fold from the pooled plasma to the final product. ELISA titers normalized to IgG were similar in the starting material and the finished product.
TABLE 2.
Anti‐SARS‐CoV‐2 titers and specific activities (n = 3, ±SD)
| Sample point | ELISA titer, 1:X | Neutralizing activity titer, IC50, 1:X | IgG, mg/ml | Normalized ELISA titer, titer/mg/ml IgG |
|---|---|---|---|---|
| Pool | 9038 ± 4037 | 110 ± 15 | 8.0 ± 0.2 | 1131 ± 497 |
| Final product | 125,089 ± 50,905 | 325 ± 76 | 103 ± 3 | 1213 ± 477 |
Quality control test results from the first three batches are presented in Table 3.
TABLE 3.
COVID‐19 hyperimmune product characteristics (n = 3, ±SD)
| Test | Average |
|---|---|
| Potency | |
| ELISA titer (1:X) | 125,089 ± 50,905 |
| ELISA level (ratio to control) | 27.1 ± 2.1 |
| Neutralization titer (IC50, 1:X) | 325 ± 76 |
| Formulation | |
| Glycine, M | 0.20 ± 0.02 |
| pH, diluted | 4.3 ± 0.1 |
| Protein concentration, % | 10.3 ± 0.3 |
| Osmolality, mOsmol/kg | 246 ± 23 |
| Molecular profile and purity | |
| Protein composition, gamma globulin % | 100 |
| Monomer + dimer, % | 100 |
| Aggregate, % | <1 |
| Fragment, % | <1 |
| Anticomplement activity, CH50/mg IgG | 0.47 ± 0.21 |
| Sodium caprylate, μg/ml | <8 |
| IgA, mg/ml | 0.038 ± 0.003 |
| IgM, mg/ml | ≤0.007 |
| Prekallikrein activator (PKA), IU/ml | <1.0 |
| FXIa, mIU/ml | <1 |
| Anti‐A titer, 1:X | 29.0 ± 5.2 |
| Anti‐B titer, 1:X | 16.0 |
| Anti‐D titer, 1:X | <2 |
In these test batches, 100% of the protein content was IgG. The IgG was present entirely as monomers and dimers with aggregates and fragments below the limits of detection. A process impurity (sodium caprylate) and plasma protein impurities were found at very low concentrations in the final product.
4. DISCUSSION
ABO distribution in the plasma used to make these batches of hIVIG was found to be similar to published distributions. 16 , 17 Modeling of the ABO distribution of units pooled was performed to control anti‐A and anti‐B levels, which are normally limited by dilution with larger batch sizes used in the manufacture of commercial immune globulin products. The modeled distribution resulted in products that all meet the anti‐A and anti‐B ≤1:64 requirements used by manufacturers of IGIV products to reduce the risk of hemolytic activity in patients. 18 , 19 Hemolytic reactions are a rare event occurring in <1 in 10,000 patients and are often without serious sequelae. These reactions are more common in non‐O blood groups and have been found to be influenced by the blood type distribution of the plasma donors. 20
Product characteristics for hIVIG were similar to those for Gamunex. The caprylate/chromatography purification process results in a highly purified IgG product that maintains the IgG activity found in the starting plasma. Additionally, experience with hIVIG preparations for rabies, tetanus, and hepatitis B provide a reasonable expectation for the safety and effectiveness of hIVIG.
During processing, the normalized SARS‐Cov‐2 antibody activity (expressed relative to IgG concentration) was maintained, as expected for a purified IgG product. Antibody neutralizing activity was increased approximately threefold from the plasma pool to the final product. This increase in neutralizing activity indicates that patients treated with hIVIG compared with an equivalent volume of convalescent plasma would receive higher neutralizing activity. Alternatively, patients treated with hIVIG could receive a smaller treatment volume compared with treatment with convalescent plasma and potentially decrease the chances for transfusion‐associated circulatory overload.
The threefold increase in neutralizing activity from the pooled plasma to the final product is smaller than the expected increase based on ELISA titer. This may be caused by contributions from IgM and IgA, which have been removed during purification of IgG.
IgM is the primary source of anti‐A and anti‐B intravascular hemolytic activity in transfusions. 21 IgM is removed to near or below the limits of detection from the hIVIG product, and anti‐A and anti‐B IgG is controlled by controlling the ABO distribution of the pools, greatly reducing the danger of this adverse event. In contrast, when patients are treated with convalescent plasma, they must be matched by donor blood type to reduce the chances of hemolysis.
Similarly, removal of IgA provides a potential therapeutic advantage for hIVIG over convalescent plasma in patients who are IgA deficient and may have been previously treated with blood products and formed antibodies to IgA. Although purified IgG products contain trace amounts of IgA and are labeled to warn for potential adverse events, reactions in IgA‐deficient patients are extremely rare. 22
A potential advantage of hIVIG over direct administration of plasma from individuals or a monoclonal antibody is the diversity of antibodies obtained from a pool of convalescent donors, which may provide a wider range of antiviral activities. 23 This diversity may be important in overcoming viral mutations, some of which have been found to have decreased sensitivity to neutralizing antibodies. 24 Antibody diversity may provide a broader range of antiviral activities by attacking different viral epitopes and enlisting different cellular mechanisms.
In summary, the processes described here describe the manufacturing of multiple batches of anti‐SARS‐CoV‐2 hIVIG that met regulatory requirements. Clinical trials to study the efficacy of hIVIG have been initiated. The first clinical study in which the hIVIG is being evaluated is the Inpatient Treatment with Anti‐Coronavirus Immunoglobulin (ITAC) trial. 25
CONFLICT OF INTEREST
The authors are employees of Grifols, which provided financial support for this study and is a manufacturer of COVID‐19 Immune Globulin.
AUTHOR CONTRIBUTIONS
All authors were involved in study design and concept of this study. All authors have reviewed and approved the final version of this manuscript.
ACKNOWLEDGMENTS
The authors thank the NIAID Integrated Research Facility, Frederick, Maryland, for antibody neutralization testing. Michael K. James, Ph.D., and Jordi Bozzo, Ph.D., are acknowledged for writing and editorial assistance. This work was sponsored and performed by Grifols Bioscience Research Group from the Bioscience Industrial Group, Research Triangle Park, NC, United States, and Grifols Therapeutics LLC, Clayton, NC, United States, with additional financial support provided by the U S Biomedical Advanced Research and Development Authority (BARDA).
Vandeberg P, Cruz M, Diez JM, et al. Production of anti‐SARS‐CoV‐2 hyperimmune globulin from convalescent plasma. Transfusion. 2021;61:1705–1709. 10.1111/trf.16378
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