Recombinant Polyclonal Antibody Research Articles

Development of antibodies against recombinant staphylococcal enterotoxin B from food poisoning cases.

Staphylococcal enterotoxin B (SEB) is the most common serotype involved in food poisoning. The aim of this study was to develop immunoassay detection methods using a recombinant enterotoxin B antigen protein to produce recombinant polyclonal antibodies in vivo. Staphylococcus aureus isolated from a food poisoning case (strain JH5800) was analyzed by polymerase chain reaction (PCR) and confirmed to contain a seb gene of 477 bp. A SEB segment was amplified, cloned, sequenced, and aligned. The PCR product corresponding to the predicted mature SEB peptide was inserted into Escherichia coli BL21 (DE-3) expression vector and expressed as a hexahistidine-SEB fusion protein. Antiserum against recombinant SEB protein was produced by immunization of Balb/c mice. In the indirect enzyme-linked immunosorbent assay (ELISA), the polyclonal antibodies produced had a titer of 1:3200. The seb gene of Staphylococcus aureus isolated from a poisoning case (JH5800) had a molecular size of about 477 bp and a band of recombinant SEB toxin was observed at approximately 30 kDa on SDS-PAGE gel. The polyclonal anti-SEB antibody titer, as revealed by indirect ELISA, was 1:3200 at 59 days. SEB recombinant protein could be used to produce polyclonal antibodies. ELISA and Western blotting were used to analyze the specificity and sensitivity of the recombinant polyclonal antibodies. Polyclonal antibodies produced could be used to detect SEB on a large-scale.

A Potent Recombinant Polyclonal Antibody Therapeutic for Protection Against New Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern.

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) possess mutations that prevent antibody therapeutics from maintaining antiviral binding and neutralizing efficacy. Monoclonal antibodies (mAbs) shown to neutralize Wuhan-Hu-1 SARS-CoV-2 (ancestral) strain have reduced potency against newer variants. Plasma-derived polyclonal hyperimmune drugs have improved neutralization breadth compared with mAbs, but lower titers against SARS-CoV-2 require higher dosages for treatment. We previously developed a highly diverse, recombinant polyclonal antibody therapeutic anti-SARS-CoV-2 immunoglobulin hyperimmune (rCIG). rCIG was compared with plasma-derived or mAb standards and showed improved neutralization of SARS-CoV-2 across World Health Organization variants; however, its potency was reduced against some variants relative to ancestral, particularly omicron. Omicron-specific antibody sequences were enriched from yeast expressing rCIG-scFv and exhibited increased binding and neutralization to omicron BA.2 while maintaining ancestral strain binding and neutralization. Polyclonal antibody libraries such as rCIG can be utilized to develop antibody therapeutics against present and future SARS-CoV-2 threats.

Immune Checkpoint Glycoproteins Have Polymorphism: Are Monoclonal Antibodies Too Specific?

Since the 2018 Nobel prize in medicine was granted to the discovery of immune escape by cancer cells, billions of dollars have been spent on a new form of cancer immunotherapy called immune checkpoint inhibition (ICI). In this treatment modality, monoclonal antibodies (mAbs) are used to block cell-surface glycoproteins responsible for cancer immune escape. However, only a subset of patients benefit from this treatment. In this commentary, we focus on the polymorphism in the target molecules of these mAbs, namely PD-1, PD-L1 and CTLA4; we explain that using a single mAb from one clone is unlikely to succeed in treating all humans because humans have a genotype and phenotype polymorphism in these molecules. Monoclonal antibodies are highly specific and are capable of recognizing only one epitope ("monospecific"), which makes them ideal for use in laboratory animals because these animals are generationally inbred and genetically identical (isogenic). In humans, however, the encoding genes for PD-1, PD-L1 and CTLA4 have variations (alleles), and the final protein products have phenotype polymorphism. This means that small differences exist in these proteins among individual humans, rendering one mAb too specific to cover all patients. Our suggestion for the next step in advancing this oncotherapy is to focus on methods to tailor the mAb treatment individually for each patient or replace a single clone of mAb with less specific alternatives, e.g., a "cocktail of mAbs", oligoclonal antibodies or recombinant polyclonal antibodies. Fortunately, there are ongoing clinical trials on oligoclonal antibodies at the moment.

Convalescent serum lines up as first-choice treatment for coronavirus

Antibodies from blood donated by people who recovered from the illness and hyper-immunoglobulins are becoming treatments of choice for COVID-19, with recombinant polyclonal antibody approaches to follow. Antibodies from blood donated by people who recovered from the illness and hyper-immunoglobulins are becoming treatments of choice for COVID-19, with recombinant polyclonal antibody approaches to follow.

Cloning, Sequencing and Production of Recombinant Polyclonal Antibodies against Egyptian Staphylococcal Enterotoxin A

Staphylococcus aureus (S.aureus) is the most common pathogen found in hospitals, community-acquired infections and colonizes up to 50% of humans, including mucous membranes and damaged skin. Some strains produce enterotoxins (Ent) as staphylococcal enterotoxin A (SEA) which is involved in 75% of food poisoning outbreaks. Few methods are sensitive and specific enough to confirm the diagnosis of staphylococcal food poisoning. In this study, a segment of Ent A-ORF with molecular weight 774 bp was amplified, cloned, sequenced and aligned with published Ent A-ORFs. Ent A-ORF was subcloned into bacterial expression vector (GST-PGEX4T1 vector) and expressed as a fusion protein with GST-tagged protein. A band size of 27 kDa of purified Ent A protein was cleaved from GST protein by thrombin. The expressed protein of Ent A was identified by strong reaction with commercialized polyclonal antibodies against Ent A. Antiserum against Egyptian recombinant Ent A protein was produced by immunization of Balb/c mice, the produced recombinant polyclonal antibodies had a titre of 1:2500 in direct ELISA. The produced recombinant polyclonal antibody was evaluated and reacted specifically in Western immunoblotting analysis and ELISA test. Finally from the obtained results, the produced recombinant protein of Ent A can be used successfully for production of recombinant polyclonal antibodies and used in large-scale detection of staphylococcal enterotoxin A.

A synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins.

Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost‐effective antivenom production based on plant‐made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin‐binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant‐made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.

Anti-VDAC3 recombinant antibody decreased human sperm motility and membrane integrity: A potential spermicide for contraception

Objective: To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3) protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded. Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly. Conclusions: Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment) and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether, an active component of the traditional Japanese medicine Yokukansan

1. Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans.2. Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms.3. The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes.4. These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS.

Should development of Alzheimer's disease-specific intravenous immunoglobulin be considered?

Recent phase II and III studies with intravenous immunoglobulin (IVIG) in patients with Alzheimer’s disease (AD) did not find evidence for the slowing of AD progression compared to placebo-treated patients, in contrast to encouraging results in pilot studies. An additional phase III trial is ongoing. If negative results are found, then further AD studies with IVIG are unlikely unless a manufacturer opts for a trial with high-dose IVIG, which would increase its anti-inflammatory effects but also the risk for adverse events. An alternative approach could be an AD-specific IVIG, supplementing IVIG with higher concentrations of selected antibodies purified from it or produced via recombinant polyclonal antibody technology. These antibodies could include those to amyloid-beta (Aβ, tau protein, inflammatory cytokines, complement activation proteins, and the receptor for advanced glycation end products. IgG fragment crystallizable (Fc) fragments containing terminal sialic acid could be added to increase anti-inflammatory effects. While this product might be more effective in slowing AD clinical progression than current IVIG, there are difficulties with this approach. Preclinical studies would be required to determine which of the antibodies of interest for supplementing current IVIG (for example, antibodies to phosphorylated or oligomeric tau) are actually present (and, therefore, available for purification) in IVIG, and the effects of the product in mouse models of AD. An Investigational New Drug application for an AD-specific IVIG would require United States Food and Drug Administration approval. If the drug would be found to benefit AD patients, meeting the increased demand for IVIG would be challenging.

