In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an over-expressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to assure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an on-line LC-MS strategy combining collision induced dissociation (CID-MS 2 ), electron transfer dissociation (ETD-MS 2 ), and CID of an isolated product ion derived from ETD (MS 3 ) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS 3 step. The on-line LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a monoclonal antibody (Herceptin) and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS 3 step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance provided not only facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The on-line LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins.

Summary The disulfides in three monoclonal antibodies (mAb), the anti-HER2, anti-CD11a, and GLP-1 with IgG4-Fc fusion protein, were completely mapped by LC-MS with the combination of ETD and CID fragmentation. In addition to mapping the 4 inter- and 12 intra-chain disulfides (total 16), the identification of scrambled disulfides in degraded samples (heat-stress) was achieved. The scrambling was likely attributed to an initial breakage between the light (Cys 214) and heavy (Cys 223) chains in anti-HER2, with the same observation found in a similar therapeutic mAb, anti-CD11a. On the other hand, the fusion antibody, with no light chain but containing only 2 heavy chains, generated much less scrambling under the same heat-stressed conditions. The preferred sites of scrambling were identified, such as the intra-chain disulfide for CxxC in the heavy chain, and the C194 of the heavy chain pairing with the terminal Cys residue (C214) in the light chain. The inter-chain disulfides between the light and heavy chains were weaker than the inter-chain disulfides between the two heavy chains. The relative high abundance ions observed in ETD provided strong evidence for the linked peptide information, which was particularly useful for the identification of the scrambled disulfides. The use of SDS-PAGE helped the separation of these misfolded proteins for the determination of scrambled disulfide linkages. This methodology is useful for comparison of disulfide stability generated from different structural designs, and providing a new way to determine the scrambling patterns, which could be applied for those seeking to determine unknown disulfide linkages.

ObjectivesTo investigate the early determinants of overweight and obesity status at age two years.MethodsA total of 1098 healthy neonates (563 boys and 535 girls) were involved in this community-based prospective study in China. Data on body weight and length were collected at birth, the 3rd and 24th month. A self-administered questionnaire was used to collect data on social demography and feeding patterns of children, etc. Three multivariable logistic regression models were employed to make various comparisons of weight status, i.e., model 1 (obesity vs. non-obesity), model 2 (combined overweight and obesity vs. normal weight, and model 3 (obesity, overweight and normal weight).ResultsPrevalences of overweight/obesity (95th >BMI ≥85th p and BMI ≥95th p, referring to WHO BMI standards) at 2 years of age are 15.8%/11.2% for boys and 12.9%/9.0% for girls, respectively. Being born with macrosomia (OR: 1.80–1.88), relatively greater BMI increment in the first 3 months (OR: 1.15–1.16) and bottle emptying by encouragement at age two (OR: 1.30–1.57) were found in all three models to be significant risk factors for higher BMI status at 2 years. Pre-pregnancy maternal BMI (OR: 1.09–1.12), paternal BMI (OR: 1.06), and mixed breastfeeding (OR: 1.54–1.57) or formula feeding (OR: 1.90–1.93) in the first month were identified as significant in models 2 and 3. Child-initiated bottle emptying at age two was observed to increase the risk of obesity by 1.31 times but only in model 1.ConclusionFetal and early postnatal growth and feeding pattern appear to have significant impacts on early childhood overweight and obesity status independent of parental BMI. Policy-based and multidisciplinary approaches to promote breastfeeding and enhancement of feeding skills of care takers may be promising intervention strategies.

Human polyclonal IgG antibodies directly against the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) are potential biomarkers and mechanistic contributors to cancer and other diseases associated with chronic inflammation. Using a sialoglycan microarray, we screened the binding pattern of such antibodies (anti-Neu5Gc IgG) in several samples of clinically-approved human IVIG (IgG). These results were used to select an appropriate sample for a multi-step affinity purification of the xeno-autoantibody fraction. The sample was then analyzed via our multi-enzyme digestion procedure followed by nanoLC coupled to LTQ-FTMS. We used characteristic and unique peptide sequences to determine the IgG subclass distribution and thus provided direct evidence that all four IgG subclasses can be generated during a xeno-autoantibody immune response to carbohydrate Neu5Gc-antigens. Furthermore, we obtained a significant amount of sequence coverage of both the constant and variable regions. The approach described here, therefore, provides a way to characterize these clinically significant antibodies, helping to understand their origins and significance.

SummaryThree different analysis platforms using LC-MS were successfully developed for pharmacokinetic (PK) studies of an antibody drug in serum. These analysis platforms can be selectively used for different types of protein drugs, which ranged from a very specific for a particular drug (antibody enrichment), to a less specific for any antibody drugs with Fc domain (protein A enrichment), and to a very generic method that can be used for any protein drugs (albumin depletion method). In this manner the three platforms will be applicable to a wide range of antibody therapeutic studies for different species. The analysis using an albumin depletion method (with SDS-PAGE) achieved the detection of the drug (to 1 ng) in an aliquot of serum (30 µL) with a 5-order magnitude of linearity. The analysis using protein A enrichment (with SDS-PAGE) achieved the detection of the drug at a 50 fold lower level (to 0.02 ng). Without using SDS-PAGE for separation, using protein A enrichment achieved the detection to 10 ng and using the anti-drug antibody enrichment achieved the detection to 0.1 ng, with a similar linear dynamic range. These three analysis platforms produced good agreement with a mimic PK study of the drug in monkey serum, as compared to ELISA approach. In addition, these analysis platforms can be selectively applied for PK studies of drugs with different requirements of development time and resources. Such as, the antibody enrichment method can be used in a high throughput manner but limited to a specific protein drug only. On the other hand, the albumin depletion method can be applied to many types of protein drugs, but with the laborious sample preparation steps (SDS-PAGE and the subsequent in-gel digestion). When anti-drug antibodies are not available for antibody drugs, or the sensitivity requirement is not stringent (e.g. > 10 ng), using protein A enrichment (without using SDS-PAGE) seems to be a good choice for PK studies which require fast throughput.