Characterization and implications of host-cell protein aggregates in biopharmaceutical processing

In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.     

Depletion of polyamines prevents the neurotrophic activity of the GABA-agonist THIP in cultured rat cerebellar granule cells

Effects of polyamine depletion by -difluoromethylornithine (DFMO) were studied on the GABA-agonist mediated enhancement of the morphological development of cultured rat cerebellar granule cells. An increase in the number of neurite extending cells and in the cytoplasmic density of organelles relevant for protein synthesis was observed upon culturing in the presence of 4,5,6,7-tetrahydro-isoxazolo[5,4-c]pyridin-3-ol (THIP) for 4 days. The intracellular concentrations of putrescine, spermadine, and spermine in these cultures were similar to the concentrations of the polyamines observed in cultures grown in a plain culture medium for 1, 2, 3 or 4 days, respectively. Upon culturing in the simultaneous presence of THIP and DFMO, the concentrations of putrescine and spermadine were reduced to less than 20% of the levels in the controls. This depletion was associated with a severely impaired morphological development of the granule cell cultures. Thus, the number of neurite extending cells was reduced to 50% of the number in the control cultures upon culturing in the presence of DFMO alone or in combination with THIP. Moreover, the THIP mediated increase in the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus and different types of vesicles was prevented by the exposure to DFMO.     

An XRF study of trace elements accumulation in kidneys of tumor-bearing mice after treatment withcis-DDP with and without selenite and selenocistamine

The effect ofcis-DDP treatment with and without selenite and selenocistamine was studied on kidneys of tumor-bearing mice. The amounts ofcis-DDP, selenite, and selenocistamine injected were chosen so as to be compatible with the treatment of humans. The animals were sacrificed at 7, 14, and 28 d after treatment. The kidneys were removed and subjected to trace element analysis by a novel X-ray fluorescence (XRF) method and for pathological assessment. The results show that following treatment withcis-DDP, K, Fe, Cu, and Zn reach a maximum level after 7 d; K, Fe, and Cu levels were significantly reduced by the addition of selenite. The level of Zn was reduced only in groups treated with selenite whereas that of K and Cu was reduced also in groups treated with selenocistamine with and withoutcis-DDP. The greatest increase in Pt and Se levels was reached 1 wk after injection withcis-DDP, with and without selenocompounds, and in the case of Pt was partly reduced by addition of selenite. Se returned to control values 2 wk after injection, although Pt was still high in all groups 2 and 4 wk after injection. The results corroborate the findings of our previous studies. The effect of selenocistamine incis-DDP treated mice was partly insufficient. The pathological examination of the kidneys did not show any differences in the effect of various additives during the study.     

The effect of dietary fat on the activity,content, rates of synthesis,and degradation and translation of messenger RNA coding for malic enzyme in mouse liver

The specific activity (units activity/mg cytosolic protein) of malic enzyme was found to be three-fold higher in the livers of mice fed a semipurified diet containing 50% ($\text{w}\text{w}$) glucose and 15% ($\text{w}\text{w}$) saturated and monounsaturated but no polyunsaturated fat (hydrogenated cottonseed oil) over an 11-day period than in the livers of mice fed a standard laboratory mouse chow (Purina) diet. In contrast, when other lab chow-fed mice were fed an isocaloric diet containing 15% ($\text{w}\text{w}$) polyunsaturated fat (corn oil), no change in the specific activity of malic enzyme occurred over a similar period of time. Rocket immunoelectrophoresis performed on cytosols from both dietary groups demonstrated that the livers of mice consuming the hydrogenated cottonseed oil diet contained approximately three times more malic enzyme protein than did the livers from the corn oil-fed animals. In mice pulse-labeled with l-[4,5-³H]leucine, the rate of hepatic malic enzyme synthesis (relative to that for total protein) was approximately twofold greater in the hydrogenated cottonseed oil-fed mice than in their corn oil-fed counterparts whereas the rate of hepatic malic enzyme degradation was similar for both groups. Immunotitration of liver malic enzyme from hydrogenated cottonseed oil-fed and corn oil-fed mice revealed identical equivalence points, demonstrating that the catalytic efficiency of mouse liver malic enzyme had not been affected by the type of dietary fat administered. When total liver RNA, isolated from the hydrogenated cottonseed oil- and the corn oil-fed animals, was translated in cell-free translation systems (wheat germ extract and reticulocyte lysate) we found that both dietary treatments had resulted in an increase in the activity of malic enzyme messenger RNA. Furthermore, there were no significant differences between the two dietary groups in this regard. These results suggest that hepatic malic enzyme specific activity in high-carbohydrate polyunsaturated fat-fed mice is regulated principally by dietary-induced changes in the rate of enzyme synthesis and not by the activity of messenger RNA coding for the enzyme.     

