Enhanced efficacy of anti-D immunoglobulin for treating ITP is not explained by higher immunoglobulin polymer content.

Several reports have suggested that low dose anti-D immunoglobulin is superior to high dose immunoglobulin for treatment of idiopathic thrombocytopenia purpura (ITP). However, some findings suggest that it is not the anti-D activity per se that is responsible for efficacy for treatment of ITP with anti-D immunoglobulin. Amongst alternative explanations for the mechanism of action is a relatively higher immunoglobulin polymer content of anti-D compared to other immunoglobulin products, which is more efficient in causing reticuloendothelial Fc receptor blockade. In order to investigate this we have evaluated the polymeric IgG content of anti-D and other immunoglobulin products. Different products showed considerable variation in immunoglobulin polymer content. There was no clear correlation between aggregate or dimer content and product type and anti-D as a class of product did not contain higher amounts of either dimer or aggregate compared to other products. Some manufacturers' products increased in polymer content on storage, but others did not show this effect. Therefore, higher immunoglobulin dimer and/or aggregate content cannot explain the increased efficacy of anti-D immunoglobulin for treatment of ITP. The role of polymeric Ig in efficacy for treatment of ITP is unclear.     

Incidence of rhesus immunisation after genetic amniocentesis.

Of 655 Rh negative women without anti-D antibody in their serum at genetic amniocentesis, 361 delivered a Rh positive infant. Prophylactic treatment with anti-D immunoglobulin was not given at amniocentesis. The women were followed prospectively, being given a screening test for antibody after amniocentesis, at delivery, and six months later. Five of these 361 women yielded a positive test result due to anti-D antibody. The immunisation rate after genetic amniocentesis was no higher than the spontaneous immunisation rate during pregnancy. Four women who had two amniocenteses in the same pregnancy and 34 women who had amniocentesis in two consecutive pregnancies with Rh positive fetuses were not immunised. Among six women with anti-D antibody in their serum before amniocentesis the titre of antibody increased in three. Amniocentesis may have worsened the outcome of these pregnancies. These results suggest that the risk of immunisation in Rh negative women is small.     

Efficacy and long term effects of antenatal prophylaxis with anti-D immunoglobulin.

OBJECTIVE--To measure the safety and efficacy of antenatal treatment with anti-D immunoglobulin. DESIGN--Open study with historical controls. SETTING--Multicentre study in 17 hospitals in West Yorkshire. PATIENTS--1238 Rh negative women who delivered Rh positive infants after 34 weeks in their first pregnancy in 1980-1 (group 1) and 2000 similar primigravidas from 1978-9 (group 2). Obstetric data were collected for 616 women in group 1 who had a subsequent pregnancy, 536 similar women in group 2, and 410 Rh positive but otherwise similar primigravidas who delivered in the same hospitals in 1978-81 (group C). INTERVENTIONS--Anti-D immunoglobulin 100 micrograms intramuscularly was given at 28 and 34 weeks to the mothers in their first pregnancy who delivered in 1980-1. END POINTS--Detection of anti-D antibody in the first or any subsequent pregnancy in groups 1 and 2. For all three groups having subsequent pregnancies gestation at delivery, birth weight, fetal survival at one month, pre-eclampsia defined as blood pressure greater than 140/90 on two occasions more than 12 hours apart, and proteinuria greater than 0.25 milligram. MEASUREMENTS AND MAIN RESULTS--Antenatal immunisation to Rh(D) occurred in six mothers in group 1 and 32 group 2. Most immunisations occurred in the first or second pregnancy. The rates of abortion, gestation at delivery, birth weight, and fetal survival were not significantly different among the three groups. The incidence of pre-eclampsia was lower in mothers given antenatal anti-D immunoglobulin, but the difference was not significant. CONCLUSIONS--Antenatal prophylaxis with anti-D immunoglobulin is effective, and the effect of giving it in the first pregnancy persists into at least the second pregnancy. It seems to be safe for the fetus in the index and subsequent pregnancies.     

Prevention of Rh haemolytic disease.

