IGF1 inclusion bodies: A QbD based process approach for efficient USP as well as early DSP unit operations.

E. coli is an attractive host organism for strong recombinant protein expression. It expresses products either as soluble protein or as inclusion bodies (IB). IBs are insoluble, mostly inactive aggregates. However, recent progress enabled the efficient refolding back into their bioactive structure. Targeted IB production processes have been designed based on their characteristic features such as high yields along with purity and their simple separation. More profound process knowledge is needed to reveal interacting parameters required for quality by design grounded process development. This enables strategies for simplifying and accelerating upstream as well as downstream procedures. We present a workflow for gathering deeper process knowledge by a design of experiment approach for improved IGF1 IB formation in relation to impurity concentration. An IB expression maximum of 19.8 mgIGF1·gDCW-1 was found at pH 6.5, 37 °C and an IPTG induction of >45 µmol gDCW-1 for 12 h. Subsequently, three refolding buffers were tested together with a nonwoven anion exchange adsorber filter module. Knowledge-based buffer selection enabled high impurity log reduction values (LRVEndotoxin = 4.9; LRVDNA = 4.8, LRVHCP = 0.1-1) as well as chromatography column guarding potential by using those adsorptive matrices. Furthermore, adsorptive filtration followed by tangential flow filtration proved to be a promising alternative for product concentration.

Enhancing Protein A performance in mAb processing: A method to reduce and rapidly evaluate host cell DNA levels during primary clarification.

The use and impact of 3M™ Emphaze™ AEX Hybrid Purifier, a single-use, fully synthetic chromatographic product, was explored to reduce host cell DNA (HC-DNA) concentration during the primary clarification of a monoclonal antibody (mAb). An approximately 5-log reduction in HC-DNA was achieved at an Emphaze AEX Hybrid Purifier throughput of 200 L/m2 . The appreciable reduction in HC-DNA achieved during primary clarification enhanced Protein A chromatography performance, resulting in a sharper and narrower elution profile. In addition, a 24× improvement in host cell protein (HCP) removal and fewer impurities nonspecifically bound to the Protein A column were observed compared to those resulting from the use of depth filtration for clarification. The use of a rapid, qualitative acidification assay to facilitate HC-DNA monitoring was also investigated. This assay involves the acidification-induced precipitation of HC-DNA, enabling the easy and rapid detection of DNA breakthrough across purification media such as Emphaze AEX Hybrid Purifier by means of turbidimetric and particle size measurements.

Development of adsorptive hybrid filters to enable two-step purification of biologics.

Recent progress in mammalian cell culture process has resulted in significantly increased product titers, but also a substantial increase in process- and product-related impurities. Due to the diverse physicochemical properties of these impurities, there is constant need for new technologies that offer higher productivity and improved economics without sacrificing the process robustness required to meet final drug substance specifications. Here, we examined the use of new synthetic adsorptive hybrid filters (AHF) modified with the high binding capacity of quaternary amine (Emphaze™ AEX) and salt-tolerant biomimetic (Emphaze™ ST-AEX) ligands for clearance of process-related impurities like host cell protein (HCP), residual DNA, and virus. The potential to remove soluble aggregates was also examined. Our aim was to develop a mechanistic understanding of the interactions governing adsorptive removal of impurities during filtration by evaluating the effect of various filter types, feed streams, and process conditions on impurity removal. The ionic capacity of these filters was measured and correlated with their ability to remove impurities for multiple molecules. The ionic capacity of AHF significantly exceeded that of traditional adsorptive depth filters (ADF) by 40% for the Emphaze™ AEX and by 700% for the Emphaze™ ST-AEX, providing substantially higher reduction of soluble anionic impurities, including DNA, HCPs and model virus. Nevertheless, we determined that ADF with filter aid provided additional hydrophobic functionality that resulted in removal of higher molecular weight species than AHF. Implementing AHF demonstrated improved process-related impurity removal and viral clearance after Protein A chromatography and enabled a two-step purification process. The consequences of enhanced process performance are far reaching because it allows the downstream polishing train to be restructured and simplified, and chromatographic purity standards to be met with a reduced number of chromatographic steps.

Angular variances for internal bond rotations of side chains in GXG-based tripeptides derived from (13)C-NMR relaxation measurements: Implications to protein folding.

The study of backbone and side-chain internal motions in proteins and peptides is crucial to having a better understanding of protein/peptide "structure" and to characterizing unfolded and partially folded states of proteins and peptides. To achieve this, however, requires establishing a baseline for internal motions and motional restrictions for all residues in the fully, solvent-exposed "unfolded state." GXG-based tripeptides are the simpliest peptides where residue X is fully solvent exposed in the context of an actual peptide. In this study, a series of GXG-based tripeptides has been synthesized with X being varied to include all twenty common amino acid residues. Proton-coupled and -decoupled (13)C-nmr relaxation measurements have been performed on these twenty tripeptides and various motional models (Lipari-Szabo model free approach, rotational anisotropic diffusion, rotational fluctuations within a potential well, rotational jump model) have been used to analyze relaxation data for derivation of angular variances and motional correlation times for backbone and side-chain chi(1) and chi(2) bonds and methyl group rotations. At 298 K, backbone motional correlation times range from about 50 to 85 ps, whereas side-chain motional correlation times show a much broader spread from about 18 to 80 ps. Angular variances for backbone phi,psi bond rotations range from 11 degrees to 23 degrees and those for side chains vary from 5 degrees to 24 degrees for chi(1) bond rotations and from 5 degrees to 27 degrees for chi(2) bond rotations. Even in these peptide models of the "unfolded state," side-chain angular variances can be as restricted as those for backbone and beta-branched (valine, threonine, and isoleucine) and aromatic side chains display the most restricted motions probably due to steric hinderence with backbone atoms. Comparison with motional data on residues in partially folded, beta-sheet-forming peptides indicates that side-chain motions of at least hydrophobic residues are less restricted in the partially folded state, suggesting that an increase in side-chain conformational entropy may help drive early-stage protein folding. Copyright 1999 John Wiley & Sons, Inc.

Efficient and scalable method for scaling up cell free protein synthesis in batch mode.

A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 microL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 microL reaction volume, the PANOx SP system produced 560 +/- 36 microg/mL of active CAT and 99 +/- 10 microg/mL of active GLH protein using the new thin film approach whereas 500 microL test tube reactions produced 250 +/- 42 microg/mL and 72 +/- 7 microg/mL of active CAT and GLH respectively. Similarly, 500 microL cell-free synthesis reactions with the Cytomim system produced 481 +/- 38 microg/mL of active CAT and 109 +/- 15 microg/mL active GLH respectively in thin films compared to 29 +/- 7 microg/mL of active CAT and 5 +/- 2 microg/mL of active GLH protein in 500 microL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume.