Effectiveness of ACB-IP 1.0 Universal Pathogen Free Concentrated Cocktail Convalescent Plasma in COVID-19 Infection

BACKGROUND The ecacy of SARS-CoV2 single donor convalescent plasma (CP) varied according to the application time and the amount of antibody that is administered. Single donor CP has some drawbacks; such as the insucient levels of neutralizing antibody activities, the requirements of blood group compatibility, and the risk of infection transmission. In this study, the safety and ecacy of pathogen inactivated, isohemagglutinin-depleted (concentrated) and pooled CP product was investigated. A total of sixteen patients were treated with either single donor CP (N:9) or pathogen-free, concentrated, pooled CP (ACB-IP 1.0) (N:7). RESULTS Five out of six single donor plasma SARS-CoV2 antibody titers remained below 12 s/co, but the antibody titers of all ACB-IP 1.0 plasma were above 12 s/co. SARS-CoV2 total antibody titers of ACB-IP 1.0 plasma were statistically higher than the antibody titers of single donor CP. Mean total plasma neutralizing antibody activity of ACB-IP 1.0 plasma (1.5421) was found statistically higher than single donor CP (0.9642) in 1:256 dilution (ρ<0.01) The mortality rates of the patients treated with ACB-IP 1.0 plasma were statistically lower (p< 0.05) than the patients treated with single donor CP. The administration of either single donor CP or ACB-IP 1.0 plasma to the patients within eight days signicantly shortened the length of hospitalization (ρ< 0.01). CONCLUSION The present study established ACB-IP 1.0 plasma product as a safe and potentially effective treatment for COVID-19, allowing rapid access to patients in need. was rst degree relatives, and trial was conducted under and Clinical Practice and 2020-06/02). Clinical information of the patients was obtained from the hospital’s electronic medical records. Demographic data, presenting symptoms as well as a radiological presentation at the onset of disease including fever, cough, fatigue, dyspnea, diarrhea, oxygen requirement, treatments received, duration of hospitalization stay, duration of Intensive care unit (ICU) stay, cycles and volume of convalescent plasma received, symptom and radiological improvements and current status of the patients were collected. Patients in the two groups were compared according to safety and ecacy, and followed up for transfusion-related reactions and ndings recorded.

WHO recommends the use of Ivermectin, thromboprophylaxis, IL-6 blocker, Lopinavir/Lipanavir, Colchicine, Vitamin D, and corticosteroid in treatment of COVID-19.(4) Nevertheless, the exact clinical bene t of these COVID-19 therapeutics are not yet determined. Hence, clinicians seek for alternative treatment options such as monoclonal antibodies and convalescent plasma. Fundamental mechanism behind monoclonal antibodies and convalescent plasma are similar with both therapies generating passive immunization for viral neutralization.(5) However, convalescent plasma may be preferable over monoclonal antibodies in terms of being effective against new strains, being accessible and cost-effective. Furthermore, convalescent plasma is the safest treatment alternative for emergency use. Convalescent plasma containing antibody from SARS-CoV2 individuals who have recovered from the disease, has been suggested as an investigational treatment option by FDA. (6) Our knowledge regarding convalescent human plasma comes from the treatment of viral infections, such as SARS-CoV, avian in uenza A (H5N1) virus, in uenza A (H1N1), MERS, and Ebola virus. (7)(8)(9)(10)(11)(12)(13) Even though there is still no consensus about its effectiveness, e cacy of convalescent plasma varied according to virus type, time of application, amount of the antibody and most importantly to the neutralizing capacity of the antibodies administered. (14) There are similar concerns regarding studies conducted in SARS-CoV2. FDA report and Mayo Clinic study involving 20,000 hospitalized patients indicated that early and adequate plasma use would reduce mortality (15,16), while PLACID and the latest published RECOVERY trial reported single donor convalescent plasma did not improve survival or other clinical outcomes. (17,18) Con icting outcomes of these studies may be due to the different times of administration, donor antibody titers (since SARS-CoV2 antibody titer was perceived to be the same as neutralizing antibody titer and the latter was therefore not estimated in most studies) and whether the convalescent plasma is strain-speci c. Variations in these parameters have made the results debatable.
Single donor plasma has some drawbacks such as requirements of blood group compatibility (17,19,20) and risk of viral infection, for instance HIV, HBV, HCV transmission by donors who do not meet the standard donor criteria in pandemia conditions. In addition, excess of procoagulant factors in standard donor plasma increases the risk of thrombosis. (21,22) These factors necessitated the search for a new convalescent plasma product. Hence, ACB-IP 1.0 cocktail plasma, where SARS-CoV2 antibody titers and neutralizing antibody activity were standardized by pooling, was designed. Furthermore, Immunoglobin M (IgM) which is responsible for most of the isohemagglutinins(23-25), was depleted by using both cryodepletion (24) and pooling method until Anti-A and Anti-B isohemagglutinin titers were below 1/8. Procoagulant factors were depleted by cryodepletion in order to eliminate the risk of thrombotic events. Furthermore, it was concentrated and safety of the plasma was improved by subjecting it to pathogen inactivation.
This study was conducted with participation of 16 patients, and product quality and clinical effectiveness of ACB IP 1.0 with standard single donor convalescent plasma were compared.

