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…
Tag: <span>characterization</span>
With the advent of whole cell-based therapeutics has come a growing standardized quality control and quality assurance of the processes employed for developing and manufacturing cellular materials, similar to the controls over traditional drugs and biologicals. Cellular therapeutics present unique process and quality control challenges due to the innate complexities of living cells, making it important to use whole cell assays to provide detailed pictures of the status and consistency of cell preparations that will be used to treat patients. This article illustrates how a cellular assay from Guava Technologies addresses these issues…
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…
Electron microscopy (EM) provides data for viral clearance studies, information on the presence and quantitation of endogenous retroviruses, and the detection and characterization of other potential contaminants. The technique is favored in this field because it is simple, reliable, and can give reliable quantitation for risk assessments. This article describes the main EM techniques currently used for testing cell cultures, culture supernatants, and bulk harvests. It also includes an in-depth description of a thin sectioning technique used to estimate virus titre in culture supernatants and bulk harvests…
The Gel Microdrop (GMD) Secretion Assay involves encapsulating cells within a biotinylated agarose matrix, followed by capture and detection of cell-secreted molecules with fluorescent markers. This technology differs from other encapsulation methods in that the small size of the microdrop (<50 ?m diameter) creates a defined microenvironment around the cell without impeding the fusion of nutrients, antibodies, or nucleic acid probes into the GMDs, or the diffusion of secreted products out of the GMDs. Large numbers of GMDs can be readily analyzed using flow cytometry, and sub-populations of rare or high-secreting cells, as small as 0.1%, can be detected and recovered in one day. This assay format is a rapid alternative to limited dilution cloning (LDC)...
As product development proceeds in the field of cellular therapies, adequate product characterization remains a challenge for both IND Sponsors and FDA/CBER. Cellular therapy products are not considered to be well-defined products, and therefore the control and characterization of each stage of the production process helps to ensure product safety and consistency. Product characterization of cellular products includes demonstration of safety, plus determination of identity, purity, potency, and product stability. Development of appropriate specifications for each of these parameters is necessary for lot release, and also provides an important database of knowledge for addressing regulatory issues, such as lot-to-lot consistency and potential issues with product comparability, should the manufacturing process, or other aspects of product development, change over time…
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…