Effect of pH, Temperature, and Salt on the Stability of <i>Escherichia coli-</i> and Chinese Hamster Ovary Cell-Derived IgG1 Fc

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of <i>Escherichia coli</i>- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both <i>E. coli</i> and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C_H2 domain unfolding first, followed by changes in the secondary structure and C_H3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the <i>E. coli</i>-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C_H2 domain melts at a temperature 4–5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ∼0.5 pH unit lower than that of the aglycosylated protein. The difference observed between <i>E. coli</i>- and CHO-derived Fc molecules primarily involves the C_H2 domain, where the glycosylation of the Fc resides

High Resolution Discovery Proteomics Reveals Candidate Disease Progression Markers of Alzheimer’s Disease in Human Cerebrospinal Fluid

<div><p>Disease modifying treatments for Alzheimer’s disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.</p></div

The demographic characteristics of the longitudinal study population, Cohort-2, at the time of CSF collection.

<p>a: Data at the first visit.</p><p>CTL = control; AD = Alzheimer's disease; N, n = number of subjects/patients; SD = standard deviation; MMSE = Mini- mental state examination; ApoE = Apolipoprotein E</p><p>The demographic characteristics of the longitudinal study population, Cohort-2, at the time of CSF collection.</p

Receiver-operator curves (ROC) for cross-sectional study (Cohort-1).

<p>Comparison of the AD samples versus the control samples was used to estimate the sensitivity, while the comparisons between two groups comprised of randomly selected but equally balanced AD and control samples were used to estimate specificity.</p

Quantitation of SME1 and SME2 by dMS (Cohort-1) and 2-way plot.

<p>SME 1 (Peptide NSEPQDEGELFQGVDPR, from neurosecretory protein VGF precursor) and SME2 (peptide VAELEHGSSAYSPPDAFK, from neuronal pentraxin receptor-1) are significantly reduced in AD patients as compared to controls. Area under the curve (AUC) intensity measurements are shown on linear scale. Horizontal bar represents the mean value, vertical error bar represents SD. (A) SME1, P < 0.0001. (B) SME2, P < 0.0005. (C) Two way plot of SME1 and SME2. The symbols (●, +) represent AD and control, respectively.</p

The demographic characteristics of the cross sectional study population, Cohort-1, at the time of CSF collection.

<p>CTL = control; AD = Alzheimer's disease; N, n = number of subjects/patients; SD = standard deviation; MMSE = Mini- mental state examination; ApoE = Apolipoprotein E</p><p>The demographic characteristics of the cross sectional study population, Cohort-1, at the time of CSF collection.</p

Twenty-five candidate disease progression biomarkers from post-hoc mixed-effect analysis of entire Cohort-2 proteomics data set.

<p>Linear mixed-effect model analysis of log area under the curve (AUC) intensity values for features quantitated by dMS in CSF samples taken annually in longitudinal cohort, Cohort-2. Details described in Methods.</p

Linear mixed effect model analysis of SME1 and SME2 and tau, p-tau, and AB42 in the longitudinal cohort, Cohort-2.

<p><b>SME1:</b> dMS feature ID 751080736 at m/z = 639.63 Da; z = 3; Mo = 1915.85 Da; AA sequence = NSEPQDEGELFQGVDPR; unique to protein sequence IPI00289501.2 Neurosecretory protein VGF.</p><p><b>SME2</b> = dMS feature ID751082515 at m/z = 635.98 Da, z = 3, Mo = 1903.90 Da, AA sequence = VAELEHGSSAYSPPDAFK, unique to protein sequence IPI00334238.1 Neuronal pentraxin receptor 1 (NPTXR).</p><p>Serial CSF samples were taken annually. Log area under the curve (AUC) intensity values for SME1 and SME2 measured by dMS and ELISA measurements for tau, p-tau, and AB42 were analyzed in the mixed-effect model described in methods.</p