Transport and metabolism of bacilysin and other peptides by suspensions of Staphylococcus aureus

L-Alanyl-L-tyrosine and glycyl-L-phenylalanine labelled with 14C competed with each other and with the dipeptide antibiotic bacilysin for transport into Staphylococcus aureus NCTC 6571 in a medium which did not support growth. They also competed with other dipeptides and several tripeptides. The fast initial transport ofthe two labelled peptides appeared to show Michaelis-Menten kinetics. Neither was transported into a bacilysin-resistant mutant of S. aureus NCTC 6571, although tyrosine was taken up by the mutant as readily as it was by the parent strain. Uptake of alanyltyrosine or glycylphenylalanine was followed by rapid hydrolysis of the peptide and the excretion of tyrosine or phenylalanine. Glycine liberated from glycylphenylalanine was partly degraded and partly incorporated into the bacterial wall. The behaviour of these dipeptides paralleled the inactivation of bacilysin by suspensions of S. aureus and the appearance of its C-terminal amino acid, anticapsin, in the extracellular fluid.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

Anatomical studies of Ustilago galls on Hordeum vulgare L.

The present paper deals with the histopathological studies of galls of Ustilago hordei (Pers.) on Hordeum vulgare L. The gall formation in the present case is the result of both hyperplastic and hypertrophic activities of the infected host cells. Also the gall formation by U. hordei on. H. vulgare is being reported for the first time through present. communication.     

Retention of the differentiated state by larvalXenopus liver cells in primary culture

Summary Electron microscopic analysis of primary cultures derived from larvalXenopus liver has shown that these cells, although they form only two-dimensional aggregates, retain and presumably also develop structural characteristics typical of liver parenchyma cells, such as bile canaliculi with microvilli and epithelial junctional complexes. As judged from structural criteria, primary cultures contain 80–90% hepatocytes. In contrast to the intact tissue, primary cultures showed excessive development of microfilaments, however.Incorporation of labeled amino acids has revealed further that the capacity for protein synthesis is maintained in culture and that synthesis of liverspecific protein albumin is maintained in vitro, even in liver cultures derived from thyrostatic tadpoles. This latter result suggests that initiation of albumin synthesis in the larval liver is probably not dependent upon thyroid hormones but rather reflects the protodifferentiated state of this tissue.     

Stimulation of microsomal prostaglandin synthesis by a vasoactive material isolated from blood plasma

Stimulation of prostaglandin synthesis by a material with coronary vasoconstrictor activity extracted from blood plasma was examined. The vasoactive material decreased the Km for arachidonate in the overall synthesis of prostaglandins by rabbit renal microsomal preparations but did not change Vmax. Increases in prostaglandin synthesis caused by the vasoactive material and L-tryptophan or L-epinephrine were additive or synergistic, whereas increases produced by the vasoactive material and hemin or hemoglobin were not. However, hemin and hemoglobin stimulated synthesis of all prostaglandins equally whereas the active material increased the synthesis of prostaglandin F_2α at the expense of other prostaglandins, both in the presence and absence of heme compounds. The increase in prostaglandin F_2α with respect to the other prostaglandins occurred in the presence of reduced glutathione. The vasoactive material attenuated inhibition of prostaglandin synthesis induced by indomethacin or aspirin but not that produced by 5,8,11,14-eicosatetraynoic acid. The interaction of the vasoactive material and indomethacin was competitive whereas hemin attenuated the effects of only low concentrations of indomethacin. Epinephrine enhanced indomethacin inhibition. These data indicate that mode of action of the vasoactive material in prostaglandin synthesis is unlike that of glutathione, aromatic amines, or heme containing compounds.     

Adventitious staminate flower formation in gibberellin treated gynoecious cucumber plants

Single gibberellin (A₄₊₇) treatments induced the appearanceof staminate floral buds in several consecutive nodes on themain stem of genetically female cucumber (Cucumis sativus L.).The staminate buds appeared next to pistillate buds which showedvarious degrees of degeneration. Similarly, repeated GA treatmentsinduced the appearance of staminate flowers in otherwise strictlyhermaphrodite plants, next to bisexual flowers. However, thebisexual buds, unlike the pistillate ones, did not show anydeleterious effects of the GA treatment. Therefore, it is inferredthat the hormonally induced staminate buds did not develop bysexual reversion of would-be pistillate or bisexual buds, butrather, represent adventitious buds which, in normally grownfemale or hermaphrodite plants, never develop. It thus seemsthat predetermined pistillate or bisexual buds do not changeinto staminate ones, while change in the reverse direction hasbeen demonstrated in the past (at least for the gynoecious ones). The effectiveness of the GA treatment in the gynoecious plantsshowed an acropetal gradient both within the affected region,as well as along the main stem. Autoradiographic histologicalexaminations showed that the course of development of the inducedstaminate floral bud did not differ from that of normally developingbuds. (Received June 16, 1977; )