**3. IVIG production**

90 Immunodeficiency

therapy [12,13]. The benefits in diseases such as childhood thrombocytopenia and Kawasaki disease refractory to or intolerant of conventional treatment have been well established [14,15]. It has been 30 years since therapeutic contribution of intravenous immunoglobulin (IVIG) administration has been proven by scientists, an increasing number of immunemediated diseases have been treated with intravenous immunoglobulin rather than corticosteroids and cytoxic drugs. IVIG has become the therapy of choice in autoimmune diseases, severe asthma, neurological diseases, transplantation, sepsis, septic shock, toxic shock syndromes and dermatologic disorders [15,16]. The recommendation of IVIG treatment in other diseases than those approved by FDA is based on limited data or some of these diseases do not have any alternative treatment regimen to compare with [16]. However, IVIG administration in the treatment of many diseases is raising the possibility of product shortages and increasing costs. Thus, concerning the shortages of products, cost and adverse reactions, definite indications for IVIG treatment are essential [12,13,16,17]. The aim of immunoglobulin therapy should be to protect the patients from frequent and severe infections finally resulting in organ damage. Advances in human immunology, has led to identify responsible genes for PID, thereby particular groups of defects are associated with susceptibility to specific types of infection [18]. Improved diagnostic precision is likely to increase more specialized management strategies of patients with PID, some of which are only supported by expert consultation. However, there are no sufficient number of studies

in PID, to optimize the quality and uniformity of management of PID**.** 

Cohn et al produced the first human immunoglobulin IgG product in1946 and it was referred as immune serum globulin (ISG)[19]. This first commercial human ISG solution tended to form aggregates during storage, therefore it was delivered via the intramuscular or subcutaneous route. After diagnosing his first patient with agammaglobulinemia in 1952, Bruton began to treat his patients by subcutaneous replacement therapy with ISG [20]. After a short time, intramuscular ISG treatment became available for all patients, but the amount of Ig used for treatment was limited and not effective enough to reduce recurrent infections and the adverse effects were also high due to IgG aggregates [21]. These disadvantages were abolished by Cohn fraction II that had been developed in 1960's by Barandum and his colleagues in collaboration with Swiss Red Cross [9,21]. The first IVIG was produced by pepsin digestion (enzymatic method: pepsin or trypsin) to reduce anticomplement activity, but this process cleaved the immunoglobulin molecule into two parts, resulting in fragments of the fc portion and Fab. Several manifacturers produced chemically modified IVIGs containing minimal anti-complement activity and no IgG fragments. Reduced bacterial opsonic activities and shortened circulating half-lives were demonstated in some antibodies of enzyme-digested or chemically modified IVIG preparations. Non-denaturating processes such as precipitation with polyethylene glycol (PEG), ion exchange chromatography, diafiltration and stabilisation of IgG at low pH, do not modify the IgG molecule and the

**2. History and recent development (IVIG)** 

half-life of IgG is generally 22-25 days [21].

IVIG preparations are derived from plasma of a huge number of human blood donors or paid plasmapheresis donors. Since IVIG preparations are blood-derived products having the risk of transmission of infectious transfusional diseases, viral safety needs to be considered [13,21,23]. The safety of IVIG products depends on donors, validated manufacturing processes and various virus clearance steps as listed below:


To produce a single product lot, sufficient number of donor recruitment and screening of viral markers (HBs-Ag, HIV-p24 antigen, antibodies to syphilis, HIV-1,HIV-2, HCV, HAV) are necessary to prevent the transmission of viruses [21].

FDA (Center for Biologics Evaluations and Research) and Plasma Protein Therapeutics Association recommended the number of donors to be minimum 15.000, but not more than 60.000. Manifacturing processes implemended in commercial IVIG preparations are the classical Cohn fractionations treated with solvent detergent, caprylate, acid or pepsin to inactivate pathogens [31-33].

Immunoglobulin, produced by cold ethanol fractionation method may contain trace amounts of contaminants such as prekallikrein activator, prekallikrein, activated coagulation factors, complement proteins, IgM, IgA, plasmin and plasminogen. Currently many manufacturers began to use purification with anion exchange (DEAE) chromatography adjusted to cold ethanol fractionations in order to obtain safe products.

Treatment at pH4 with trace amounts of pepsin is also validated by some manifacturers. Both, alcohol fractionation and acid treatment procedures eliminate other proteins and inactivate dangerous live viruses such as HIV, Hepatitis B, HCV.

