RESUMO
Relapsing polychondritis (RPC) is a rare autoimmune condition that often mimics recurrent external otitis. This multisystemic disease primarily affects cartilaginous structures in the body, with the ear pinna being the most commonly impacted. RPC is associated with elevated inflammatory markers and antinuclear antibodies (ANA), and it can lead to chondral destruction. Our case is a 74-year-old Caucasian male with a history of peripheral vascular disease (PVD) who presented to the clinic with recurrent, painful swelling of the right upper ear for 14 days despite multiple antibiotics and nonsteroidal anti-inflammatory drugs (NSAIDs). He had chronic sensorineural hearing loss in the same ear. He was seen multiple times with identical symptoms in the last seven months and was diagnosed with otitis externa. He denied arthritis, fatigue, rash, abrasion, allergies, trauma, or fever. He was prescribed antimicrobials, alternating NSAIDs, and methylprednisolone with temporary relief. He is only on statins and has an unremarkable family history. He was afebrile with normal vital signs. On physical examination, he was not in acute distress and had a normal voice but had a diffusely erythematous, tender, swollen right ear pinna and external canal sparing the lobe. The rest of the physical examination was unremarkable. Laboratory results showed elevated C-reactive protein (CRP) of 100 mg/L (normal range: <3 mg/L) and erythrocyte sedimentation rate (ESR) of 200 mm/hour (normal range: <20 mm/hour). ANA titer is 1:160 with a homogenous pattern, but other autoantibodies were negative. No red flags were noted on the complete blood count (CBC) or comprehensive metabolic panel (CMP), and his rapid plasma reagin (RPR) test was negative. In this patient, prednisone 60 mg daily was initiated as monotherapy, and rheumatology was also consulted. The patient sought consultation due to recurrent and persistent upper ear infections despite antibiotic treatment and was ultimately diagnosed with a rare medical condition called relapsing polychondritis. Following this treatment, the auricular chondritis improved promptly. The steroid dosage was then slowly tapered and maintained at 10 mg daily to prevent flare-ups. Subsequently, after the initiation of corticosteroids, inflammatory markers trended down to normal levels.
RESUMO
Detection of host cell protein (HCP) impurities is critical to ensuring that recombinant drug products, including monoclonal antibodies (mAbs), are safe. Mechanistic characterization as to how HCPs persist in drug products is important to refining downstream processing. It has been hypothesized that weak lipase-mAb interactions enable HCP lipases to evade drug purification processes. Here, we apply state-of-the-art methods to establish lipase-mAb binding mechanisms. First, the mass spectrometry (MS) approach of fast photochemical oxidation of proteins was used to elucidate putative binding regions. The CH1 domain was identified as a conserved interaction site for IgG1 and IgG4 mAbs against the HCPs phospholipase B-like protein (PLBL2) and lysosomal phospholipase A2 (LPLA2). Rationally designed mutations in the CH1 domain of the IgG4 mAb caused a 3- to 70-fold KD reduction against PLBL2 by surface plasmon resonance (SPR). LPLA2-IgG4 mutant complexes, undetected by SPR and studied using native MS collisional dissociation experiments, also showed significant complex disruption, from 16% to 100%. Native MS and ion mobility (IM) determined complex stoichiometries for four lipase-IgG4 complexes and directly interrogated the enrichment of specific lipase glycoforms. Confirmed with time-course and exoglycosidase experiments, deglycosylated lipases prevented binding, and low-molecular-weight glycoforms promoted binding, to mAbs. This work demonstrates the value of integrated biophysical approaches to characterize micromolar affinity complexes. It is the first in-depth structural report of lipase-mAb binding, finding roles for the CH1 domain and lipase glycosylation in mediating binding. The structural insights gained offer new approaches for the bioengineering of cells or mAbs to reduce HCP impurity levels.Abbreviations: CAN, Acetonitrile; AMAC, Ammonium acetate; BFGS, Broyden-Fletcher-Goldfarb-Shanno; CHO, Chinese Hamster Ovary; KD, Dissociation constant; DTT, Dithiothreitol; ELISA, Enzyme-linked immunosorbent assay; FPOP, Fast photochemical oxidation of proteins; FA, Formic acid; F(ab'), Fragment antibodies; HCP, Host cell protein; IgG, Immunoglobulin; IM, Ion mobility; LOD, Lower limit of detection; LPLA2, Lysosomal phospholipase A2; Man, Mannose; MS, Mass spectrometry; MeOH, Methanol; MST, Microscale thermophoresis; mAbs, Monoclonal antibodies; PPT1, Palmitoyl protein thioesterase; ppm, Parts per million; PLBL2, Phospholipase B-like protein; PLD3, Phospholipase D3; PS-20, Polysorbate-20; SP, Sphingomyelin phosphodiesterase; SPR, Surface plasmon resonance; TFA, Trifluoroacetic acid.
