healthmedicinet

Experimental design

General

We prepare buffers for SEC and LS experiments from ultrapure water having a resistivity
of 18 M? cm as provided by, for example, a Millipore filtration system (Merck Millipore).
All buffers are filtered through a 0.22-?m filter with the aid of a vacuum filtration
system before addition of detergent. This helps avoid both clogging of the SEC column
and scattering artefacts. In the course of an SEC run, the applied sample is diluted,
which simplifies data analysis because no intermolecular interactions have to be considered
38], 39]. However, precautions need to be taken to avoid dilution of the detergent below its
CMC, which would result in aggregation and precipitation of the protein. To ensure
the presence of detergent micelles during all stages of the experiment, we typically
use 2 mM and 5 mM micellar detergent in buffer and sample, respectively. Some co-solutes
and co-solvents such as denaturants increase the CMC 36]; therefore, buffer and sample detergent concentrations have to be adapted under denaturing
conditions. We determine the buffer’s refractive index and the refractive index increment,
d/dc, of the detergent in this buffer using a table-top refractometer. Because of the
wavelength dependence of the refractive index 40], it is important that the wavelength at which the buffer refractive index and d/dc values are obtained is close to that of the RI detector used in triple-detection
SEC 39]. However, differences within the wavelength range typically employed (i.e., 630–690 nm)
are negligible 30].

Concentration determination

For concentration determination based on the RI signal, knowledge of the refractive
index increment d/dc of all species of interest is essential. For proteins, literature values of 0.185–0.187 mL/g
28], 31] are often used. However, the d/dc value depends on a number of factors, including buffer composition, wavelength, and
temperature. The software SEDFIT (http://www.analyticalultracentrifugation.com/default.htm) contains a convenient tool providing a more accurate estimate of the d/dc values of proteins on the basis of their amino acid composition 41]. For some detergents, literature values are available 42], 43]; for more complex buffer systems or uncommon detergents, d/dc values can be determined as described in the procedure below. We typically use 10–15
different detergent concentrations distributed both above and below the detergent’s
CMC, with the exact values depending on the detergent used. For concentration determination
based on UV absorbance, the extinction coefficient has to be known. Extinction coefficients
for proteins can also be estimated from amino acid compositions with the aid of SEDFIT
44] or online tools such as ProtParam (http://web.expasy.org/protparam/). For some proteins, however, the extinction coefficient estimated from primary structure
differs significantly from the experimental value; in such cases, the extinction coefficient
has to be determined experimentally for correct mass calculation. All detergents mentioned
in this protocol do not absorb significantly in the UV range, but some detergents
do, including the popular Triton series. In these cases, the extinction coefficient
of the detergent has to be considered in data analysis. If no literature values are
available, the extinction coefficient can be determined from UV absorbance spectra
recorded at different detergent concentrations, analogously to the procedure for d/dc determination described in the protocol part below.

Sample

To ensure good separation by SEC, the sample volume should be as small as possible
and should not exceed the maximum volume provided by the manufacturer (typically,
0.5–2 % of the total column volume). We usually apply 50–100 ?L of sample to a column
of ~24 mL.

Calibration

When triple-detection SEC is used to determine molar masses, no system calibration
for relating molar mass to elution volume is needed. Nevertheless, running a sample
protein of known molar mass may be used to calibrate the system for peak broadening
effects, which is caused by offsets in elution time among different detectors, volume
differences among measurement cells of different detectors, and normalisation of all
LS signals to the 90° signal 30]. Peak-broadening and interdetector-delay corrections have to be performed once after
the system has been set up and after each change affecting detector sequence or interdetector
volumes (i.e., length or diameter of tubing or flow cells). The detector with the
broadest signal peak should be chosen as the reference detector for peak-broadening
correction. This is usually the RI detector, which also contributes the most to additional
interdetector signal broadening. Therefore, it is preferable to install the RI detector
as the last detector in line. Normalisation of all LS signals to the 90° signal has
to be performed for a newly installed LS detector or after exchanging the detector’s
flow cell. We also recommend normalisation of the LS detector each time a new solvent
is used that has a refractive index significantly different (~10 % deviation) from
that of the solvent for which the last normalisation was performed.

Solvent refractive index

Knowledge of the refractive index of the solvent not only is important for deciding
when new calibration is required but also is indispensable for data analysis. According
to Equation (3), the optical constant K is proportional to the square of the refractive index of the solvent ₀
. In the above examples, with a buffer refractive index of ~1.33 in the absence of
urea and ~1.38 in the presence of 6 M urea, disregarding this difference would result
in systematic errors in the values of the derived molar masses of the PDC and the
protein of ~8 % and, by error propagation, a larger error in the molar mass of the
detergent. Solvent refractive indices can be measured using a table-top refractometer
operating at a wavelength identical to or, at least, close to that of the online LS
and RI detectors. Additionally, some online RI detectors can determine not only the
differential RI but also the absolute RI of the solvent. The value of the solvent
refractive index can be corrected before data analysis by changing the experiment
configuration referring to the solvent settings in ASTRA.

Instruments

The protocol presented here was developed for an 1100/1200 Agilent HPLC system fitted
with a G1365B UV absorbance detector and extended by a miniDAWN TREOS light scattering
detector and an Optilab T-rEX refractive index detector, both from Wyatt Technology
(see Equipment). Measurements were controlled through the programs ChemStation and
ASTRA V from Agilent Technologies and Wyatt Technology, respectively. Note, however,
that PDC composition can be determined with the aid of any setup providing triple-detection
by UV absorbance, LS, and RI, and data analysis can be performed with any spreadsheet
program 45] following the procedure for basic LS analysis.

Materials

Bovine serum albumin (BSA) (Carl Roth, cat. no. 8076, purity 98 %)

Ethylenediamine-N,N,N’,N’-tetraacetic acid (EDTA) (Carl Roth, cat. no. 8040, purity 99 %)

KCl (Sigma, cat. no. 9333, purity 99.0 %)

LDAO (Sigma, cat. no. 40234; purity 99.0 %, molar mass 229.4 g/mol).

NaCl (VWR, cat. no. 27810, purity 99.5 %)

Tris buffer (Carl Roth, cat. no. 5429, purity 99.9 %)

Equipment

Buffer vacuum filtration system (Carl Roth, cat. no. XT09.1) equipped with filter
paper with 0.22-?m pores (Sartorius, cat. no. 18407-50-N).

Liquid-chromatography system; the system should include a pump providing a stable
flow rate, a degasser unit, and a UV absorbance detector; available, for example,
from Agilent Technologies, Bio-Rad Laboratories, Buck Scientific, GE Healthcare, Hitachi,
Jasco Analytical Instruments, Perkin Elmer, Shimadzu Scientific Instruments, Thermo
Scientific, or Waters Corporation.

LS detector; in principle, a detector employing a single scattering angle is sufficient
for proteins with molar masses up to ~10
⁶
 g/mol. For optimising the signal/noise ratio, a multi-angle LS detection system with
at least three detection angles is advantageous. LS detectors are available, for example,
from Malvern Instruments or Wyatt Technology.

Refractometer, for instance, Abbemat 500 from Anton Paar or other table-top refractometer
providing at least 5 digits.

RI detector, for example, OPTILAB T-rEX from Wyatt Technology.

Size-exclusion column, preferably with a wide separation range (e.g., 3–70 kg/mol
such as Superdex 75 10/300 GL from GE Healthcare) to ensure good separation of PDCs
and protein-free detergent micelles; available from Agilent Technologies, GE Healthcare,
Tosoh Bioscience, and others.

Table-top centrifuge; Eppendorf centrifuge 5340 R or other centrifuge allowing centrifugation
of samples with volumes of several millilitres.