Improved quality standards for plasma products and new blood borne pathogens such as SARS forced the scientists to develop and integrate new specific viral inactivation methods. RNA virus with lipid envelope, DNA virus with lipid envelope and non-lipid enveloped virusus must all removed by viral inactivation procedures. The heat and chemical treatment processes are able to remove and/or inactive blood-borne pathogens:

Immunoglobulin Treatment of Immunodeficient Patients 93

Octagam

Gammagard liquid Flebogamma 5% DIF

Flebogamma 5% Flebogamma 5% DIF

Carimune NF Privigen

> **IgA µg/mL**

100 310-380

720 192-1074

Trace 274

<2.2. 636

270 1250

37 240-300

**Osmolarity mOsm/kg** 

Octagam Privigen

**Antimicrobial processes** 

 Cold ethanol fractionation Solvent-detergent

Dialfiltration, Ultrafiltration Solvent-detergent

 Kistler&Nitchman Fractiotion, trace Pepsin, pH 4.0 Nanofiltration

Ultrasantrifuge, Ion exchange chromotography, Solvent-detergent

Ultrasantrifuge, Ion exchange, chromotography, Solvent-detergent

 Cohn-Oncley fractionation, Ion exchange chromotography, Nanofiltration, Solvent-detergent, pH 4 filtration

**Virus inactivation/removal procedure Product**

**Solvent-detergent inactivation** Gammagard S/D

**Heat inactivation(10h at 60 C)** Vivaglobulin

**Low pH incubation in final container(21 day)** Gamunex

**Table 1.** Dedicated virus inactivation procedures used in IVIG production [22]

**Sodium Content mEq/mL**

**Dosage form** 

Octagam Octapharma 5 %Liquid 0.03 Maltose

Gamimune N Bayer 10%Liquid Trace Glycine

3, 6 ,9,12% lyophilized

lyophilized powder

lyophilized powder

Baxter 10% liquid none glycine

CSL Behring AG

Baxter 5%

Baxter 10%

**Product Manufacturer** 

Carimune NF liquid

Gammagard 5% S/D

Gammagard 10% S/D

Gammagard S/D10% (KIOVIG)

**Removal by nanofiltration** Gammagard liquid

**pH4 incubation (in process)** Flebogamma 5% DIF

**Low pH incubation at elevated temperature in final container** Gammagard liquid **Pepsin treatment** Carimune NF **Caprylic acid virus inactivation** Gamunex

**/PH** 

PH 5.1-6

pH4.25

1.67 per gram protein PH 5.3

<20 sucrose

0.145 2% glucose PH 6.8

0.145 4% glucose PH 6.8

PH 4.85

**Stabilizing agent** 


Transmission of Prion diseases such as Creutzfeldt–Jakob disease (CJD) or variant CJD by administration of blood products is also possible, since the incubation period of the disease is too long leading to difficulties in risk determination. Because of this possibility, donors who have spent more than 6 months in the United Kingdom from 1986 to the present are not allowed to donate blood or plasma in the United States and Europe [21]. Some researchers demonstrated that depth filtration step that is common in all IVIG production procedures and nanofiltration removed hamster scrapie protein reactivity. The Finish Red Cross Blood Transfusion Service (FRC BTS' Helsinki, Finland) had developed a liquid 5% IVIG product (IVIG-L) in which a nanofiltration step was incorporated into the production process [34]. Van der Meer JWM et al. evaluated efficacy and safety of that nanofiltered liquid IVIG product and showed that IVIG-L was efficacious and pharmacokinetic properties were comparable to other IVIG preparations. In addition relatively low level of adverse reactions and the absence of seroconversion were observed. Thus, this liquid form product is considered to be safe and well tolerable. Over the past years, improved manifacturing processes and integrated specific viral inactivation steps have increased the safety and quality of IVIG products (Table 1). Commercially available products represent recent advancements in IVIG product formulation, but potential transmission of emerging pathogens can still not be ruled out completely.

Currently licensed IVIG preparations are supplied either in lyophilized powder or premixed solution, contains 95% IgG at a concentration of 16.5% (165 mg/ml), all the IgG subclasses, multiple IgG allotypes (Gm and Km), minimal anti-complement activity, broad spectrum of antibodies against viruses and bacteria, and no difference in therapeutic efficacy. Half-life of immunoglobulins is approximately 21-25 days. The osmolarity varies between 253 mOsm/L for a 5% IgG product to1250 mOsm/L for a 10% product. The final sterile product contains varying amounts of sodium, glycine, polyethylene glycol, D-mannitol, D-sorbitol, sucrose, glucose or maltose, glycerol as the stabilizer, and thiomersal as the preservative and has a pH of 6.8 (Table 2).


