Porcine Gamma Globulin Salt Fractionation

BSA Cell Culture Grade Grade 35% BSA Liquid
BSA Cell Culture Grade Grade 35% BSA Liquid

Swine Gamma Globulin Fraction Properties

Description
SWINE Gamma Globulin Fraction – D613-0100
Species of Origin
Swine
Physical State
Lyophilized
Shipping Condition
Ambient
PSC Type
Protein
PSC Format
Native Protein
Label
Unconjugated
Concentration
12.5 mg/mL by UV absorbance at 280 nm
Buffer
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Reconstitution Volume
10.0 mL
Reconstitution Buffer
Restore with deionized water (or equivalent)
Preservative
0.01% (w/v) Sodium Azide
Stabilizer
None

E Immunohistochemistry

Another analytical use for antibodies is to localize particular bioregulators, synthesizing enzymes, or degrading enzymes in tissues, cells, or parts of cells. This approach, called immunohistochemistry (or immunocytochemistry), employs an antibody made in one species, say a mouse, against the specific molecule (antigen) you wish to localize in the brain of a song sparrow or other animal. There are several variations of this technique based on a simple procedure (Fig. 2.7). First, you make the mouse antibody that would be a mouse gamma-globulin protein and apply that to sections of song sparrow tissue placed on a microscope slide.

Theoretically, the antibody will bind only in cells that contain the antigen. In another mammalian species, you make an antibody against mouse gamma-globulin and conjugate that with an enzyme known as a peroxidase. This is also applied to the tissue sections. This second antibody will bind to the gamma-globulin, which has bound previously to the song sparrow antigen. Next, you add a substrate for the peroxidase enzyme that results in a colored product that will be localized in the cell containing the antigen-gamma-globulin antigamma-globulin-peroxidase complex. This complex can then be viewed with the microscope.

Abstract

Immunoglobulin is a highly diverse autologous molecule able to influence immunity in different physiological and diseased situations. Its effect may be visible both in terms of development and function of B and T lymphocytes. Polyclonal immunoglobulin may be used as therapy in many diseases in different circumstances such as primary and secondary hypogammaglobulinemia, recurrent infections, polyneuropathies, cancer, after allogeneic transplantation in the presence of infections and/or GVHD. However, recent studies have broadened the possible uses of polyclonal immunoglobulin showing that it can stimulate certain sub-populations of T cells with effects on T cell proliferation, survival and function in situations of lymphopenia. These results present a novel and considerable impact of intravenous immunoglobulin (IVIg) treatment in situations of severe lymphopenia, a situation that can occur in cancer patients after chemo and radiotherapy treatments. In this review paper the established and experimental role of polyclonal immunoglobulin will be presented and discussed as well as the manufacturing processes involved in their production.

Introduction

In 1890 von Behring firstly communicated their work in serum application against diphtheria [1] and tetanus [2]. Since then, intravenous immunoglobulin (IVIg) has been object of intense investigation in terms of function, clinical application, molecular structure and its modification, and purification.

Human immunoglobulin G (IgG) has been used to treat people with inherited immunoglobulin deficiencies since 1952 when Bruton infused it in a child with undetectable “gamma globulin” levels and who suffered from recurrent pneumococcal infections [3]. Subcutaneous infusions of gamma globulin at monthly intervals induced serum measurable gamma globulin levels and completely eliminated pneumococcal infections. Human IgG soon became the standard treatment for patients with primary antibody deficiencies who develop chronic bacterial infections [3]. Later, in 1981, the use of IVIg for the treatment of autoimmune diseases was first described [4]. Nowadays, IVIg is the major product on the global blood product market and its consumption is multiplying every year in North America, Europe and Asia for licensed and unlicensed/off-label uses.

The active substances in IVIg preparations are polyclonal natural antibodies synthesized, in response to immune stimuli (antigens and T cells), by plasma B cells.

Immunoglobulins are a group of closely related glycoproteins composed of 82%–96% protein and 4%–18% carbohydrate. These glycoproteins of about 150 kDa are present in plasma at a mean concentration of 7 to 12 g/L depending upon individual variations and level of environmental exposure to antigens. The immunoglobulin G (IgG), a major effector molecule of the humoral immune response in man accounts for about 75% of the total Igs in the plasma of healthy individuals. The Igs of the other classes (IgM, IgA, IgD and IgE) each of which has specific properties and functions, constitute the other 25% of the Igs [5].

