Control of Antibody Impurities Induced by Riboflavin in Culture Media During Production

Abstract

During the manufacturing of protein biologics, product variability during cell culture production and harvest needs to be actively controlled and monitored to maintain acceptable product quality. To a large degree, variants that have previously been described are covalent in nature and are easily analyzed by a variety of techniques. Here, we describe a noncovalent post translational modification of recombinantly expressed antibodies, containing variable domain tryptophans, that are exposed to culture media components and ambient laboratory light. The modified species, designated as conformer, can be monitored by hydrophobic interaction chromatography and often exhibits reduced potency. We studied conformer formation and identified key elements driving its accelerated growth using an IgG2 monoclonal antibody. Conformer is a result of a noncovalent interaction of the antibody with riboflavin, an essential vitamin added to many production cell culture formulations. Chemical and physical factors that influence the impact of riboflavin are identified, and methods for process control of this product quality attribute are addressed in order to prevent loss of antibody potency and potential safety issues. Identifying therapeutic antibody drug candidates with the potential to form conformers can be performed early in development to avoid this undesirable product quality propensity.

Assessing Photostability of mAb Formulations In Situ Using Light-Coupled NMR Spectroscopy

“…While aromatic residues absorb light in the UV–B and −C wavelengths (<320 nm), resulting in direct protein photodegradation, , biopharmaceutical formulations are not exposed to such wavelengths under typical indoor lighting (320–700 nm). However, various molecules acting as photosensitizers present in biopharmaceutical formulations, for example, histidine (His) buffer degradation products, ,, riboflavin contaminants from cell culture, citrate buffer-Fe 3+ complexes, , or polysorbates, , may absorb UV-A or visible light and trigger protein degradation via direct reaction of photosensitizers with protein residues or via formation and subsequent action of reactive oxygen species (ROS) . Additionally, oxidation products of tryptophan , or tyrosine residues absorb UV-A light, potentially promoting further protein degradation.…”

Establishing a novel and sensitive assay for bioactivity determination of anti-CD25 antibodies

“…T cells expressing the IL-2R release pro-inflammatory cytokines after binding with IL-2. Basiliximab has a strong and specific affinity for CD25 on the surface of activated T-cells to play the role of restraint T cell activation, proliferation, and response in transplant recipients [ [9] , [10] , [11] ]. Furthermore, Basiliximab has been shown to improve long-term graft and patient survival via lessening the speed of acute cellular rejection after solid organ transplantation.…”

Photo-Oxidation of Therapeutic Protein Formulations: From Radical Formation to Analytical Techniques

“…Hence, most photo-oxidation processes observed in therapeutic protein formulations are not based on proteinogenic sensitizer such as Trp or Tyr, since these solely absorb light in the UV-C (200-280 nm, FUV) and UV-B (280-315 nm) regions. Impurities from cell culture such as B vitamins [23] might introduce photooxidation processes. Worth mentioning, wavelengths in the UV-A and UV-B region are usually absorbed by commercially available window glass or medicinal containers such as glass vials or polyethylene infusion bags [24].…”