Materials And Methods
According to the criteria speci ed in the COVID-19 Immune Plasma Procurement and Clinical Use Guidelines of FDA(8), donor candidates who were eligible according to apheresis donor criteria were invited to Therapeutic Apheresis Center of Acıbadem Altunizade Hospital. Blood products were taken from the donor plasma candidates. (Supplement-1) Effect of administration of convalescent plasma samples obtained from 9 Turkish Red Crescent donors and 7 ACB-IP 1.0 products, where each one of the 7 plasma samples was prepared from 8 different donors, were compared according to outcome of the patients.
Plasma Collection: ACB IP 1.0 was obtained from donor plasma using Trima Accel (Trima Accel, Terumo BCT, Inc. Colorado, USA) device. 400 ml -600 ml plasma was collected according to the patient's height, weight, and blood count results. During this process, an ISBT code was obtained from the Turkish Red Crescent.
Pathogen Inactivation: Plasma collected by plasmapheresis was connected to Cerus Intercept Blood System plasma treatment bags (Intercept Blood System Pathogen Reduction System, Cerus, CA, USA) using bag joining device (Terumo TSCD-II TSCD Welders, Terumo BCT, Inc. Colorado, USA). Prior to pathogen inactivation process, 2 ml of 2 tubes of witness samples were removed from the collected plasma. Witness samples were stored at -40 °C. According to the manufacturer instruction, plasma was initially treated with Amotosalen followed by photochemical irradiation with UVA at 320-400 nm wavelengths in Intercept INT100 illuminator (INTERCEPT INT100 Illuminator, Cerus, CA, USA). Following inactivation, plasma was passed through the adsorption lter to remove unreactive amotosalen and free photoproducts, and then it was divided into 2 or 3 equal volumes, depending on the volume of the collected plasma. Following this process, 2 ml of 2 witness sample tubes were separated and stored at -40 °C. Pathogen inactivated plasma was stored at -40 °C for labeling until the pooling process prior to clinical usage.
Isohemagglutinin Assay: Ready to use (commercial) A and B kits (ID-Diacell ABO, Bio-Rad Laboratories, Inc., Cressier, Switzerland) were provided by Bio-Rad and gel centrifugation method was performed according to the manufacturer's protocol (NaCl, Enzyme Test and Cold Agglutinins, Bio-Rad Laboratories, Inc., Cressier, Switzerland).
IgM Detection: IgM titers were obtained by using Siemens Advia 1800 Chemistry System. The photometric method was performed according to the manufacturer's protocol.

Isohemagglutinin Depletion and Concentration of Plasma:
Following obtaining plasma from the donor, Anti-A and Anti-B hemagglutinin titers were determined. Management of Isohemagglutinin titer was carried out in two separate steps. Firstly, isohemagglutinins, most of which were of IgM nature, were reduced by cryodepletion, while simultaneously concentrating the product. Secondly, isohemagglutinin titer was reduced by pooling of Anti-A and Anti-B free plasma and the plasma containing them.
Cryodepletion Method (23,24) Plasma samples from the apheresis product of 200 ml were transferred to plasma storage bags (Teru ex, Terumo BCT, Inc. Colorado, USA) frozen in a deep freezer at -40 °C. One bag consisted of eight donors' apheresis plasma and volume in each bag was approximately 1600 ml. Frozen samples were kept at + 4°C for defrosting for approximately 8-12 hours. Liquid plasma was separated from cryoprecipitate by centrifugation. Witness samples were taken and stored at -40 °C. The bag where cryodepleted pool would be produced was connected to the plasma bag (Terumo BCT, Inc. Colorado, USA) using the bag joining device (Terumo TSCD-II TSCD Welders, Terumo BCT, Inc. Colorado, USA).

Plasma Pooling:
Plasma bag was placed in the extractor and the cryopoor plasma was transferred to the pooling bag. Finally, pooling was achieved by mixing the low SARS-CoV2 antibody titer with high antibody titer plasma prior to cryodepletion and the total product was packaged in 200 ml bags and stored at -40 °C until clinical use.
Following one hour of incubation at room temperature, 10000 Vero cell/well was placed in a 96 Well Flat Bottom plate with 100 µl of complete DMEM (4% FBS + 1% PSA). Supernatants were removed after 72 hours of incubation, 50 μl of MTT solution and 50 μl serum-free media were added to the remaining cells.
Following incubation at 37 ° C for 4 hours, 100 μl of Isopropanol dispersion was added to each well and placed on a shaker for 10 min. Results were obtained by ELISA Reader at an absorbent value of 570 nm. Neutralizing antibody activity was studied in 1:64, 1:128 and 1:256 dilution based by cell viability index.
Endotoxin Analysis: Gel-clot technique was used for detecting or quantifying endotoxins (Gel-Clot Endotoxin Test, Division of Charles River Laboratories Inc, CA).

Microbiological Quality Control:
Pooled convalescent plasma was placed on to Bactec Fx device for microbiological quality control analysis (Bactec FX, Becton Dickinson, New Jersey, USA). A total of 16 hospitalized adults were screened for enrollment and included in the study if they had positive reverse-transcriptase-polymerase-chain-reaction (RT-PCR) for SARS-CoV2 and radiologically con rmed pneumonia.

Sars-CoV2
A total of 16 patients were treated with two different convalescent plasma products. Nine patients were treated with single donor convalescent plasma and seven were treated with pathogen-free, concentrated, pooled convalescent plasma between 01 March 2020 and 31 December 2020.
Written informed consent was obtained from all patients or their rst degree relatives, and trial was conducted under the principles stated in the Declaration of Helsinki and Good Clinical Practice guidelines and approval of Acıbadem University ethics committee (Approval No: 2020-06/02).
Clinical information of the patients was obtained from the hospital's electronic medical records. Demographic data, presenting symptoms as well as a radiological presentation at the onset of disease including fever, cough, fatigue, dyspnea, diarrhea, oxygen requirement, treatments received, duration of hospitalization stay, duration of Intensive care unit (ICU) stay, cycles and volume of convalescent plasma received, symptom and radiological improvements and current status of the patients were collected. Patients in the two groups were compared according to safety and e cacy, and followed up for transfusionrelated reactions and ndings recorded.
Statistical Analysis: SARS-CoV2 Antibody titers, neutralizing antibody activities, and duration of hospitalization were analyzed by employing Mann Whitney test. Mean age of the groups was compared by the Kolmogorov-Smirnov test. Survival differences between the two plasma groups were analyzed by Chi-Square test. Fisher exact test was used to compare the categorical variables such as radiological presentation, co-existing disease and oxygen supplement requirement among the groups. Results were analyzed with a %95 con dence interval and a signi cance level of p=0.05 was used for all statistical analysis.