**Table 1.** Dedicated virus inactivation procedures used in IVIG production [22]

92 Immunodeficiency

presence of stabilizers.

viruses.

Improved quality standards for plasma products and new blood borne pathogens such as SARS forced the scientists to develop and integrate new specific viral inactivation methods. RNA virus with lipid envelope, DNA virus with lipid envelope and non-lipid enveloped virusus must all removed by viral inactivation procedures. The heat and chemical treatment

a. Pasteurisation: Based on heating to 60°C in an aqueous solution for 10 hours in the

b. Solvent/Detergent: The solvent/detergent consists of an organic solvent (ether, 0.3% trin-butylphosphate (TNBT) and 0.2% detergent (Tween 80, sodium cholate or triton-100).

d. Low pH-incubation: This incubation at elevated temperatures completely removes

Transmission of Prion diseases such as Creutzfeldt–Jakob disease (CJD) or variant CJD by administration of blood products is also possible, since the incubation period of the disease is too long leading to difficulties in risk determination. Because of this possibility, donors who have spent more than 6 months in the United Kingdom from 1986 to the present are not allowed to donate blood or plasma in the United States and Europe [21]. Some researchers demonstrated that depth filtration step that is common in all IVIG production procedures and nanofiltration removed hamster scrapie protein reactivity. The Finish Red Cross Blood Transfusion Service (FRC BTS' Helsinki, Finland) had developed a liquid 5% IVIG product (IVIG-L) in which a nanofiltration step was incorporated into the production process [34]. Van der Meer JWM et al. evaluated efficacy and safety of that nanofiltered liquid IVIG product and showed that IVIG-L was efficacious and pharmacokinetic properties were comparable to other IVIG preparations. In addition relatively low level of adverse reactions and the absence of seroconversion were observed. Thus, this liquid form product is considered to be safe and well tolerable. Over the past years, improved manifacturing processes and integrated specific viral inactivation steps have increased the safety and quality of IVIG products (Table 1). Commercially available products represent recent advancements in IVIG product formulation, but potential transmission of emerging

Currently licensed IVIG preparations are supplied either in lyophilized powder or premixed solution, contains 95% IgG at a concentration of 16.5% (165 mg/ml), all the IgG subclasses, multiple IgG allotypes (Gm and Km), minimal anti-complement activity, broad spectrum of antibodies against viruses and bacteria, and no difference in therapeutic efficacy. Half-life of immunoglobulins is approximately 21-25 days. The osmolarity varies between 253 mOsm/L for a 5% IgG product to1250 mOsm/L for a 10% product. The final sterile product contains varying amounts of sodium, glycine, polyethylene glycol, D-mannitol, D-sorbitol, sucrose, glucose or maltose, glycerol as the stabilizer, and thiomersal as the preservative and has a

The process lasts for 6 hours and destroys infectivity of lipid-enveloped viruses. c. Nanofiltration: This procedure is effective to remove small non-enveloped (B19V, HAV)

processes are able to remove and/or inactive blood-borne pathogens:

lipid-enveloped viruses like HIV, HBV/HCV).

pathogens can still not be ruled out completely.

pH of 6.8 (Table 2).



Immunoglobulin Treatment of Immunodeficient Patients 95

• >20 days of half life • >90 % monomeric IgG

• Low adverse effects • Trace IgA concentration • Stabile in solution

**4. Mechanism of action** 

• Low price

antagonists

• Effective IgG subclasses, a profile similar to that of human plasma • Complete Fc functions, complement fixation, opsonophagocytosis

immunomodulatory effects of IVIG infusion are shown below [35-37]:

• Changes in solubility and rate of clearance of immune complexes

• Functional blockade of Fc receptor on splenic macrophages

• Neutralization of circulating autoantibodies

• Inhibition of complement-mediated damage

• Neutralization of superantigens

• No pyrogenic and vasoactive agents (kinin or plasmin), protein aggregates

Human immunoglobulin is obtained from a large number of donors and exceeding 2.000 donors is preferred. IVIG contains large spectrum of antibody specificities such as antibodies to foreign (non-self) antigens, to self-antigens (natural autoantibodies) and to other antibodies (idiotypic antibodies which represents antibody repertoire of each donor [35]. That is the reason of the differences between immunoglobulin batches [13,21,35]. The mechanism of activity of the substituted IgG is easily understood for immunodeficiency disorders considering common pathogen-specific IgG antibodies are replaced by those from the donor pool [35]. Thereby, regular intravenous immunoglobulin therapy reduces the incidence of infection in these patients compared to their infection rates before IVIG treatment [7-13]. Immunomodulatory effect of IVIG therapy depends on several mechanisms. Proposed early

