The last 30 years have seen rapid and dramatic developments in recombinant DNA technology and the related biological sciences. In 1972, Paul Berg’s group used restriction enzymes to cut DNA in half and then used ligases to stick the pieces of the DNA back together. By doing this, they produced the first recombinant DNA. Within a year, the first genetically engineered bacterium existed. A little more than ten years later, recombinant human insulin was approved for diabetic patients and became the first recombinant healthcare product. Before the end of the 1980s, the first gene therapy trial had occurred. Today, a large number of recombinant proteins are used as marketed drugs and even more are in clinical trials targeting a wide range of diseases…
Tag: <span>chromatography</span>
Short Monolithic Columns — An Enabling Technology for the Purification of Proteins, DNA, and Viruses
A growing number of separations’ scientists and process developers are looking beyond protein A sorbents for capture and initial purification of monoclonal antibodies. A variety of strategic and operational goals have prompted examination of alternative immunoglobulin-selective sorbents. Most broadly, many workers wish to eliminate design considerations associated with leached protein A. Also cited is a preference for sorbents that can withstand stringent cleanup using 1 M sodium hydroxide. In some applications, it is desirable to avoid the low-pH elution conditions typically employed with protein A sorbents — conditions that can foster aggregate formation. In still other cases, the target antibody may bind poorly to protein A. Finally, there may be interest in evaluation of immunoglobulin-selective sorbents less costly than protein A sorbents…
Cation exchange chromatography (CEX) is a versatile method for separation of proteins based on exploiting differences in positive electrostatic charges. In CEX, proteins are bound to the negatively charged stationary phase (cation exchangers) and then eluted using a salt gradient. Typically, the liquid-phase pH in CEX is lower than the isoelectric points (pI) of the proteins. CEX has been used to monitor various post-translational modifications such as glycosylation, deamidation, phosphorylation, truncation, oxidation, C-terminal and N-terminal clipping, and N-terminal cyclization. Some of these variants may exhibit different bioactivity. Therefore, it is important to characterize protein variants and monitor the stability of these variants throughout the process of drug discovery, development, and manufacture. Characterization of complex proteins such as antibodies, has traditionally been performed using slab gel-based techniques such as isoelectric focusing (IEF). This technique is qualitative and time consuming. It also generates large quantities of chemical waste from the staining process…
Over the past decade there has been a steady increase in the number of biotherapeutics requiring high doses and long term administration. Most notable among these are monoclonal antibodies (MAbs) and fusion proteins comprised partially of antibody molecules. Column chromatography is a commonly applied purification method for downstream processing of biotherapeutics, and there is considerable pressure to process much greater volumes at a faster rate. For recombinant proteins and MAbs, a variety of chromatographic methods are employed, including affinity, ion exchange, hydrophobic interaction, and to a lesser extent, immobilized metal affinity and gel filtration. Improving process control for chromatography operations is essential for biopharmaceutical manufacturers to process larger volumes and overcome capacity shortfalls. As the past ten years have seen increasing volumes of MAb-based drugs, there have been significant innovations to address growing productivity requirements. Dominant among these has been high throughput media capable of isolating product at faster rates than previously achievable…
lation components that stabilize the molecule in order to provide the desired product storage stability. Generally, an aqueous formulation is preferred; however, the instability of proteins, both physical (e.g. aggregation) and chemical (e.g. deamidation and oxidation), often necessitates the development of lyophilized formulations. In these formulations, selection of the appropriate stabilizing cryoprotectants, lyoprotectants, and bulking agents is critical. Accelerated stability studies are typically used to evaluate the effect of a single factor at a time in order to identify the optimum pH, buffer, and stabilizing excipients. This approach is limited in that many independent time-consuming experiments must be run, the results are obtained only at the evaluated set points, and additional experiments are required to assess potential interactions between the evaluated factors…
Recombinant monoclonal antibodies (rMAbs) are the predominant biotherapeutic protein under development today. FDA requires the structure characterization if rMAbs and other recombinant proteins to grant marketing approval. Characterizing such complex, inherently heterogeneous molecules is a significant analytical challenge that requires a broad array of physico-chemical tests. This article reports the use of reversed phase high-performance liquid chromatography (RP-HPLC) with on-line electrospray ionization mass spectrometry (ESI-MS) to rapidly determine the glycoform composition and the heavy chain C-terminal lysine heterogeneity of an intact rMAb. In addition, a novel multidimensional chromatographic platform was developed to investigate the two-dimensional, size exclusion chromatography (HPSEC) separation of the rMAb followed by RP-HPLC (HPSEC-RP-HPLC) with on-line ESI-MS analysis. Such analyses can characterize, identify, and confirm the structure of an intact rMAb…
Monoclonal antibodies constitute a significant percentage of the protein-based therapeutic molecules currently in clinical trials. The broad applicability and proven commercial success for this class of molecules suggest a larger future market potential. The current biopharmaceutical manufacturing capacity is widely anticipated to be a rate-limiting factor in the growth of the biotech sector. Because antibody therapeutics represent such a large part of this market, and because the therapeutic dosages of antibodies tend to be greater than most biopharmaceuticals, there is an immediate need for novel antibody manufacturing approaches that deliver significantly greater productivity…