Results
Laboratory Results:

Results of the Isohemagglutinin Titers:
Pre-pooling cryodepletion process caused a statistically signi cant reduction of 35% in Anti-A titers, 44% in Anti-B titers, and 11% in IgM levels (Table 1, Figure   1). Following the pooling process, maximum Anti-A titers in ACB-IP 1.0 was reduced to 1/4 and Anti-B titers to 1/8, while maximum Anti-A titers were 1/64 and Anti-B 1/128 in single donor plasma. Results show that cryodepletion and pooling process effectively control isohemagglutinin levels, giving ACB-IP 1.0 a universal character (blood-group free convalescent plasma).

Results of the SARS-CoV2 Antibody Titers in Plasma:
Five out of six single donor plasma SARS-CoV2 antibody titers remained below 12 s/co, but antibody titers of all ACB-IP 1.0 plasma were above 12 s/co. Mean antibody titer of single donor plasma was 9.2083 and mean antibody titer of ACB-IP 1.0 plasma was 32.700. SARS-CoV2 total antibody titers of ACB-IP 1.0 plasma were statistically higher in comparison with the antibody titers of single donor plasma (Figure 2).
Mean total plasma neutralizing antibody activity of ACB-IP 1.0 plasma (1.5421) was found to be statistically higher than single donor plasma (0.9642) in 1:256 dilution (ρ=**0.0087) (Figure 3).  Table-2. Six out of nine single donor convalescent plasma patients were male. Median age of the patients was 65 years and none of them had a history of smoking. Two male patients aged 54 and 63 had no coexisting disease.

No correlation was found between SARS-CoV2 antibody titers and neutralizing antibody activity in both groups
Treatment of all patients was performed according Covid-19 treatment algorithms. Five patients received dornase-alpha and three patients received IL-6 blocker.
One of the patients had two cycles of single donor convalescent plasma total of 400 ml volume. Volume loading due to transfusion was detected in one patient. No other reaction was observed.
Mean duration of hospital stay was 41 days (11-101 days) and mean duration of ICU stay was 35 days (7-90 days). Following the treatments and convalescent plasma administration, four out of nine patients had clinical improvement, three patients had radiological improvement, one of the patients was not evaluated. Five out of nine patients died in total.
Clinical characteristics of SARS-CoV2 infected patients who received pathogen-free, concentrated, pooled, convalescent plasma is presented in Table-3. All of the patients treated with pathogen-free, concentrated, pooled convalescent plasma were males. Median age of the patients was 51 years and none of them had a history of smoking. Two patients aged 50 and 58 had no coexisting disease.
Treatments of all seven patients were performed according to Covid-19 treatment algorithms. One patient received IL-6 blocker and one patient had dornasealpha treatment.
One of the patients had two cycles of pooled convalescent plasma total of 400 ml volume. No transfusion-related reactions had been observed. The mean duration of the hospital stay was 44 days (12-172 days) and the mean duration of ICU stay was 30 days (0-139 days). Following treatments and convalescent plasma administration, four patients had clinical improvement and ve patients had radiological improvement. Seven patients in total were discharged from the hospital.
Mortality rate of the patients treated with ACB-IP 1.0 plasma were statistically lower (p: 0.033) than the patients treated with single donor plasma. (Figure 4) Median length of hospital stay was 41 days for single donor plasma patients, and 44 days for ACB-IP 1.0 plasma patients and there was no signi cant difference. Administration of single donor plasma or ACB-IP 1.0 plasma to the patients within eight days from presentation signi cantly shortened the length of hospitalization in comparison with administration of either plasma later than eight days from presentation (ρ= 0.0021) ( Figure 5). On the other hand, a number of studies failed to show bene cial effect of convalescent plasma in SARS-CoV2 pneumonia patients. (17,18,32) One of these studies was PLACID trial, even though it had a number of design faults, such that there was no neutralizing antibody titer measurement of the donor plasma and also convalescent plasma was administered to the patients later than 3 days, contrary to what was recommended by the FDA. V.A. Simonovich et al. also showed no relationship between SARS-CoV2 antibody titers, early plasma administration and clinical e cacy.(32) However, hypoxia was one of the patient selection criteria and treatment was not commenced within 72 hours of the disease onset and neutralizing antibody titers could only be assessed in 56% of the donors in this study.