PRODUCTION AND CHARACTERIZATION OF MONOCLONAL AND POLYCLONAL ANTIBODY AGAINST RECOMBINANT OUTER MEMBRANE PROTEIN

There are many studies related to immunological and molecular methods for diagnosis of Vibrio cholera (V. cholerae ). However, most assays dependent on enrichment of culture of bacteria, which need more time and involves the use of costly equipment and reagents. In this study Balb/c mice were immunized with recombinant Outer Membrane Protein (rOMPw) of vibrio cholerae and splenocytes of hyper immunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using rOMPw as coating antigen. The monoclonal antibodies from ascitic fluids were purified and its reaction with rOMPw was assessed by ELISA. Polyclonal antibodies were also produced by immunization of rabbits with the above mentioned antigen. The rabbit sera w as affinity purified using Hi-Trap protein G column . The result showed that monoclonal antibody specific to rOMPw has been successfully generated. The monoclonal antibody reacted with recombinant OMPw in ELISA and immunonoblat method. Rabbit polyclonal antibody was also bound to rOMPw by ELISA. The results of agglutination test with whole bacteria also showed that both mouse monoclonal and rabbit polyclonal antibodies reacted with whole vibrio cholera but not other related bacteria . The purpose of this study was to check out if ant i OMPw antibodies could use as diagnostic assay for detect ion of V. cholerae . Our results demonstrated that anti recombinant OMPw monoclonal and polyclonal antibodies are able to diagnose whole bacteria in pure culture using agglutination test but not by home ma de immunochromatic strip test.

Should development of Alzheimer¿s disease-specific intravenous immunoglobulin be considered?

Recent phase II and III studies with intravenous immunoglobulin (IVIG) in patients with Alzheimer’s disease (AD) did not find evidence for the slowing of AD progression compared to placebo-treated patients, in contrast to encouraging results in pilot studies. An additional phase III trial is ongoing. If negative results are found, then further AD studies with IVIG are unlikely unless a manufacturer opts for a trial with high-dose IVIG, which would increase its anti-inflammatory effects but also the risk for adverse events. An alternative approach could be an AD-specific IVIG, supplementing IVIG with higher concentrations of selected antibodies purified from it or produced via recombinant polyclonal antibody technology. These antibodies could include those to amyloid-beta (Aβ, tau protein, inflammatory cytokines, complement activation proteins, and the receptor for advanced glycation end products. IgG fragment crystallizable (Fc) fragments containing terminal sialic acid could be added to increase anti-inflammatory effects. While this product might be more effective in slowing AD clinical progression than current IVIG, there are difficulties with this approach. Preclinical studies would be required to determine which of the antibodies of interest for supplementing current IVIG (for example, antibodies to phosphorylated or oligomeric tau) are actually present (and, therefore, available for purification) in IVIG, and the effects of the product in mouse models of AD. An Investigational New Drug application for an AD-specific IVIG would require United States Food and Drug Administration approval. If the drug would be found to benefit AD patients, meeting the increased demand for IVIG would be challenging.

Skin changes associated to hypothyroidism

Antibody therapeutics are one of the fastest growing classes of pharmaceuticals, with an annual US market over $20 billion, developed to treat a variety of diseases including cancer, auto-immune and infectious diseases. Most are currently administered as a single molecule to treat a single disease; however there is mounting evidence that cocktails of multiple antibodies, each with a unique binding specificity and protective mechanism, may improve clinical efficacy. Here, we review progress in the development of oligoclonal combinations of antibodies to treat disease, focusing on identification of synergistic antibodies. We then discuss the application of modern antibody engineering technologies to produce highly potent antibody preparations, including oligoclonal antibody cocktails and truly recombinant polyclonal antibodies. Specific examples illustrating the synergy conferred by multiple antibodies will be provided for diseases caused by botulinum toxin, cancer and immune thrombocytopenia. The bioprocessing and regulatory options for these preparations will be discussed.

Immune Blot Analysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with Recombinant Polyclonal Antibody

To detect the mammalian target of rapamycin (mTOR) expressed in Cashmere goat fetal fibroblasts (GFb), mTOR gene was cloned from Inner Mongolia Cashmere goat (Capra hircus) and expressed in Escherichia coli followed by immunizing mice with the purified recombinant protein as an immunogen to produce the anti-goat mTOR recombinant polyclonal antibody. Antiserum was collected from the immunized mice after the fifth immunization and its titer was determined with enzyme-linked immunosorbent assay (ELISA). The results showed that the recombinant polyclonal antibody had a titer 1:200 000 and could react with the mTOR expressed in GFb cells with a specific and sensitive affinity. Western blot showed that mTOR expression and phospho-mTOR (Ser 2448) activity were inhibited when GFb cells were treated with CCI-779, an mTOR specific inhibitor.