Between 1970 and 1976 in the Yorkshire region the incidence of Rh antibodies in Rh-negative pregnant women fell by 70%. This decrease occurred in both old (long-standing) and new (first-affected) cases, which emphasised that the reduction in numbers was as much due to fewer pregnancies among Rh-negative mothers as to administration of anti-D immunoglobulin. Nevertheless, the incidence has begun to level out. The continued incidence of first-affected cases is caused by three main factors: failure of administration of anti-D immunoglobulin after normal deliveries and abortions; a steady incidence of antibodies in primigravidae; and cases in which administration of anti-D immunoglobulin had failed to protect. Administering anti-D antenatally might reduce the incidence of new cases among primigravidae who are sensitised before anti-D is normally given. Even without routine antenatal administration of anti-D, the incidence of severely affected Rh babies in the Yorkshire region could be reduced to one or two isolated cases a year in a population of three to four million by administering anti-D after all Rh-negative deliveries and after every abortion.     

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for sup-pressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human trans-formed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.     

Reduced severity of Rh-haemolytic disease after anti-D immunoglobulin.

A total of 2459 Rh-negative women who received anti-D immunoglobulin after a Rh-positive pregnancy were followed up in at least one subsequent pregnancy. There was a failure of protection rate of 1-6%. Follow-up of 53 subsequent infants of mother in whom protection had failed showed that the infants were less severely affected than would have been expected. This was confirmed by a comparative statistical analysis of the present series and a series of first affected cases before anti-D immunoglobulin was available, using the antibody titre during pregnancy and the haemoglobin levels at delivery.     

Is an expression system for producing therapeutic antibodies with immunosuppressive properties found at last? Comment to letter by Dr. Quagliaroli

The prophylaxis of the hemolytic disease of the newborn — a mandatory procedure in obstetrics — requires significant amounts of plasma-derived polyclonal anti-D immunoglobulin. Despite numerous attempts, the proper technology for mass production of effective monoclonal anti-D is still not available. LFB Biotechnologies is currently performing clinical trials with recombinant anti-D antibody that has low fucose content and is expressed in the cells of rat myeloma YB2/0. It was shown that this drug is well tolerated, accelerates fast clearance of D+ red blood cells, and can inhibit anti-D immune response in Rhesus-negative volunteers.     

Haemolytic Disease of the Newborn Caused by Anti-Lan Antibody

The first recorded example of anti-Lan associated with haemolytic disease of the newborn is reported. This emphasizes the importance of screening for atypical antibodies early in pregnancy, even though prophylactic use of anti-D immunoglobulin will eventually reduce the incidence of haemolytic disease due to anti-D antibody.     

The Effect of Anti-D IgG on D-positive Recipients

When one standard prophylactic dose of anti-Rh(D) immunoglobulin (Connaught) (and in one case two doses) was injected into 15 D-positive volunteers it caused no reaction and no measurable alteration in readings of hemoglobin, hematocrit, reticulocytes or bilirubin, although some of the volunteers briefly became weakly direct Coombs''-positive. It is argued that since any isohemagglutinin that may contaminate anti-D IgG must be much less in amount than the anti-D it contains, the contaminating antibody cannot carry a greater potential for harm to a recipient having the corresponding antigen than the anti-D for a D-positive recipient, which is zero. It is suggested that the presence of anti-DC or G in so-called anti-Rh(D) IgG may, in fact, be a mark of excellence. The application of these facts to selection of plasma donors and to tests required before giving prophylactic treatment to women is briefly stated.     

Virus reduction in an intravenous immunoglobulin by solvent/detergent treatment, ion-exchange chromatography and terminal low pH incubation

Virus reduction by several steps in the manufacturing process for the intravenous immunoglobulin Vigam^®, has been investigated. The solvent/detergent step based on treatment with 0.3% tri-n-butyl phosphate and 1% polysorbate 80 at 37 °C, was confirmed to be effective for a range of enveloped viruses. Virus infectivity was undetectable i.e. >6 log inactivation within 30 min of the standard 6 h process. This was consistent over the range of conditions tested i.e. solvent/detergent and protein concentration, temperature and pH. The ion-exchange chromatography step in the process was also able to remove some viruses. Virus spiked followed by blank column runs confirmed the effectiveness of the sanitisation step for ensuring there was no virus cross contamination between column runs. The terminal low pH incubation step was also able to inactivate enveloped viruses, as well as some non-enveloped viruses. The combination of these three steps ensures a high margin of virus safety for this product.     

Process development for the recovery and purification of recombinant protein G.

