See aslo antibody variants in Antibody purification
Unpredictability in Antibody Formulations/stabilization
mAbs generally have reasonable stability, and about half of all mAb drug products are in liquid dosage form. Goswami (Antibodies 2031, 2, 452-500).
If one examines the amino acid sequences for IgG1 based antibody drugs, only a small segment of these proteins are dramatically different one one another, being the Fv segments that involved in antigen binding. One might assume that by finding a stable formulation for one of these antibody drugs, such a formulation would be good for most if not all, similar antibodies. Instead, each antibody seems to have a unique persoanlity related to its requirements for stability; a phenomenon derived form the fact that the small differences between these antibodies are fucoused on surface epxosed amino acid differences that stipulate antigen specificity. Thus, the interfacial surface of each antibody is unique and requires specific formulation components to provide maximal stability and retention of activity. (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706).
Despite acknowledging the similarity in structures, Wang repeatedly states that the differences amoung antibody sequences affect the stability of antibody pharmaceuticals. One formulation excipient stabilizing a specific antibody may not be suitable for another because of the differences in their sequence. (Wang, “Antibody Structure, Instability, and Formulation, 96 U. Pharm. Sciences 1-26 (2007) (Ex. 2007); See also IPR2016-01018, Patent 9,114,166B2 which discusses Wang as evidentially support that one of ordinary skill in the art would hnot have had a reasonable expectation of success in formulating a stable, liquid, high cconcentration D2E7 formulation as required by the claims. ).
Liquid Formulations
Developing conditions to keep proteins stable in a liquid form for a pharmaceutically relevant storage time (e.g., two years) is not a simple task. With many proteins, it is not possible, especially considering the time constraints for product development, to develop sufficiently stable aqueous formulations. (Rational design of stable protein formulations, 13 Pharmaceutical Biotechnology (John F. Carpenter & Mark C. Manning eds., 2002) See as IPR2106-01018, Patent 9,114,166B2 which discusses this book).
The development of stable liquid antibody formulations, especially those at a concentration high enough to be suitable for [subcutaneous] administration, was far from routine (Wang, Instability, Stabilization, and Formulation of Liquid Protein Pharmaceuticals, 185 Int’l J. Pharmaceutics 129-188 (1999).
Types of changes which can occur to Antibodies
Chemical degrdation of antibodies may occur in a number of different ways. The type of degradation affects the primary sequence and may also lead to significant changes in the higher order structure. Examples of chemical degradation include deamidation, oxidation, isomerization, clipping/fragmentation and cross-linking. Degradation of the molecule may interfere with the intended biological activity, as the site of degradation may involve a domain that is critical for biological function. There are cases in which the formation of aggregates was followed by loss of biological activity, as for example antiviral activity of interferon-beta-1b. Another major impact of protein aggregation is immunogenicity, which may result from the multipolicity of the epitope and/or change in conformation. Anitobides formed can be nuetralizing in nature and thus may simply result in the loss of efficacy of the adminsitered therapeutic. if the level of nuetralizing antibody is very high, it may result in the neutralizaiton of the essential endogenous protein, for example, the nuetralization of endogenous erythropoietin by the antibody, formed against recombinant human erythropoietin has been reported to cause red cell aplasia Goswami (Antibodies 2031, 2, 452-500)
According to the International Conference on Harmonization (ICH) guidance document, drug substance heterogeneity defines its quality, and the degree and profile should be monitored and characterized to ensure lot to lot consistency. Microheterogeneity of mAbs thus is a concern for production, expecially for downstream processing. Characterization of the product is necessary for determining variants. (Burg, WO2011/009623).
A number of authors have shown that charge variants of therapeutic proteins can have vastly different bioactivity (Pabst, Biotechnol. Prog 2008, 24, 1096-1106).
Oxidation: methionine and cysteine residues are frequently a site of oxidation in protein drugs and this is also the case for antibody drugs. Specific methionine reisudes within the Fc domain may be prone to oxidation, resulting in the production of methionine sulfoxide. While cysteines are present in teh Fc framework as disulfide pairs, unpaired cysteines in the variable region may also be sites of oxidaiton. Besides methionine and cysteine residues, oxidation of histidine, tyrosine, tryptophan and phenylalamine reisudes can also occur. (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706).
Modifications that form acidic species
Acidic species are antibody variants that elute earlier than the main species of the antibody during CEX or later than the main peak during AEX analysis. The main causes for the formation of acidc spces include sialic acid, deamidation, glycation (Du, “Chromatographic analysis of the acidc and basic species of recombinant monoclonal antibodies” Merck Research Laboratories (2012).
–Deamidation of side chains of asparagines aand glutamine residues:
Deamidation is probably the most common type of chemical degradation encountered in mAb based biotherapeutics. Goswami (Antibodies 2031, 2, 452-500). Glutamine and asparagine residues show a propensity for deamidation. Initial detection of deamidation in antibody preparations is typically dientified by differences in charge distribution of content using methods such as isoelectric focusing (IEF) of high performance cation exchange chromatography (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706).
Deamidation in proteins is often responsible for charge heterogeneity of recombinant proteins. The uncharged side chains of these amino acids are modified to an isoglutamate and iso-asparatate residue to a glutamate and aspartate residue. Thus, an additional charge is introduced to the protein per modification. For recombinant monoclonal antibodies, deamidation can occur at any stage from inside the cells, after secretion, during purification, during storage and under different conditions of stress. Deamidated and other acidic variants of the her2 antiboides, for example, were found to exist in seven different variants and the variants could be separated by cation exchange chromatography using a liner increase in ionic strengh for elution. (Burg, WO2011/009623).
Glycosylation pattern changes: can also occur in the course of cell culture (Burg, WO2011/009623).
Factors which Influence Antibody Stability:
Nonspecific protein-protein interactions have been driven by the need for developing stable liquid formulations of antibodies. Antibody self-interactions are linked to problems such as protein aggregation and high viscosities. These problems are often exagerated at high protein concetnrations, which are often needed in liquid formulations to meet patient dose requirements. Problems can be minimized by manipulating the colution condtiions, such as choice of buffers, pH, ionic strength, and the inclusion of other small molecule additives sucha s sugards, amino acids and other excipients. (Curtis “The role of electrostatics in protein-protein interactions of a monoclonal antibody” 2014).
Isoelectric point:
An inherent protein proeprty of itneret is its isoelectric point (IEP). The IEP is defined as the pH at which net charge becoems azero. It is a rule of thumb to define the formulation’s pH not too close to IEP as this increases the risk of preciptiation. The IEP of prtoeins can be theorecctically derived from amino acid sequence or determined experimentally using different methods including isoelectric focusing (IEF) of IEF capillary electrophoresis (iCE). (Stefan fischer “Assessmetn of net charge and prtoien-protein interactions of different monoclaonl antibodies” J. Pharma Sciences, 100(7) 2011).
Techniques used to Increase Antibody Stability
A number of additive have been identified that can reduce the rate of protein aggregation including urea, guanidinium chloride, amino acids (in particular glycine and arginine), various sugars, poly alcohols, polymers (including polyethylene glycol and dextrans), surfactants and even antibodies themselves. (Dougherty, Advanced Drug Delivery Reviews 58 (2006) 686-706).
(1) Addition of Amino Acids: See Buffer conditions
(2) Lyophilization:
Because immunoglobulin is unstable in the form of a solution, it has been formulated not as a liquid preparation but as a drug preparation. However, the dry prepration is accompanied by the problem that it cannot be administered easily because of the necessity of dissolving it in distilled water for injection.
(3) Addition of Carbohydrate/Polyols or Poly-alcohols: See buffer Conditions
The modification of the carbohydrate chains and thus the glycosylation of proteins is done in order to improve the erum half life of recombinantly expressed proteins such as antibodies. Sialic acids are the most prevalent terminal monosaccharides on the surface of eukrayotic cells and it is generally believed that the more a glycoportein is sialyated the longer is its serum half life during ciruculation. Goletz (US2010/0028947).
(4) Addition of Sorbital and Mannitol: See buffer conditions
(7) Addition of Polymers: See buffer conditions
(8) Non-Ionic surfactants: See buffer conditions
Formulation of IVIG
Deamidation and isomerization are favored at neutral and basic pH. Thus it is preferable to formulate biopharmaceuticals at slightly acidic pH. In fact, out of 28 commerically approved mAbs, 20 are formulated at pH less than or equal to 6.5. (Goswami, Antibodies, 2013, 2, 452-500, 2013)
IVIG has enhanced stability in the liquid state at pH 4.25. There is a trend for modern IVIG preparation to be formulated as liquid at high protein concentrations (typically 100 mg/ml) within a low pH range (pH 4.5-5.5) in the presence of stabilizers like polyols (sorbitol), sugars (maltose, glocose) or amino acid (glycine, proline, isoleucine) and without sodium chloride addition. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28.