BSA Cell Culture Grade Grade 35% BSA Liquid
BSA Cell Culture Grade Grade 35% BSA Liquid

The basic Ig molecule has a four-chain structure, comprising two identical heavy (H) chains and two identical light (L) chains, linked together by inter-chain disulfide bonds. Intra-chain disulfide bonds are responsible for the formation of loops, leading to the compact domain-like structure of the molecule. The amino terminal portions of the H and L chains, characterized by a highly variable (V) amino acid composition, are referred to a VH and VL, respectively. The constant part (C) of the L chain is designated as CL, while that of the H chain are further divided into three distinct subunits: CH1, CH2 and CH3. IgG possesses dual functions characterized by the capacity to recognize and react specifically with the antigen and to perform a series of non-specific effector functions in which the antigen is rendered harmless and eventually eliminated. This functional dichotomy of IgG is reflected in the structure of the molecule that comprises two variable regions responsible for antigen binding (Fab), and a constant region (the Fc or crystallisable fragment) that mediates the specific effector functions.

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The Fab fragments are composed of a light chain and part of a heavy chain, whereas the Fc fragment has various regions (CH), which exhibit several functions such as binding of complement component 1 (C1) and interactions with the Fc receptor on macrophages or neutrophils and other immune cells. The activation of the effector function is initiated by an aggregation of the IgG molecule on the surface of the antigen. Such activation exposes molecular structures that can activate the complement system or induce opsonization by phagocytes [6]. Immunoglobulins, B and T cells are the key mediators of adaptive immunity. Deficiencies in either of these two arms of immunity can lead to a heightened susceptibility to bacterial, fungal or viral infections. Primary immunodeficiency (PID) disorders, such as agammaglobulinaemias, hyperimmunoglobulin M (IgM) syndromes and common variable immunodeficiencies (CVID), are either caused by defined gene mutations or remain molecularly undefined [7,8]. In addition, hypogammaglobulinaemic phenotypes, termed secondary immunodeficiencies, can arise for example from viral infections, B cell malignancies, bone marrow transplantation or after immunosuppressive therapy [7]. For most of the primary and secondary Ig deficiencies, intravenous Ig replacement therapy (IVIg) is the treatment of choice.

The polypeptide chains of Igs are encoded by three non-linked cluster of autosomal genes, one cluster coding for H chains of all classes and subclasses, a second one for kappa (k) light chains and a third one for lambda (λ) light chains. These three genes clusters are called the H-, k-and λ gene families respectively. In humans the H gene family is on chromosome 14, the k gene family is on chromosome 2 and the λ gene family is on chromosome 22. Molecular genetic studies have revealed the arrangement of gene segments within the H chain and L chain families. Each H chain is encoded by 4 distinct types of gene segments, designated VH (variable), DH (diversity), JH (joining) and CH. The V region of the H chain is encoded by the VH, DH and JH segments. The L chains are encoded by the 3 gene segments, VL, JL and CL segments.

The C gene segments of the H and L chains encode for the constant regions. Nine immunoglobulin H chain isotypes are found in humans: IgM, IgE, IgG (with subclasses IgG1, IgG2, IgG3 and IgG4) and IgA (with subclasses IgA1 and IgA2).

The CH gene segments determine the class and/or subclass of the H chain, whereas VH, DH and JH regions determine the antigen-recognizing part of the Ig molecule. The H and L chains constant genes lie 3′ to the VH, DH, JH, and VL, JL genes, respectively. During maturation of progenitor B cells to mature B cells an active H chain exon is formed by VH, DH and JH, and that of L chain formed by VL and JL somatic gene rearrangements (recombined VHDHJH and VLJL) which codes for antigen binding variable region of IgG, followed by linkage to a certain CH gene locus. This is transcribed to mRNA and subsequently translated to an immunoglobulin H chain molecule. The CH gene closest to the JH locus is the Cμ gene (IgM), which is the first isotype gene to be expressed. The other CH genes can subsequently be expressed by downstream switching mechanisms with simultaneous deletion of the original isotypic CH genes. The DNA rearrangements that underlie isotype switching and confer their functional diversity on the humoral immune response are directed by cytokines, especially those released by effector CD4 T cells [9].