• Modulation of production and release of proinflammatory cytokines and cytokine

On the other hand, IVIG infusion downregulates IVIG-reactive B cell clones in long-term. Serum IL-6, IL-8, IL-1Ra and TNFalpha concentrations were increased in patients with primary immunodeficiencies following IVIG infusion, without any difference in serum ILbeta, IFNgamma or IL-2 levels. Understanding these immunomodulatory effects of IVIG is essential to define IVIG indications in autoimmune disorders [35-37]. In severe infections regarding increased catabolism of IgG, IVIG can be added to antibiotic treatments [16, 17]. The concentration of IgG is very important for its pro-inflammatory or anti-inflammatory properties. Low-dose IVIG has proinflammatory properties, but high dose IVIG has antiinflammatory effects. The proinflammatory properties are dependent on complement activation or binding of the Fc fragment of IgG to IgGspecific (FcγR) on effector cells of the innate immunity leading to receptor clustering, activation of intracellular signaling pathways and finally to cell activation. The anti-inflammatory effect of IgG is still not clear, but IgG is known to inhibit the differentiation and maturation of human dendritic cells (DCs), expression

**Table 2.** Commercial IVIG Products and properties (Data from Immune Deficiency Foundation, October 2011 and reference [17, 22])

All the available IVIG preparations approved by FDA and EMEA should at least have the following features:

• Sterile >4000[5000-10000]donors

• >20 days of half life

94 Immunodeficiency

Flebogamma DIF

Venoglobulin

Gammar-PIV Centeon,

Endobuln Baxter

Privigen CSL Behring AG

Gamunex- C Talecris

Omr-IgG-am Omrix

following features:

L.L.C., Kankakee

Iveegam Immuno US lyophilized

Immuno France

Biotherapeutics

Biopharmac euticals Ltd

October 2011 and reference [17, 22])

• Sterile >4000[5000-10000]donors

S

**Product Manufacturer** 

**Dosage form** 

Liquid

10% Liquid

5%

Grifols 5% , 10%

Alpha 5 %

**Sodium Content mEq/mL** **Stabilizing agent** 

**Antimicrobial processes** 

Cold alcohol fractionation, PEG, Ion exchange, chromotography, PH4 treatment, Solvent-detergent, double sequential nanofiltration

PEG, Ion exchange Chromotography, Solvent-detergent,

Cold ethanol fractionation, heat10 hours 60°C

Polyetilene glycol/trypsin

Solvent-detergent

pH 4 filtration

fractionation, CH9 filtration, pH 4.0 incubation, Depth filtration, Chromotography, Nanofiltration ,

fractionation, caprylate precipitation, Sepharose chromatography, Cloth and depth filtration Final container pH 4.25 ±0.25incubation

Cold ethanol fractionation, S/D, 24 h @ pH 4, pH 5.5 ± 0.4,

None Octanoic acid

None Cohn-Oncley

**IgA µg/mL** 

5%: < 50 10%: < 100

24

100

25 258

5 >240

≤25 Isotonic (320)

46 258

**Osmolarity mOsm/kg** 

240-370

**/PH** 

<0.032 D-sorbitol PH 5.0-6.0

 Albumin (human) D-sorbitol

0.05 Glucose, NaCl

(human) Sucrose PH 6.8

Polyetylene glycol(PEG),

lyophilized 0.085 Albumin

lyophilized 3mg Glucose,

IgVena Sclavo Liquid Maltose Solvent-detergent

amount

amount

Liquid 50 mg/mL; 100

**Table 2.** Commercial IVIG Products and properties (Data from Immune Deficiency Foundation,

All the available IVIG preparations approved by FDA and EMEA should at least have the

mg/mL maltose

Liquid Trace

Liquid Trace


### **4. Mechanism of action**

Human immunoglobulin is obtained from a large number of donors and exceeding 2.000 donors is preferred. IVIG contains large spectrum of antibody specificities such as antibodies to foreign (non-self) antigens, to self-antigens (natural autoantibodies) and to other antibodies (idiotypic antibodies which represents antibody repertoire of each donor [35]. That is the reason of the differences between immunoglobulin batches [13,21,35]. The mechanism of activity of the substituted IgG is easily understood for immunodeficiency disorders considering common pathogen-specific IgG antibodies are replaced by those from the donor pool [35]. Thereby, regular intravenous immunoglobulin therapy reduces the incidence of infection in these patients compared to their infection rates before IVIG treatment [7-13]. Immunomodulatory effect of IVIG therapy depends on several mechanisms. Proposed early immunomodulatory effects of IVIG infusion are shown below [35-37]:


On the other hand, IVIG infusion downregulates IVIG-reactive B cell clones in long-term. Serum IL-6, IL-8, IL-1Ra and TNFalpha concentrations were increased in patients with primary immunodeficiencies following IVIG infusion, without any difference in serum ILbeta, IFNgamma or IL-2 levels. Understanding these immunomodulatory effects of IVIG is essential to define IVIG indications in autoimmune disorders [35-37]. In severe infections regarding increased catabolism of IgG, IVIG can be added to antibiotic treatments [16, 17].

The concentration of IgG is very important for its pro-inflammatory or anti-inflammatory properties. Low-dose IVIG has proinflammatory properties, but high dose IVIG has antiinflammatory effects. The proinflammatory properties are dependent on complement activation or binding of the Fc fragment of IgG to IgGspecific (FcγR) on effector cells of the innate immunity leading to receptor clustering, activation of intracellular signaling pathways and finally to cell activation. The anti-inflammatory effect of IgG is still not clear, but IgG is known to inhibit the differentiation and maturation of human dendritic cells (DCs), expression of co-stimulatory molecules like CD80 and CD86, both leading to lower self antigen processing and presentation [8]. Fc and F(ab′)2 fragments of IgG molecule are both able to suppress of DCs. Antibodies with the intrinsic capacity to recognize foreign antigens or common pathogen-specific IgG antibodies are replaced by those from the donor pool [35].

Immunoglobulin Treatment of Immunodeficient Patients 97

The guidelines for clinical Investigation of human normal Immunoglobulin for Intravenous administration of the European Medicines Agency (EMA/CHMP/BPWP/94033/2007 rev.2) and FDA recommended that an immunoglobulin product is effective if treated patients experience less than 1.0 serious infection per year [21,34]. A new IVIG product must have 'intact IgG' which means pharmacokinetic properties of Immunoglobulin G is similar to

2. Prevention of bacterial infections in patients with hypogammaglobulinemia and

4. Prevention of infections, pneumonitis, and acute graft-versus-host disease (GVHD)

5. Reduction of serious and minor bacterial infections, to decrease the frequency of

6. Increase of platelet counts in idiopathic thrombocytopenic purpura to prevent or

IVIG therapy has been evaluated in a number of clinical conditions mentioned above and categorization of evidence, basis of recommendation and strength of recommendation have

III From nonexperimental descriptive studies such as comparative, correlation or case

**Table 3.** Categorization of evidence and basis of recommendation and strength of recommendation [17]

Benefits Diseases Evidence Strenght of

IV From expert committee reports or opinions or clinical experience of respected

B Based on category II evidence or extrapolated from category I evidence C Based on category III evidence or extrapolated from category I or II evidence D Based on category IV evidence or extrapolated from category I, II or III evidence

recurrent bacterial infections caused by B-cell chronic lymphocytic leukemia

endogeneous IgG and available other immunoglobulin preparations.

IVIG, has been licensed by FDA for only 6 clinical indications [8,22,23]:

3. Prevention of coronary artery aneurysms in Kawasaki disease

**6. Indications of IVIG treatment** 

1. Treatment of primary immunodeficiencies

after bone marrow transplantation

hospitalisation in children with HIV

been established (Table 3 and Table 4) [16].

Categorization of evidence and basis of recommendation Ia From meta-analysis of randomized controlled studies Ib From at least one randomized controlled study

IIa From at least one controlled study without randomization IIb From at least one one other type of quasiexperimental study

control bleeding

control studies

authorities or both

**Strenght of recommendation** A Based on category I evidence

At a lower dose, administered generally to patients with immunodeficiencies, however, IVIG exerts a contrasting effect. DCs of patients with common variable immune deficiency (CVID) differentiated in the presence of IVIG and presented with an up-regulated expression of CD1a and the co-stimulatory molecules CD80, CD86 and CD40 [38,39]. Defective functions of DCs have been associated with predisposition to several pathological conditions. CVID patients display high susceptibility to recurrent infections and autoimmune diseases that could be due in part to impaired DC functions [38,39].

Advantages of IVIG administration are the following:


Unfortunately, there are also some disadvantages of IVIG administrations:

