Effects of Arginine on Photostability and Thermal Stability of IgG1 Monoclonal Antibodies

Abstract

This study demonstrates that arginine is a highly effective solvent additive which significantly reduces the light induced aggregation of four IgG1 type monoclonal antibodies (named as IMC-1A, IMC-1B, IMC-1C and IMC-1D) as measured by size exclusion chromatography. All experiments were performed in a phosphate buffer system containing either sodium chloride or arginine hydrochloride. The protein samples were exposed to light in a photo chamber according to ICH (International Conference on Harmonization) guidelines. Thermal unfolding transition temperature (T(m)) of IMC-1A as determined by differential scanning calorimetry (DSC) was significantly decreased ( approximately 3.3 degrees C) in the presence of arginine hydrochloride as compared to in sodium chloride. However, a noticeable increase in thermal stability was observed for IMC-1B, IMC-1C and IMC-1D in the presence of arginine hydrochloride. The photostability of all these molecules was significantly enhanced by arginine hydrochloride and both a direct and inverse correlation was observed between conformational stability and photostability. To our knowledge, this paper for the first time, demonstrates that arginine hydrochloride considerably reduces the light induced aggregation of monoclonal antibodies. Arginine hydrochloride is also known to increase protein solubility and its ability to extensively reduce light induced aggregation makes it a potential solvent additive for the formulation development of therapeutic proteins.

Effect of pH and Light on Aggregation and Conformation of an IgG1 mAb

“…For reference purposes, typically the first melting transition is assigned as either the CH2 domain or the Fab, with the third peak being assigned as the CH3 domain. 25,53 Whether or not this holds for this antibody, it can still be observed from an overlay of the three unexposed samples that there is clearly a shift towards higher temperatures with increasing pH, going from approximately 65°C for pH 3. C obtained for the 1 mg/mL pH 3.5 solution exposed for 60 minutes to 302 nm light as compared to that of unexposed material, but failed to show significant differences at 10 mg/mL (see Figure 7).…”

“…23,24 Type I photooxidation proceeds via singlet or triplet excited states of amino acids, or via photoionization. 25 Type II photooxidation involves intermediary singlet oxygen ( 1 O 2 ) 23,24 but is generally considered less efficient for protein amino acid residues. 23 Direct photo-oxidation of proteins by a Type I process typically occurs at λ ≤ 320 nm and is only significant at λ > 320 nm if the protein contains a bound co-factor or functional group that is able to absorb light at higher wavelengths.…”

“…30 For comparison, the light-induced cleavage of glutathione disulfide led predominantly to thiyl radicals. 30 It is well documented that radical mediated damage to proteins may lead to a variety of potentially negative consequences such as fragmentation 23,38 , aggregation 25,38,39 , oxidation [40][41][42][43] , changes to surface hydrophobicity, conformational changes 39 and even denaturation. 38,39 It may also ultimately result in the loss of activity or simply generate new reactive species propagating the radical reactions.…”

“…25,38,[41][42][43] Common to all of these studies was photodegradation resulting in the oxidation of tryptophan 38,43 and methionine 38,41,42 with differences noted in specific regions of each antibody. Qi et al observed substantial oxidation of light chain Trp94 in the CDR resulting in multiple oxidation products along with secondary oxidation of Met261, Met437 and His294 of the heavy chain.…”

“…As a result equilibrium thermodynamics could not be used to obtain accurate thermodynamic parameters. 25 Despite this limitation, DSC and fluorescence measurements allowed for a number of observations to be made. Three to four distinct unfolding transitions are observed for all thermograms, and in general are considered to be apparent melting transitions.…”