2. IVIg’s Composition and Industrial Production Methods

Intravenous immunoglobulins (IVIgs) are a therapeutic preparation of pooled normal polyspecific human IgGs obtained from large numbers of healthy donors. The preparation contains antibodies to microbial antigens, self antigens (natural autoantibodies) and anti-idiotypic antibodies which recognize other antibodies [10]. These categories are not mutually exclusive [11].

Plasma used in the production of IVIg comes from two origins: approximately 20 percent is from blood donors, and the other 80 percent is from plasma donors [12]. Individual plasmas are pooled; the pool size is a minimum of 1000 donors, but may be up to 100,000 donors [5,13,14,15]. The maximum number of donors in pools is treated as proprietary information by each manufacturer. The many thousands of donors who contribute to a typical pool of plasma used for isolation of immunoglobulin represent a wide range of antibody specificities against infectious agents [13,15] such as bacterial, viral and also a large number of self antigens reflecting the cumulative exposure of the donor population to the environment.

Techniques developed by Cohn and his coworkers [16,17] in the USA at the beginning of World War II led to the development of the separation of plasma proteins into individual stable fractions with different biologic functions. The basis for Cohn’s fractionation was to use low concentrations of alcohol, reducing the pH and lowering ionic strength. The procedure was performed at low temperature, which reduced the likelihood of contamination and made large-scale fractionation possible. This method, further refined in cooperation with J. L. Oncley [17], is basically still in use and, with some additional steps, yields Ig for intravenous and subcutaneous use [6].

The products are licensed as 50 mg/mL (5%) or 100 mg/mL (10%) IgG solutions for infusion, but may vary slightly from lot to lot and significantly from manufacturer to manufacturer [18].

Preparations of IVIg consist of intact IgG molecules with a distribution of IgG subclasses corresponding to that of normal serum. Subclass distribution may vary between preparations, with some products having less than physiological levels of IgG3 and/or IgG4. IVIg also contains small, but variable, amounts of other proteins and products, notably, and depending on the commercial preparation, albumin, IgA (content varying from less than 5 μg/mL to more than 700 μg/mL), IgE, IgM, sugars, salts, trace amounts of solvents, detergents and buffers may contribute to tolerability difficulties [19]. The monomer and/or dimer content may vary between preparations and up to 3% non-active polymers may be found. Several of these proteins and products may affect the tolerability of IVIg infusions, notably salt, sugar and/or IgA content; volume, pH, osmolality and rate of infusion. The half-life of IVIg after intravenous infusion or intramuscular injection is approximately 2–3 weeks. This can, however, vary depending on the immune status of the patient.

After blood collection (and posterior separation of cellular components) or plasma collection, the plasma is typically stored at ≤ −20°C for several months. All donations are screened for virus infections (Hepatitis B, Hepatitis C and Human immunodeficiency virus) [20,21]. A specified acceptable maximum titre of ABO blood groups antibodies is present, reducing the risks of haemolytic reactions due to the presence of ABO antibodies or antibodies to other blood group system in IVIg [22]. The quality of each individual plasma donation through using good collection practices is crucial to optimize pathogen safety and to ensure the preservation of protein functional activities. Collecting plasma for fractionation are inspected by National Regulatory Authorities (NRAs) and audited by fractionators. In Europe, details on plasma collection procedures, testing, handling and transportation are assembled into a “Plasma Master File” (PMF) [5,21,23].

Recently, the manufacturing processes have much evolved ensuring good in vivo tolerance and minimizing side effects, in particular transmission of infectious agents and improved recovery [5,24]. The manufacturers have strived to develop processes which do not treat the proteins as harshly as reducing and alkylating. These gentler processes result in benefits to the patients [24]:

  • Improved or increased efficacy;

  • Shorter and easier to manage processes will increase consistency;

  • More efficient processes will increase supply;

  • Increased purity leads to better tolerability and efficacy;

  • An optimized formulation, such as a liquid increases convenience and may improve tolerability.

 

 

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