Gamma irradiation is a well-established process for reducing or eliminating the bacterial and viral load in medical devices, biologics, and other products such as animal sera. This process can lead to alterations in both the materials being treated and the product containers in use. High-energy radiation produces ionization and excitation in materials, generating energy-rich ions which undergo dissociation, abstraction, and additional reactions in a sequence that may lead to chemical alterations. The resulting chemical stabilization process, which occurs during, immediately following, and occasionally days after irradiation, often leads to physical and chemical cross-linking or chain scission. The physical changes to materials can include embrittlement, discoloration, odor generation, stiffening, softening, and enhancement or changes in chemical structure. This paper discusses how and why irradiated polymeric materials, including those of biological origin, may change their structure and effectiveness during and after exposure to gamma irradiation, and the potential impact of these changes on serum during irradiation…
Category: <span>Biologics Production</span>
Medicago manufactures influenza vaccine virus-like particles (VLPs) in an unusual production platform consisting of Nicotiana benthamiana plants. During the in vitro adventitious agent test (AAT) of certain Medicago B strain influenza vaccine VLP test samples, positive hemagglutination of guinea pig red blood cells was observed on day 14, but not on day 28. The positive result in the assay was surprising because the production process uses no animal-derived raw materials and contains a viral inactivation step. Plant-associated viruses would not be expected to infect the mammalian cell-based assay. No cytopathic effects or hemadsorption of red blood cells was observed in these AATs. The positive hemagglutination was observed at 2–8°C, but not at 36–38 °C, and only in a few of the six detector cell lines used in the assay. Because this is quite an unusual pattern of responses for an AAT, Medicago and the contract testing lab, Eurofins Lancaster Laboratories (ELLI) investigated the positive responses thoroughly for the presence of an adventitious agent or an alternative explanation not involving a viral contaminant. Investigation results indicated that the hemagglutinating activity associated with the vaccine test sample itself was responsible for the positive hemagglutination response. The positive hemagglutination on day 14 of these AATs was deemed an assay artifact, and preventive actions were taken to prevent recurrence of this type of false positive response…
Bead matrices have been used in affinity chromatography to purify molecules in multiple applications. For instance, the hepatitis B surface antigen (HBsAg) is one of the molecules purified by this technique for human vaccine development programs. However, the use of monolithic supports have emerged as the advantageous choice for affinity chromatography based on convective mass transfer, a high number of channels, and low backpressures at high flow rates. For this reason, several experiments were conducted to determine the suitability of CB.Hep-1 monoclonal antibody (mAb) immunosorbent developed on carboxyimidazole (CDI)-monolithic supports (ligand concentrations: 0.5, 1.0, and 7.0 mg/mL) for HBsAg particle purification. Key results from this study show the highest amounts of HBsAg adsorbed (3059.31 ± 865.71 µg HBsAg/mL immunosorbent, n = 2), and HBsAg eluted (2884.50 ± 541.01 µg HBsAg/mL immunosorbent, n = 2), were estimated in the 1.0 mg/mL-CDI-CB. Hep-1 mAb monolithic support immunosorbents. In addition, the ligand leakage was always < 3 ng mAb/µg HBsAg (approved limit) in the 1.0 mg/ mL-CDI-CB.Hep-1 mAb immunosorbents. Experiments also evidenced the high purity and molecular homogeneity of purified HBsAg particles (< 95 %) across 20 purification cycles. Therefore, the ligand concentration could be reduced up to 1.0 mg/mL, which would enable a notable decrease in the mAb amount required for vaccine manufacturing, as compared to bead matrices (4.0 mg/mL). This study demonstrated that CDI-CB.Hep-1 mAb monolithic support immunosorbents are best suited for assessing the large-scale purification performance of HBsAg particles for human vaccine development programs at low ligand concentration and high flow rates...
Growth performance testing in cell culture is an effective approach to making serum suitability and purchase decisions. An independent commercial testing lab conducted two separate and sequential growth promotion studies to underscore the need for pre-purchase lot performance testing with: (1) FBS; and (2) FBS alternatives. Results from both studies are presented here to compare and contrast:
• FBS lots to each other
• FBS alternatives lots to each other
• FBS alternatives lots to FBS
FBS alternatives are included because they are often overlooked as a cost-effective substitute for FBS, providing, in many cases, equivalent performance. It is advisable to avoid preconceived notions concerning the quality or performance of a serum product without considering the culture system, culture conditions, and the subject cells, which can all have a material impact on its performance in cell culture.
Test – then decide…
The continued use of animal serum as an important component in biotechnology manufacturing processes has raised questions regarding both the reliability of geographic origin and possible adulteration of product. The International Serum Industry Association (ISIA) has implemented a traceability certification program designed to demonstrate traceability from slaughterhouse or abattoir to the end-user. This is based on an audit performed by an independent, approved third-party auditor according to an approved audit plan, using a detailed audit checklist. Recent advances have led to the development of a complementary testing program to determine geographic origin of material. The methodology described in this paper differentiates fetal bovine serum from newborn calf serum on the basis of biochemical composition…
Glycosylation drives protein quality and therapeutic benefits in protein-based therapies. Recently, there has been a push in the pharmaceutical industry to improve the consistency and quality of the glycan patterns on therapeutic proteins like monoclonal antibodies. Post-translational modification begins in the endoplasmic reticulum but is finished in the Golgi where more complex glycans are added. In this study, the addition of lipids via a novel mechanism provided by the medium supplement, Cell-Ess®, improves the consistency in glycan patterns so that they are more reproducible between product batches. The effect of media supplementation with Cell-Ess on the variation of glycan patterns was measured in two different media formulations across two separate experiments. Supplementation with Cell-Ess resulted in a statistically significant reduction in the variation of glycoforms when measured by the standard error of the mean. In addition, to improved consistency, there were increased higher glycoforms or galactosylation. There was also significantly more total galactosylation and significantly fewer lower glycoforms for antibodies produced by CHO cells supplemented with Cell-Ess. These data taken together suggest that the addition of lipids via Cell-Ess results in a more functional Golgi and an associated improvement of protein quality and consistency…
Because the Lambda MINIFOR bioreactor provides good mixing of cell culture, nutrients, and gases without any damaging hydrodynamic forces by using a bio-mimicking “fish-tail“ disc stirrer, it can be successfully applied for the cultivation of bacteria and yeast, insect, plant, and mammalian cells. However, reports on its application in mouse hybridoma cell culture using protein-free media is non-existent in the scientific literature. Therefore, this study describes preliminary findings of the Lambda MINIFOR bioreactor suitability in mouse hybridoma cell culture and antibody production using the SP2/O-Ag14-CB.Hep-1 mouse hybridoma cell and the PFHM-II protein-free medium as models. Results verified 2.45 × 106 viable cells/mL as the highest cell concentration, 86% as maximum cell viability, 0.0156/h as the exponential growth rate, 44 h as cell population doubling time, a stable phenotype measured by limiting dilution after 2.5 months, no antibiotic and antifoam requirements, 71.4% of IgG SDS-PAGE purity in the cell culture harvested supernatant, 38.68 ± 22.29 µg/mL, 39.23 ± 10.66 pg/cell/day, up to 99.5% of purity (sample measured by SDS-PAGE and SE-HPLC) after an IgG capture step based on protein A-Sepharose, a low pH incubation, and size-exclusion chromatography, no molecule aggregation, specificity for the CKTCTT epitope (located in the HBsAg “a” determinant), an IgG affinity constant equal to 1.11 × 1010 M-1, and < 78 pg mouse DNA/mg of IgG. In conclusion, this study corroborated a cumulative CB.Hep-1 mAb production of 1.77 g/15 days and validated the usefulness of the Lambda MINIFOR bioreactor in mouse hybridoma cell culture in protein-free media for research applications...
Plantibody purification is not as efficient as antibody purification from serum, ascites, or mammalian cell cultures. It is characterized by the application of inefficient plantibody solid-liquid extraction systems, low plantibody recovery, and short lifetimes of expensive chromatography matrices. To overcome it, several protocols of liquid-liquid aqueous two-phase extraction (ATPE) combined with affinity chromatography were previously studied to purify the CB.Hep-1 monoclonal antibody, which showed an unexpectedly high recovery. However, a study of ATPE combined with several affinity chromatography matrices to purify plantibodies has not been reported so far. Therefore, a combination of the best ATPE protocol with five specific affinity chromatography matrices to purify a plantibody for vaccine manufacturing is described in this study. Positive outcomes from plantibody recovery (%), specific activity (%), yield (mg purified IgG/L of leaf extract), and productivity (mg purified IgG/L of leaf extract/h) were achieved. Plantibody purity did not show statistical differences among all samples (> 97%, p < 0.05), and protein A leakage was thousands of times smaller than toxic protein A for non-human primates. In summary, the combination of ATPE (10% PEG 4000/15% K2PO4, pH 5.5) with two specific affinity resins were well-suited for large-scale plantibody purification from tobacco plant leaves...