A highly productive chemically defined fed-batch process was developed to maximize titer and volumetric productivity for Chinese hamster ovary cell-based recombinant protein manufacturing. Two cell lines producing a recombinant antibody (cell line A) and an Fc-fusion protein (cell line B) were used for development. Both processes achieved product titers of 10 g/L on day 18 under chemically defined conditions. For cell line B, the use of plant derived hydrolysates combined with the optimized chemically defined medium increased the titer to 13 g/L. Volumetric productivities were increased from a base line of about 200 mg/L/d to about 500 mg/L/d under chemically defined conditions and as high as 700 mg/L/d with cell line B using plant derived hydrolysates. Peak cell densities reached greater than 20E6 vc/mL, and cell viabilities were maintained above 80% on day 18 without the use of antiapoptotic genes or temperature shift. A rapid compound screening method was developed to effectively test positive factors within 72 h. Peak volumetric oxygen uptake rates (OUR) more than tripled from the baseline condition. Oxygen demand continued to increase after maximum cell density was reached with a maximal OUR of 3.7 mmol/L/h. The new process format was scaled up and verified at 100 L pilot scale using reactor equipment of similar configuration as used at manufacturing scale.

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A␤1-42 is involved in Alzheimer's disease (AD) pathogenesis and is prone to glycation, an irreversible process where proteins accumulate advanced glycated end products (AGEs). N ⑀-(Carboxyethyl)lysine (CEL) is a common AGE associated with AD patients and occurs at either Lys-16 or Lys-28 of A␤1-42. Methyglyoxal is commonly used for the unspecific glycation of A␤1-42, which results in a complex mixture of AGE-modified peptides and makes interpretation of a causative AGE at a specific amino acid residue difficult. We address this issue by chemically synthesizing defined CEL modifications on A␤1-42 at Lys-16 (A␤-CEL16), Lys-28 (A␤-CEL28), and Lys-16 and-28 (A␤-CEL16&28). We demonstrated that double-CEL glycations at Lys-16 and Lys-28 of A␤1-42 had the most profound impact on the ability to form amyloid fibrils. In silico predictions indicated that A␤-CEL16&28 had a substantial decrease in free energy change, which contributes to fibril destabilization, and a increased aggregation rate. Single-CEL glycations at Lys-28 of A␤1-42 had the least impact on fibril formation, whereas CEL glycations at Lys-16 of A␤1-42 delayed fibril formation. We also tested these peptides for neuronal toxicity and mitochondrial function on a retinoic acid-differentiated SH-SY5Y human neuroblastoma cell line (RA-differentiated SH-SY5Y). Only A␤-CEL16 and A␤-CEL28 were neurotoxic, possibly through a nonmitochondrial pathway, whereas A␤-CEL16&28 showed no neurotoxicity. Interestingly, A␤-CEL16&28 had depolarized the mitochondrial membrane potential, whereas A␤-CEL16 had increased mitochondrial respiration at complex II. These results may indicate mitophagy or an alternate route of metabolism, respectively. Therefore, our results provides insight into potential therapeutic approaches against neurotoxic CEL-glycated A␤1-42. A␤1-42 peptides are implicated in the pathogenesis of Alzheimer's disease (AD). 5 A␤1-42 peptides are released extracellularly after being enzymatically processed from the amyloid precursor protein and are prone to aggregation. Aggregated A␤1-42 eventually forms amyloid plaques and causes neuronal dysfunction (1). As aging is a major risk factor for AD development, this natural process of aging also ubiquitously produces advanced glycated end products (AGEs), which are a type of post-translational modification (glycation). Several seminal studies provide evidence of increased AGE modifications that are associated with AD brains (2-4). Other disease states, which affect cerebral sugar homeostasis as a consequence of aging, such as Type II diabetes mellitus, may also contribute to the formation of AGEs within the brain (5). Glycation is an irreversible and nonenzymatically driven process that occurs through a Maillard reaction that conjugates sugars and proteins (6, 7). A particular in vivo sugar derivative, methylglyoxal (MG), is a glycating agent capable of forming different AGEs on A␤1-42 at lysine residues 16 (Lys-16) and 28 (Lys-28) (8, 9). The increase in in vivo MG production was observed in AD patients (10). Several ke...