Current and future approaches to ensure the viral safety of biopharmaceuticals.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
K Brorson
Publications and source records attributed to K Brorson.
An overview of quantitative PCR assays for biologicals: quality and safety evaluation.
Applications of real time quantitative PCR (Q-PCR) to the evaluation of biologicals quality and safety are discussed in the following areas: (i) endogenous retrovirus load quantification in production cell cultures, (ii) viral clearance studies, (iii) lot release testing, (iv) detection of specific viral contaminants for raw material screen and in-process control, (v) mycoplasma and bioburden detection, (vi) host cell DNA quantification, and (vii) genetic stability characterization of production cell lines.
Animals↗
Mutational analysis of avidity and fine specificity of anti-levan antibodies.
Using the polyfructose, bacterial levan, as a model polysaccharide, we analyzed how V regions affect binding in anti-polysaccharide mAbs. Previously, panels of mAb were constructed from bacterial levan-immunized BALB/c and CBA/Ca mice. The BALB/c mAb were mostly germline VHJ606:Vkappa11, and a subset contained presumed somatic mutations in the complementarity-determining regions (CDRs) that correlated with increases in avidity for the beta(2-->1) inulin linkage of levan. The CBA/Ca mAb were more heterogeneous in V gene usage, but a subset of inulin-nonreactive mAb were VHJ606:Vlambda and had VH sequence differences in the CDRs from the VHJ606 regions of the BALB/c mAb. In this report, VHJ606 Abs containing various combinations of specifically mutated H and L chains were produced by engineered transfectants and tested for inulin avidity and levan binding. Two presumed somatic mutations seen in CDRs of the BALB/c hybridomas were shown to directly cause marked increases in avidity for inulin (VH N53H, 9-fold; VL N53I, 20-fold; together, 46-fold) but not for beta(2-->6) levan. Exchange of either positions 50 or 53 in VH or the H3 loop between the BALB/c and CBA/Ca mAb resulted in either fine specificity shift or total loss of bacterial levan binding. Three-dimensional models of the V regions suggested that residues that affect binding to inulin alone are near the edge of the CDR surface, while residues involved with binding both forms of levan and affecting fine specificity are in the VH:VL junctional area.
Amino Acid Sequence↗
xid affects events leading to B cell cycle entry.
X-linked agammaglobulinemia patients and X-linked immunodeficient (xid) mice possess mutations in the Bruton's tyrosine kinase (Btk kinase) gene and display defects in B cell development and activation by sIg cross-linking. Btk is an early activation kinase in sIg-cross-linked B cells. xid does not ablate Btk protein kinase activity, and immediate signal transduction events, such as tyrosine phosphorylation, occur in sIg-activated xid B cells. These cells do not subsequently progress into cell division and have a high rate of apoptosis, which has been shown to correlate with an absence of sIg-mediated induction of the bcl-xL protein. To establish the point where Btk activity is critical for progression beyond immediate signaling, we examined early and late events in sIg-cross-linked xid B cells. Induction of proto-oncogenes and nuclear factors occurred normally in xid cells. However, induction of cyclins and increased GAPDH mRNA was not observed in xid cells. Degradation of the cyclin inhibitor p27Kip1 occurred normally in xid cells. After 24 h of culture with anti-mu, the remaining live, nonapoptotic xid cells were enlarged, viable, and primed for subsequent stimulation by LPS. Our data suggest that the Btk kinase is not essential for several G1 events and that the failure of sIg-activated xid B cells to enter cell cycle correlates with a defect of cyclin induction. Moreover, these data suggest that Btk is important not only for immediate events following B cell activation and control of apoptosis but also for subsequent events leading to cyclin activation.
Agammaglobulinaemia Tyrosine Kinase↗
Molecular regulation of the IL-2 gene: rheostatic control of the immune system.
The delivery of costimulation and the effects of the anergic state impinge on IL-2 production via different molecular mechanisms. The strongest experimental support at this stage suggests that CD28 signaling effects mRNA stability of several lymphokine genes including IL-2. While there may also be transcriptional effects of CD28 signals in human cells, controversy surrounding relevant TCR mimics must be addressed. In the case of clonal anergy, however, transcriptional non-responsiveness is evident when anergic cells are restimulated with TCR and costimulatory signals. This repression affects predominantly AP-1 activity. So far, the nature of the repression has not been identified.
Animals↗
Transactivation by AP-1 is a molecular target of T cell clonal anergy.
Anergy is a mechanism of T lymphocyte tolerance induced by antigen receptor stimulation in the absence of co-stimulation. Anergic T cells were shown to have a defect in antigen-induced transcription of the interleukin-2 gene. Analysis of the promoter indicated that the transcription factor AP-1 and its corresponding cis element were specifically down-regulated. Exposure of anergic T cells to interleukin-2 restored both antigen responsiveness and activity of the AP-1 element.
Animals↗
Analysis of MHC class I gene expression in adult bone marrow and fetal liver of the BALB/c mouse.
The BALB/c mouse has at least 29 class I genes encoded in the Qa, Tla, and Hmt (histocompatibility dependent on a maternally transmitted factor) regions of the MHC. The pattern of expression of these class Ib MHC genes is not well characterized, although some of their products such as the serologically detectable Qa-2 and TL Ag are expressed mainly in lymphoid and hematopoietic tissues. In this study, the expression pattern of BALB/c class I genes has been analyzed in adult bone marrow and fetal liver. cDNA libraries were synthesized from these tissues, and isolated class I cDNA clones were characterized by hybridization with oligonucleotide probes and by nucleotide sequence analysis. Of the 29 total class Ib genes, transcripts of five including Q6d, Q7d, T9c, T10c, and the 37 gene were isolated from the bone marrow cDNA library. Four of these can encode proteins; the sequence of the T10c gene demonstrates it is most likely a pseudogene. A non-overlapping set of three class Ib cDNA clones was obtained from the fetal liver, including T13c, the Thy 19.4 gene, and a previously uncharacterized class I gene provisionally designated as FL 57.2. Although the majority of H-2Dd cDNA clones that were analyzed lack introns, many of the class Ib cDNA clones contain intron sequences. This suggests that the expression of some of these genes may be regulated at the level of RNA splicing. The T13c gene encodes the thymus leukemia Ag in BALB/c mice. We have confirmed that the T13c gene is expressed in fetal liver by flow cytometric analysis of cells stained with anti-TL mAb.
Animals↗
Expression of the thymus leukemia antigen in mouse intestinal epithelium.
The Qa and Tla regions of the mouse major histocompatibility complex contain a series of genes encoding proteins with structural similarity to the class I transplantation antigens of the same complex. In contrast to the genes encoding the transplantation antigens, the Qa and Tla genes show very little polymorphism. Function(s) of the proteins encoded by the Qa and Tla loci remain an enigma. Recently, the protein products of the Qa and Tla loci, often referred to as class Ib major histocompatibility complex molecules, have been proposed to present antigen to gamma delta T cells. In mice, gamma delta T cells have been found concentrated in several epithelial barriers and in the skin; yet, expression of serologically detectable Tla antigens is believed restricted to thymocytes, activated T lymphocytes, and some T-cell leukemias. Here we report that luminal epithelial cells of the mouse small intestine express the thymus leukemia antigen (TLA). We also find that, unlike T cells in Peyer's patches, a significant fraction of intestinal epithelial lymphocytes also express TLA. RNA prepared from intestinal cells contains transcripts of the T18d gene, which encodes TLA. These data extend the known expression profile of TLA molecules to mature lymphocytes and to nonhematopoietic cells. These data also demonstrate the specific expression of TLA on antigen-presenting cells in a site enriched for T cells that express gamma delta T-cell antigen receptor.
Animals↗
Genetic and molecular mapping of the Hmt region of mouse.
We have mapped a new region of the mouse major histocompatibility complex (MHC) that contains the nuclear gene, Hmt, for the maternally transmitted antigen, Mta. The Hmt region of chromosome 17 lies between a recombinational breakpoint distal to Tla and another proximal to Tpx-1, thus including Pgk-2. A novel MHC class I gene fragment, R4B2, was cloned and mapped to this region as was another new class I gene, Thy19.4. Both lie proximal to Pgk-2, within the distal inversion in t-haplotypes. The presence of several other MHC class I genes in the Hmt region is predicted from analysis of the recombinants that define the region.
Amino Acid Sequence↗
The isolated major histocompatibility complex class I alpha3 domain binds beta2m and CD8alphaalpha dimers.
The MHC class I molecule plays a crucial role in cytotoxic lymphocyte function. The heavy chain of the MHC class I molecule can form many non-covalent interactions with other molecules on multiple domains and surfaces. We have generated an isolated alpha3 domain of a murine MHC class I molecule and evaluated the contribution of this domain to binding with the MHC class I light chain, beta2m, and CD8. The alpha3 domain binds beta2m at a thousand-fold higher concentration than the whole MHC, and binds CD8alphaalpha with a dependence on the alpha3 CD loop. Our results are relevant for models of MHC folding and CD8-MHC function. The study of individual domains of complex molecules is an important strategy for understanding their dynamic structure and function.
Binding Sites↗
Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing.
Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/mL) were substantially above the detection limit of the TM-PERT assay ( approximately 10(6) pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.
Animals↗