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…
Tag: <span>monoclonal antibody</span>
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…
Monoclonal antibodies (MAbs) destined for use in drug-specific assays must meet specific binding criteria, and therefore require much more in the way of development than many protein-specific antibodies. This article describes one way to facilitate the development of well-characterized, high specificity MAbs. Essentially the same techniques have also been used for producing MAbs that are cancer markers and MAbs that are specific for infectious agents. Monoclonal antibody development, as in other endeavors, requires clearly defined goals and an examination of proposed methods to attain them. This has been succinctly stated by the phrase “Start with the end in mind.” Unfortunately, the vast majority of hybridoma development could be more appropriately described by Yogi Berra, “If you don’t know where you are going, you will wind up somewhere else.” In most laboratories, fusions are carried out with the goal of reducing the amount of cell culture and handling required and using the minimum amount of screening. Little or no consideration is given to ensuring that single clones are subjected to the screening tests. Under these conditions, it is little wonder that dozens of fusions can be carried out, each with the same result — no specific antibody found…
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…
Foreign substances, such as disease-causing viruses and other infectious agents are recognized by the body’s immune system as invaders. Parts of the spectrum of our defenses are antibodies that bind these antigens and direct their destruction by other elements of the immune system. Antibodies have two very useful characteristics. First, they are extremely specific; that is, each antibody binds to one particular antigen. Second, the body retains the memory of encounters with antigens, enabling it to reproduce an antibody response to a later attack by that that same agent or disease. These properties make it possible to develop vaccines. It is the first trait of antibodies, their specificity, which makes monoclonal antibody (MAb) technology so valuable. Not only can antibodies be used therapeutically, to protect against disease, they also can help diagnose a wide variety of illnesses and can detect the presence of infectious agents (including biological threat agent). The use of MAbs specific for biological warfare agents in fielded assays requires maintaining a high-quality and economical supply of these reagents…
Bioreactor productivities are highly dependent on the process used to cultivate mammalian cells. These productivities directly affect the manufacturing plant capacity, and thereby the economics of production of monoclonal antibodies (MAbs). Historically, companies have chosen bioreactor process strategies that emphasize simplicity of scale-up at the expense of productivity, and conducted manufacturing using well-characterized and relatively straightforward batch processes. Such processes have successfully produced small or moderate quantities (ranging from ~100 g to ~ 1 kg per lot) of the desired antibody. Given the anticipated demand for large-scale quantities of MAbs (and the high stakes for the companies investing in these new biological entities), it is worthwhile to revisit these past selection strategies and see if — and under what conditions — they remain optimal today…
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…
Over 25 years have elapsed since Kohler and Milstein electrified the immunology community with their article describing the reliable preparation of monoclonal antibodies (MAbs) by fusing immune splencytes with immortalized myeloma cells. This discovery not only garnered the pair of scientists a Nobel Prize, but also led to the development of a technology which has yielded a number of important therapeutic, prophylactic, and diagnostic products for in vivo human use, and hundreds of in vitro diagnostic products. Some of these products proved to be significant in meeting previously unmet medical needs, and a few have been commercial successes. But the path, from Kohler and Milstein’s discovery to commercial products, was discontinuous and a bit bumpy, and the technology continues to evolve…
Glycosylation, a posttranslational modification that adds sugars to proteins, is required by many proteins to function properly. Glycosylation can modulate the biological activities of monoclonal antibodies (MAbs), including certain effector functions in the Fc region of IgG antibodies. Monoclonal antibodies produced at higher expression levels by mammalian cell culture may contain small amounts of nonglycosylated heavy chain (NGHC). Recent cell culture mini-reactor studies have shed light on the process parameters that most affect the occurrence of NGHC, and have greatly minimized NGHC levels in IgG MAb products…
The characterization of a batch cell culture process to produce a monoclonal antibody from a GS-NS0 mouse myeloma cell line is described. Productivity and cellular metabolism were monitored during scale-up to both characterize the process and aid in assessing cell culture stability. During fermentation scale-up studies, it was found that as culture generation number increased, productivity declined. In both flask and bioreactor cultures, declining production started abruptly at approximately generation 60. In this study, we assessed whether the decline in productivity was due to genetic instability of the cell line, which resulted in the generation of a non-producer sub-population, or a shift to a less productive state of cellular metabolism…