| Frequently Asked Questions for XstalBio CD Technology General Information
(1) What is solid-state circular dichroism?
Circular dichroism (CD) is normally applied to study protein structure in solutions but rarely on suspensions due to artefacts arising from particle sedimentation, differential light scattering (DLS) and absorption flattening (AF). These effects can be minimised by optimising the experimental technique so that it is possible to acquire useful structural information from the spectra. The solid-state CD technique enables measurement and quantification of spectra from suspensions by (i) using a rotating cell holder that spins the quartz cell thereby keeping the particles in suspension, (ii) minimising DLS effects by optimising the optical configuration inside the sample chamber and (iii) introducing an semi-empirical correction for AF.
This technique is specifically aimed at probing the conformational integrity and stability of solid formulations of biopharmaceuticals such as antigens adsorbed onto adjuvants, proteins encapsulated within particles, insoluble protein aggregates, etc. However, it can be applied to any form of particulate provided it meets certain criteria (vide infra). The main advantage is the ability to analyse secondary and tertiary structure of protein particulates in situ, which hitherto has not been possible.
(2) Are there any limitations on particle size?
The answer is yes and no! We have successfully applied the technique to investigate proteins or enzymes adsorbed on particles ranging from adjuvants of sub-micron size to polymer particles as large as 1 mm in diameter. The limitation is usually not the particulate size but interference from absorption by the particle material in the spectral region of interest and the presence of non protein moieties such as nucleic acids.
(3) Can this technique applied for any type of protein particulate suspensions?
Yes. Solid-state CD has been successfully applied to investigate structural stability of enzymes or protein antigens adsorbed onto adjuvant, coated on microcrystals, suspensions of lyophilised powder in organic solvents, protein aggregates and protein encapsulated into membranes (cf. section III).
The main success criteria are (a) the additive or carrier in the formulation should exhibit minimal absorption in the region of interest viz. 190 nm to 310 nm; (b) only one protein should be present; and (c) the protein concentration in the suspension needs to be accurately known or estimated
(4) Could you let us know the concentration of protein that you need for CD analysis using your technique?
Structural analysis using CD requires protein concentration between the range 100 ug to 1.5 mg/ml. Outside this range the spectra is either noisy or the signal is saturated, which obstructs acquiring data at far UV region. Often biopharmaceutical formulation contains protein less than 100 ug/ml and in that case samples are concentrated by centrifuging prior to measurement or by using a cell of longer pathlength.
(5) How much sample do you require?
A minimum of 2 ml of protein suspension is required for CD analysis in both the far and near UV region. It is preferable to have 5 ml or more suspension (sample recoverable) to check the reproducibility of the spectra.
(6) What else do you need?
(a) Protein particulate suspension as stated in (3) and (4)
(b) Adjuvant free native protein solution
(c) Buffer used for sample preparation
(d) Protein free adjuvant suspension of similar w/w as the formulation
(e) Brief instructions about storage conditions and handling
(f) Information concerning protein MW, residue number and if possible total number of aromatic residues (Phe, Tyr and Trp).
(7) Is it possible to study protein aggregates?
Yes. This technique has been successfully applied to study the structure of both soluble and insoluble protein aggregates (cf. section III).
(8) How many mgs of an aggregated protein would you need?
From previous studies it has been found that a protein concentration between 0.5 mg to 1.5 mg/ml sufficient for CD analyses. Due to its turbid nature high concentration is not preferable but it is important to know the total amount of protein in the sample.
(9) What about sensitivity?
We have found it is possible to detect the presence of less than 5% insoluble aggregates from spectral changes in the near UV region. But it should be noted that CD is protein specific and the sensitivity will largely be influenced by the molecule under investigation.
(10) Do you have any information comparing a protein adsorbed onto different adjuvant, say, aluminium phosphate vs. aluminium hydroxide?
Yes. Please refer section III.
(11) Are there any limitation on protein loading?
Usually not (cf. section III) but it is important to know the total protein content (both adsorbed and free form) in suspension.
(12) Can I use rectangular sample cells for acquiring CD spectra using your technique?
No. The rotating cell holder is designed to use for cylindrical CD cells only.
Data analysis
(1) Can you elaborate on the absorption flattening correction?
AF arises due to flattening of the suspension CD spectra in relation to that of solution of the same chromophore. This effect is proportional to concentration and molar extinction coefficient of the absorbing species. A correction for this phenomenon can be introduced if the flattening coefficient (Q), which is the ratio of the absorbance of the suspension to that of solution, is known. Because measuring the absorption of the particulates is not possible we calculate this parameter from the apparent absorption of the sample measured as high tension voltage (HTV).
(2) What programmes do you apply to calculate secondary structural information from far UV CD?
We routinely use CDSSTR, SELCON3, CONTIN, VARSLC and K2D for structural analysis. Source codes for these programmes are available freely on the web but alternatively one could use DICHROWEB (http://public-1.cryst.bbk.ac.uk/cdweb/html/) an interactive online tool for CD spectra analysis.
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