Can aberrometry provide rapid and reliable measures of subjective depth of focus following multifocal intraocular lens implantation?

Purpose: To determine whether the ‘through-focus’ aberrations of a multifocal and accommodative intraocular lens (IOL) implanted patient can be used to provide rapid and reliable measures of their subjective range of clear vision. Methods: Eyes that had been implanted with a concentric (n = 8), segmented (n = 10) or accommodating (n = 6) intraocular lenses (mean age 62.9 ± 8.9 years; range 46-79 years) for over a year underwent simultaneous monocular subjective (electronic logMAR test chart at 4m with letters randomised between presentations) and objective (Aston open-field aberrometer) defocus curve testing for levels of defocus between +1.50 to -5.00DS in -0.50DS steps, in a randomised order. Pupil size and ocular aberration (a combination of the patient’s and the defocus inducing lens aberrations) at each level of blur was measured by the aberrometer. Visual acuity was measured subjectively at each level of defocus to determine the traditional defocus curve. Objective acuity was predicted using image quality metrics. Results: The range of clear focus differed between the three IOL types (F=15.506, P=0.001) as well as between subjective and objective defocus curves (F=6.685, p=0.049). There was no statistically significant difference between subjective and objective defocus curves in the segmented or concentric ring MIOL group (P>0.05). However a difference was found between the two measures and the accommodating IOL group (P<0.001). Mean Delta logMAR (predicted minus measured logMAR) across all target vergences was -0.06 ± 0.19 logMAR. Predicted logMAR defocus curves for the multifocal IOLs did not show a near vision addition peak, unlike the subjective measurement of visual acuity. However, there was a strong positive correlation between measured and predicted logMAR for all three IOLs (Pearson’s correlation: P<0.001). Conclusions: Current subjective procedures are lengthy and do not enable important additional measures such as defocus curves under differently luminance or contrast levels to be assessed, which may limit our understanding of MIOL performance in real-world conditions. In general objective aberrometry measures correlated well with the subjective assessment indicating the relative robustness of this technique in evaluating post-operative success with segmented and concentric ring MIOL.

Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.