The domains of protein G from streptococcus which bind immunoglobulin G have been cloned and expressed in Escherichia coli (Fahnestock et al., 1986). Because protein G binds to several animal immunoglobulin G's, it has many immunochemical applications. This report describes process development for large-scale production of this recombinant protein G (also known as GammaBind G). In 200 l cultures of E. coli, this protein G variant was released from the cell into the culture medium by heating at 80 degrees C for 10 min. The concentration was monitored by either a competitive enzyme-linked immunoassay or a liquid chromatographic assay. Cross-flow microfiltration with 0.22 micron membrane was used to remove the cells. The protein G-rich permeate from the cross-flow microfilter was purified by affinity chromatography using a 5 l column of IgG-Sepharose 6 Fast Flow, which yielded 16-18 g of protein G per column cycle. The pools of purified protein G were concentrated and desalted using ultrafiltration. The salt-free protein G was then lyophilized as bulk product. The overall recovery through the entire process was 50-64%. The analysis of the final product included sodium dodecyl sulfate polyacrylamide gel electrophoresis, UV-visible spectrum, high performance gel filtration, endotoxin level and binding efficiency to human IgG Sepharose.     

Standardization of U.S. Reference Rh0 (D) Immune Globulin by quantitative automated hemagglutination

An automated hemagglutination procedure was used to assess the relative potency of US Reference Rh0 (D) Immune Globulin, Lot 3, with respect to the International Reference Preparation, WHO Anti-D immunoglobulin, Lot 68/419. A value of 300 international units (IU) of anti-D per ampoule has been assigned to Lot 68/419. In 25 assays, the mean value for Lot 3 was 820 IU anti-D per milliliter when tested in parallel with Lot 68/419.     

Deaths from rhesus haemolytic disease in England and Wales in 1982 and 1983.

Examination of death certificates and the clinical notes of the patients concerned showed that the number of deaths from rhesus (D) haemolytic disease in England and Wales was 44 and 34 during 1982 and 1983, respectively, a substantial decrease from the figure of 106 for 1977. Of the 78 women whose infants died in 1982 and 1983, 49 had not received anti-Rh immunoglobulin after previous pregnancies with Rh positive infants; most of these deaths would presumably have been prevented had postnatal anti-Rh immunoglobulin been given. In 13 women anti-D was detected during, or immediately after, a first pregnancy, and in 15 women rhesus immunisation developed despite administration of anti-Rh immunoglobulin postnatally. One or two apparent failures of treatment may have been due to underdosage, but it must be concluded that about one third of the deaths in 1982 and 1983 could have been prevented only by giving anti-Rh immunoglobulin antenatally as well as postnatally.     

High dose intravenous immunoglobulin repertoire versus anti-D. Disproportions in quantity and quality.

Here we consider certain therapeutic effects that intravenous administration of pooled high dose immunoglobulin and anti-D IgG share. Despite million-fold difference in doses such an effect occurs at least in idiopathic thrombocytopenic purpura (ITP). We postulate that spontaneous bleeding events may remit even when platelet numbers show refractoriness. We also mention the possible sparing of anti-D antibody-coated red blood cell (RBC) destruction and, finally, an acceleration of fibrotic involution. Fc receptors (FcRs) play a central role; beyond the well-established interactions with the immunoglobulin Fc fragment, FcRs are supposed to display special cognitive properties that enable them to pick out the therapeutic molecules from the recipient's IgG pool. Such subtle selection suggests some disarray in the host. On the other hand it may explain why the often-encouraging outcome of IVIG therapy remains unpredictable.     

A chemiluminescence assay for erythrophagocytosis

A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized with anti-D IgG immunoglobulin by mononuclear leukocytes is described. The mononuclear leukocytes were obtained by apheresis enriched by centrifugation through a density gradient and stored in liquid nitrogen before use. The total reaction mixture, consisting of mononuclear leukocytes-luminol-erythrocytes (either anti-D IgG sensitized or unsensitized controls) was 500 μl, light detection was by an LKB 1251 luminometer. Peak luminescence was seen between 35–45 minutes, the reaction being exhausted by 120 minutes. Determination of the reproducibility of the assay gave intra- and inter-assay coefficients of variation of 5% and 13% respectively. We found the chemiluminescent response to be affected by the number of erythrocytes used in the assay and by the composition of the medium in which the cells were resuspended, particularly the pH at the initiation of the assay. We also compared the chemiluminescence assay to a microscopic phagocytic assay and found the results virtually identical. However, the former chemiluminescence assay was much easier to perform, marginally more sensitive, less laborious and eliminated any possibility of subjective error.     