Final Results From a Phase II Trial with the First in Class Recombinant Polyclonal Antibody Product Rozrolimupab in Primary Immune Thrombocytopenia

Abstract 527▪▪This icon denotes a clinically relevant abstract Background:Rozrolimupab is the first in-class fully human, recombinant antibody mixture for the treatment of ITP. Rozrolimupab comprises 25 unique human antibodies with documented binding capacity to the Rhesus D antigen on red blood cells, and constitutes a promising, modern counter-part to the blood-derived immunoglobulin products currently used in the treatment of ITP. The antibody mixture constituting rozrolimupab is produced by a novel single batch manufacturing strategy. Objectives and Endpoints:The objectives of the trial were to investigate the safety and efficacy of a single dose of rozrolimupab in RhD positive, non-splenectomized adult patients with primary ITP. The primary endpoint was incidence and severity of Adverse Events (AEs) during the 6-week trial period. Secondary endpoints included identification of the optimal dose of rozrolimupab and percentage of patients responding to treatment at 72 hours, 7 and 15 days. Methods:Patients from 27 sites in 11 countries were enrolled in this dose escalation (75 μg/kg – 300 μg/kg), multicentre, open label trial. Inclusion criteria included confirmed presence of thrombocytopenia with two individual pre-dose platelet counts of < 30 × 109/L. The patients received a single iv dose of rozrolimupab, and were followed for 6 weeks with frequent evaluations for safety and efficacy. Response was defined as platelet count ≥ 30 ×109/L and increase in platelet count from baseline by > 20 × 109/L. Results:A total of 61 ITP patients were treated with rozrolimupab: 75 μg/kg (11 patients), 100 μg/kg (10 patients), 125 μg/kg (10 patients), 150 μg/kg (5 patients), 200 μg/kg (6 patients), 250 μg/kg (6 patients) and 300 μg/kg (13 patients). Data from the 300 μg/kg cohort were not available for this abstract, but will be included in the final presentation.Given the small size of this study, the dose cohorts differed in a number of baseline characteristics. 60–90% of patients were females in all dose cohorts except in the 75 μg/kg cohort where 73% were males. Median platelet count before trial entry ranged from 11.5 to 22.3 × 109/L, and median time from first ITP diagnosis ranged from 5 to 68 months. In the individual dose groups, up to 83% of the patients responded at day 7. Up to 50% and 60% of the patients responded at 24 and 72 hours, respectively, and some responses lasted for the duration of the 6 weeks follow up period. Platelet responses also included patients who had baseline platelet counts below 10 ×109/L. A total of 51 AEs considered related to trial drug were reported in 20 patients. The most frequently reported adverse reactions were headache (7 events in 5 patients) and pyrexia (5 events in 4 patients). All but 2 of the adverse reactions (one headache, one extravascular haemolysis) were of mild or moderate intensity. Four of the 51 adverse reactions were categorized as Serious Adverse Events: One mild haemoglobin decrease in the 100 μg/kg cohort, one extravascular haemolysis in the 200 μg/kg cohort (mentioned above) and two mild to moderate, transient increases in D-dimer with no clinical symptoms in the 250 μg/kg cohort. Laboratory data showed a mean maximum decrease in haemoglobin ranging from 1.1 g/dL to 2.7 g/dL in individual cohorts with a slight dose dependency. A drop in haemoglobin of ≥ 3 g/dL was seen in 2 patients, one of 3.1 g/dL in the 100 μg/kg cohort and one of 4.5 g/dL in the 200 μg/kg cohort. Conclusions:Rozrolimupab is well tolerated with a favourable safety profile. In the individual dose groups, up to 83% of patients responded at day 7 including patients who had baseline platelet counts below 10 ×109/L. Final safety and efficacy data from all dose groups in the completed Phase II trial of rozrolimupab in primary ITP patients will be presented. Disclosures:Wiktor-Jedrzejczak:Celgene: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria, Research Funding. Flensburg:Symphogen: Employment. Petersen:Symphogen: Employment.

Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use

The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well-characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost-efficient cell banking and single-batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled-up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic-, protein-, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition.

Cloning and Prokaryotic Expression of &lt;i&gt;Apple Stem Pitting Virus&lt;/i&gt; CP Gene in Ya Pear in &lt;i&gt;E. coli&lt;/i&gt;

Abstract In order to clarify evolutionary relationship and genetic diversity of apple stem pitting virus (ASPV), and preparation the corresponding special antiserum. In this study, total RNA was extracted from phloem tissue of Ya (Y) pear which infected by ASPV and used as template for cDNA synthesis. The coat protein (CP) gene of ASPV was cloned and sequenced. The CP gene of Ya pear was cloned into expression vector PET-28a, and transformed into E . coli BL21 (DE3), and SDS-PAGE electrophoresis analysis. The results showed that the complete CP gene consisted of 1 194 nucleotides and encodes a polypeptide of 397 amino acid (aa). Comparison of the amino acid sequences of Ya pear CP gene with other ASPV isolates showed approximately 70% similarity. Phylogenetic tree showed that all isolates of the coat protein (CP) genes of ASPV isolates at AA sequence revealed three groups. All ASPV isolates from apple were clustered to group I, whereas pear were clustered to groups â…i (except NC_003462) and the Ya pear were clustered into group â…¢. Results of SDS-PAGE showed that specific expression of a 42 kD fusion protein was achieved by the inducing of 1 mmol/L IPTG. The complete CP gene and prokaryotic expression vector of Ya pear provided additional baseline data for preparation recombinant polyclonal antibody of ASPV and further study of ASPV molecular biology.