Discussion
Largest trial conducted so far, RECOVERY Group trial, also indicated that even if convalescent plasma is used in early onset of disease, it does not reduce respiratory support requirement, hospital stay, and mortality.(18) However; convalescent plasma in this study was again administered later than 72 hours following the onset of disease. Also the convalescent plasma was not strain-speci c because a new SARS-CoV-2, B.1.1.7 dominated most regions of UK at the time of the study. Hence, it is likely that modi ed antigenicity further reduced neutralizing capacity of the plasma. Furthermore, all convalescent plasma were chosen based on high anti-spike concentration. This was on the assumption that IgG concentration shows correlation with the neutralizing antibody titer.
(18) Although, Salazar et al. showed that there is a correlation between antibody titers and neutralizing capacity, they only reported this correlation in only 80% of single donor plasma. The fact that the correlation between IgG concentration and the neutralizing antibody titer was not shown in %20 of single donor plasma is a high ratio that can make the results questionable. (33) Results of the current study for single donor plasma showed heterogeneity of SARS-CoV2 antibody titers and neutralizing antibody activity. However, ACB-IP 1.0 plasma which is a pooled concentrated plasma product, showed higher SARS-CoV2 antibody titers and neutralizing antibody activity. There was no correlation between SARS-CoV2 antibody titers and neutralizing antibody activities in single donor plasma or ACB-IP 1.0 plasma. This may have been due to only SARS-CoV-2 S1 spike protein antigen being used for antibody detection in this study. More reliable results could possibly have been obtained using nucleocapsid and matrix protein antigen in addition to the spike protein antigen for antibody detection.
Presence of re-infected cases despite high antibody titers con rms that this is not a reliable parameter for protection against Covid. ACB-IP 1.0 plasma in this study had less variance of SARS-CoV2 antibody and neutralizing antibody titer, and hence more standardized convalescent plasma could be obtained.
Findings of this study showed signi cantly decreased in mortality in ACB-IP 1.0 plasma in comparison with single donor convalescent plasma. There was also a reduction of length of hospital stay in both groups if the plasma was administrated within 8 days onset of disease (p=0.0021). Failure to show signi cant difference at 3 days may have been due to the small number of patients in this study group.
Another, signi cant clinical result was di culty in accessibility of single donor plasma, which requires blood group compatibility, especially in the early stages of the pandemic due to the lack of su cient available donors. However, as a result of cryodepletion and pooling processes, ACB-IP 1.0 convalescent plasma does not require blood group compatibility, and hence can be employed immediately. Furthermore, by containing pooling of plasma from various convalescent individuals, ACB-IP 1.0 plasma may be more advantageous in the treatment of variant mutations.
Single donor plasma also has the disadvantage of increased risk of transfusion-transmitted infections due to individuals who may not meet the standard donor criteria. With the help of pathogen inactivation process, transfusion-transmitted infections are minimized. However, due to its small scale, no data could be obtained to determine the advantage of pathogen inactivation in this study and a randomized, controlled study with a larger patient group is required.    Effects of cryodepletion on Isohemaglutinin and IgM levels Comparison of the mortality rate between the two different plasma groups (*p=0.033)