Addressing a whole bioprocess in real-time using an optical biosensor-formation,recovery and purification of antibody fragments from a recombinant<Emphasis Type="Italic"> E. coli</Emphasis> host

The use of biosensor technology is described to address in real-time the production and subsequent purification of a bioactive recombinant protein product. The product, D1.3 Fv antibody fragment, was expressed in Escherichia coli and purified via two process routes, one for extracellular and one for intracellular product material. The cells were harvested by centrifugation in a solid bowl CARR Powerfuge and stored at –70°C. Clarification of the supernatant was performed by depth filtration, followed by affinity chromatography for final purification of the extracellular product. To purify the intracellular product the harvested cells were resuspended and homogenised. Removal of debris in the CARR Powerfuge was followed by depth filtration and affinity chromatography. In this work we have shown the rapid determination of bioactive product levels, and the impact this has on improved accountability and confidence is demonstrated in process mass balances on the product using the data acquired during process operation.     

International collaborative study to assess candidate reference preparations to control the level of anti-D in IVIG for use in Europe and the United States.

Regulatory requirements to control the level of anti-D in intravenous immunoglobulin (IVIG) products with European and United States (US) licences are to be introduced. A reference preparation of IVIG containing anti-D at 0.0475 IU/ml and having a nominal titre of 8 using the proposed direct haemagglutination reference method was deemed suitable to define the anti-D limit. This preparation, code 02/228, and a negative control IVIG preparation, code 02/226, were established by the World Health Organization as International Reference Reagents (IRRs). As stocks of the IRRs are limited, new larger fill stocks of positive and negative reference preparations, codes 04/132 and 04/140, respectively, were produced. The results from an international collaborative study involving 16 laboratories showed that preparations 04/132 and 04/140 are indistinguishable from the corresponding IRRs 02/228 and 02/226, respectively, using the proposed direct haemagglutination reference method. Stocks of 04/132 and 04/140 have been shared with the European Directorate for the Quality of Medicines (re-coded as 23613 and 23614, respectively) and with the Center for Biologics Evaluation and Research of the United States Food and Drug Administration (re-coded as CBER Lots 1B and 1N-b, respectively) for use as European and US Biological Reference Preparations, respectively.     

Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations

Proteolytic fragmentation is the only suggested cause of potency losses during storage of liquid human polyclonal anti-D Ig. Besides the effect of fragmentation, we have investigated the potential contribution of neutralising anti-idiotypic antibodies (anti-Ids). Potency changes during storage and/or upon pH reduction in anti-D IgG batches with or without addition of plasminogen and urokinase were quantitatively analysed by the autoanalyser (AA) method or by a special procedure of flow cytometry (FC). Moreover, simultaneous changes of the molecular size distribution pattern have been determined by size exclusion chromatography. In contrast to the AA procedure, the particular FC methodology was found to be almost insensitive to proteolysis comprising up to 30% of total IgG. Data interpretation was based on the assumption that both assays cannot detect Ids with neutralised paratopes. In the absence of detectable neutralisation (functional absence of anti-Ids), it could be demonstrated that the anti-D IgG subpopulation is more sensitive to fragmentation by endogenous protease as compared to the unrelated bulk. However, both methods detected batch- and assay-dependently variable potency losses during storage. Moreover, the increase of potency induced by pH reduction correlated with the increase of monomeric IgG, essentially on the expense of dimers. This finding was interpreted to indirectly indicate the neutralising action of anti-Ids known to be the major driving force of dimer formation in polyclonal IgG. A more or less pronounced pH-dependent potency increase was also detectable in three arbitrarily selected batches of two other manufacturers. The data allows to assume that anti-Id-mediated neutralisation can significantly contribute to losses of anti-D potency. In addition, it turned out that anti-D plasma itself can be the source of anti-Ids.     

Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein

An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.     

Glycosylation and functional activity of anti-D secreted by two human lymphoblastoid cell lines

Cell lines BTSN4 and BTSN5 were produced by the Epstein-Barr Virus (EBV) transformation of B-lymphocytes from the same human donor. Both secrete an anti-D monoclonal of the IgG1 subclass but these antibodies display vastly different effector activities. Specifically, anti-D from BTSN4 has a far greater activity in both monocyte-and lymphocyte-mediated ADCC reactions and causes a higher percentage of rosettes to be formed with monocyte-like U937 cells. This variation in functional activity is shown to coincide with changes in the structure of the sugar chains attached to the asparagine-297 site on the immunoglobulin heavy chain.Abbreviations ADCC antibody dependent cellular cytotoxicity - EBV Epstein-Barr virus - GlcNAc glucosamine - Gal Galactose - Man Mannose - Fuc Fucose - SA Sialic acid