Automated multi-attribute method sample preparation using high-throughput buffer exchange tips.

RATIONALE: The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability. METHODS: In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system. RESULTS: No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates. CONCLUSIONS: This automated protocol enables the preparation of up to 96 samples with less "at-bench" time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

Impact of depth filtration on disulfide bond reduction during downstream processing of monoclonal antibodies from CHO cell cultures.

Monoclonal antibody interchain disulfide bond reduction was observed in a Chinese Hamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452-461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (Δ P) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of Δ P and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6-phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP + )/NADPH-dependent manner.

Aggregation of human serum albumin during a thermal viral inactivation step.

A terminal pasteurization step has been used for some plasma-derived protein products such as human serum albumin (HSA), which consists of heating the protein in solution at 60 degrees C for 10 h. Native and denaturing SDS-PAGE and dynamic light scattering were used to follow the stability of HSA during this process. It appears that a thermally unstable fraction, comprised primarily of haptoglobin, is involved in the formation of soluble aggregates of HSA. Therefore, it appears that aggregation during heat treatment is not due to conformational instability of HSA itself, but arises from unfolding of a thermally labile protein impurity. As haptoglobin aggregates, it entraps some HSA, which is present at much higher concentrations. This study emphasizes that, in a complex mixture of naturally occurring proteins, one thermally labile species can trigger aggregation of more stable proteins.

Impact of bulking agents on the stability of a lyophilized monoclonal antibody.

The impact of the bulking agents on the stability of lyophilized protein pharmaceuticals is not typically considered, as they usually crystallize, preventing them from stabilizing the protein. In this study, combinations of sucrose with mannitol or glycine were evaluated for their effects on antibody stability (deamidation and aggregation) and solid-state properties (water content, residual antibody structure, and T(g) values). While sucrose remains the primary stabilizing agent for the antibody in the solid state, inclusion of some amorphous glycine leads to a significant increase in stability. Mannitol displays little, if any, stabilizing ability in this system. In formulations where infrared spectroscopy could be applied, maintenance of secondary structure is an important factor in predicting storage stability. This study demonstrates that bulking agents can impact protein stability in the solid state. By remaining partially amorphous, bulking agents can sometimes lower degradation rates, while at the same time, providing a crystalline matrix producing elegant cakes. It appears that bulking agents may belong to a larger class of low molecular weight plasticizers, which appears to be able to increase solid-state stability of proteins despite lowering the glass transition temperature.

Infrared spectroscopic studies of protein formulations containing glycine.

Glycine is extensively used as an excipient in protein formulations. However, it absorbs significant infrared (IR) radiation in the conformationally sensitive amide I region (1700-1600 cm(-1)) of proteins. Furthermore, glycine can form a number of polymorphs, as well as an amorphous phase. Each of these forms possibly exhibits a different IR absorption spectrum. Accurate subtraction of glycine signals, in order to obtain reliable amide I spectra, was found to be possible only if the protein-to-glycine ratio was >/=1:1. In those cases, the solid-state conformation of the protein could be determined. In addition, a new method for estimating the degree of crystallinity of freeze-dried glycine is described, using IR bands in the 1350-1300 cm(-1) region.

Effects of potassium bromide disk formation on the infrared spectra of dried model proteins.

The most common method of sample preparation for Fourier transform infrared spectroscopic analysis of proteins in the solid state is compression after mixing with potassium bromide (KBr). Recently, questions have arisen as to whether proteins are conformationally altered by this process. In this study, the amide I Fourier transform infrared spectra of two model proteins, lysozyme and alpha-chymotrypsinogen, were measured before and after compression in KBr, and the effects of moisture exposure and the ratio of KBr to protein were examined. Contrary to earlier reports, compaction of the KBr/protein mixtures did not foster aggregation, and only minor apparent structural alterations were observed.

Lipid unsaturation determines the interaction of AFP type I with model membranes during thermotropic phase transitions.

We have previously shown that antifreeze protein (AFP) type I from winter flounder interacts with the acyl chains of lipids in model membranes containing a mixture of dimyristoylphosphatidylcholine (DMPC) and the plant thylakoid lipid digalactosyldiacylglycerol (DGDG), most likely through hydrophobic interactions. By contrast, in studies with pure phospholipid membranes, no such interaction was seen. DGDG is a highly unsaturated lipid, which renders these studies quite different from the previous studies of AFP-membrane interaction where the lipids were saturated or trans-unsaturated. Therefore, it seemed possible that either the digalactose headgroups or the unsaturated DGDG acyl chains, or both, may be important for interactions of membranes with AFP type I. To distinguish between these possibilities, we catalytically hydrogenated the DGDG to obtain a galactolipid with completely saturated fatty acyl chains. The results with the hydrogenated DGDG were strikingly different from those obtained previously with the unsaturated DGDG; the clear binding of AFPs to the bilayer appeared to be lost. Nevertheless, the temperature-dependent folding of AFP type I was inhibited in the presence of liposomes containing either the unsaturated or the hydrogenated DGDG. The results indicate that the liposomes and protein still interact, even following hydrogenation of the acyl chains, perhaps at the membrane-solution interface.

Effects of conformation on the chemical stability of pharmaceutically relevant polypeptides.

Control of chemical instability in protein pharmaceuticals continues to be a critical issue in developing stable formulations. While the effects of pH, buffer composition, ionic strength and temperature remain the most effective methods for controlling hydrolysis and oxidation reactions, it appears that conformational control may also be important. Addition of excipients to maintain native structure and reduce the propensity of the protein to denature and/or aggregate is already a central theme in stabilizing proteins (Arakawa et al., 1993). The same additives have now been found to slow both deamidation and oxidation, whether in solution or in the solid state. What is emerging is an additional approach for producing protein pharmaceuticals that maintain native structure and activity during long-term storage.