Development of Mass Spectrometry Based Techniques for the Identification and Determination of Compositional Variability in Recombinant Polyclonal Antibody Products

Recombinant polyclonal antibodies are a new class of protein biologics, combining a defined number of target-specific antibodies, developed for therapeutic use across various indications. Development, manufacture, and release of recombinant polyclonal antibodies as well characterized biological products have required development of new chemistry, manufacturing, and control (CMC) technologies. Sym001 is a recombinant polyclonal antibody product containing 25 unique antibodies specific for the Rhesus D antigen. Sym001 drug substance is manufactured using a single batch technology, Sympress. Here, we describe the development of two novel mass spectrometry based methods that allows identification of individual antibodies in the Sym001 drug substance, through the determination of unique marker peptides or antibody light chains. The two methods provide an unambiguous identification of the 25 unique antibodies comprised in the Sym001 drug substance. Furthermore, the light chain liquid chromatography-mass spectrometry (LC-MS) method has been developed to allow the determination of the relative distribution of the 25 antibodies. The light chain LC-MS method has demonstrated linearity, specificity, precision, and accuracy, thus qualifying it for use in the quality control of recombinant polyclonal antibodies for human use. The development of such quantitative methods is central for the development and quality control of additional therapeutic recombinant polyclonal antibody products.

Single-Batch Production of Recombinant Human Polyclonal Antibodies

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the SympressTM I technology. The SympressTM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the SympressTM I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the SympressTM II technology. Here we describe proof-of-principle data demonstrating the feasibility of the SympressTM II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.

Sym001, the First Recombinant Polyclonal Rhesus-D Specific Antibody Product, Was Safe and Well-Tolerated in a Placebo-Controlled Randomized Phase I Trial.

Sym001 is the first compound in a new class of biopharmaceuticals in clinical development for the treatment of Immune Thrombocytopenic Purpura (ITP), and the prevention of hemolytic disease of the newborn by anti-D prophylaxis (ADP). Sym001 is a recombinant polyclonal antibody product consisting of 25 different anti-Rhesus D specific (RhD) antibodies. The primary objective of this clinical study was to assess the safety of Sym001 following a single intravenous (iv) dose in RhD+ and RhD− healthy male volunteers. Secondary objectives were to assess the potential immunogenicity and pharmacokinetic (PK) profile of Sym001. Fifty-nine RhD+ subjects and 18 RhD− subjects were randomized to receive up to 12.5 μg/kg or 75 μg/kg, respectively, in seven dose cohorts as shown below:Number of SubjectsCohortDose (μg/kg)RhD+ Sym001RhD+ PlaceboRhD− Sym001RhD− Placebo10.25520021.0520034.07272412.57272525720065072007757200Treatment with Sym001 was well tolerated and there were no serious adverse events (SAEs), no AEs of severe intensity and no AEs that resulted in discontinuation of the study in either the RhD+ or RhD− population. The frequencies of treatment-emergent AEs (TEAE), i.e. AEs occurring after drug administration, were similar among Sym001 and placebo treated subjects and there was no dose-dependent pattern of TEAEs. No significant decrease in mean hemoglobin level compared to baseline was observed at any dose level during the study, and no decrease in hemoglobin > 2 g/dL in any subject was seen. No other laboratory findings were suggestive of clinically significant hemolysis. In addition, no clinically significant changes in vital signs or electrocardiograms (ECGs) were observed, and there were no significant changes in clinical chemistry variables that did translate into SAEs or were assessed as being related to study medication. No immunogenicity to Sym001 was detected and analysis of PK is in progress. In conclusion, Sym001 was found to be safe and well-tolerated in healthy volunteers in doses up to 75 μg/ kg. Sym001 is now being evaluated in a red blood cell (RBC) challenge trial for the ADP indication and in a Phase II trial for treatment of patients with ITP.