Dialysis membrane with a molecular-weight cut-off (MWCO) of 12–14 kg/mol (e.g., Spectrum
Laboratories, cat. no. 132706)

5-mL QuixSep Micro Dialyzer capsules (Carl Roth, cat. no. 0671.1)

Screw cap with septum suitable for screw-top vial (Carl Roth, cat. no. LC13.1)

1.5-mL screw-top vial (Carl Roth; cat. no LC03.1)

ASTRA software, version V (Wyatt Technology) or other software for operating LS and
RI detector and recording data.

ChemStation software package (Agilent Technologies) or other software for operating
HPLC system and UV detector.

Spreadsheet program such as Microsoft Excel, Libre Office, Open Office, etc. if required
for more sophisticated analysis (see Results and Discussion).

Reagent setup

The reagent setup described below is applicable to system calibration with BSA and
characterisation of OmpLA/LDAO complexes as detailed in “Separation and characterisation
of PDCs and protein-free micelles” and shown in Fig. 3. Note that system calibration can be performed with any sample providing a well-resolved
peak. For other protein/detergent combinations, the reagent setup will have to be
adjusted to meet specific requirements. Use of buffer stocks is recommended for preparation
of buffered solutions containing different detergent concentrations.

Solution A: Prepare 1.5 L buffer solution containing 50 mM Tris, 100 mM KCl, 2 mM EDTA, adjust
the pH to 8.3 at RT, and filter the buffer through a 0.22-?m filter using a vacuum
filtration system.

Solution B: Prepare 500 mL buffer containing 50 mM Tris and 50 mM NaCl and adjust the pH to 7.4
at RT. Filter the buffer through a 0.22-?m filter using a vacuum filtration system.

From these buffer stock solutions, all required buffers are prepared by addition of
concentrated detergent or protein and detergent stocks.

For d/dc determination, a detergent concentration series is needed. To this end, a detergent
(here, LDAO) stock solution is used to minimise non-systematic errors.

Solution C (LDAO stock solution): Weigh 57.35 mg LDAO and dissolve it in 10 mL solution A to a final concentration
of 25 mM LDAO.

System calibration is performed using the BSA monomer peak in the absence of detergent.

BSA solution: Weigh 2 mg protein and dissolve it in 2 mL solution B. To remove aggregates and dust,
filter the protein solution through a 0.22-?m syringe filter or centrifuge it for
10 min at 20’000?g.

Because refolded OmpLA is usually stored in a buffer different from the one used in
the protocol below, buffer composition as well as protein and detergent concentrations
have to be adjusted prior to triple-detection SEC measurements.

OmpLA stock solution: Refolded OmpLA (typically at a concentration of 3–4 mg/mL) is dialysed to adjust
the detergent concentration to 5 mM and to complex traces of Ca
²⁺
that would induce dimerisation of OmpLA. To this end, proceed as follows:

Solution D (dialysis buffer): Weigh 573.5 mg LDAO and dissolve it in 500 mL solution A to a final concentration
of 5 mM LDAO.

Solution E (running buffer): Weigh 458.8 mg LDAO and dissolve it in 500 mL solution A to a final concentration
of 4 mM LDAO.

OmpLA sample: Dialyse at least 1 mL of the OmpLA stock solution with the aid of a Micro Dialyzer
capsule fitted with a dialysis membrane with a cut-off of 12–14 kg/mol against a 100-fold
excess volume of solution D overnight at RT. Determine the final protein concentration
and prepare a 500-?L sample of ~3 mg/mL OmpLA by dilution with solution D. To remove
aggregates and dust, centrifuge the sample for 10 min at 20’000?g and 10 °C.

Protocol

Determination of refractive index increment

1| From solutions A and C, prepare an LDAO dilution series at 15 different concentrations
with 1 mL of each concentration (e.g., 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1,
0.5, and 0 mM) (30 min).

2| Record the refractive index of each sample and determine d/dc using either one of the following two procedures (2-3 h).

(A)
Use of a standalone table-top refractometer. Use a table-top refractometer measuring at a wavelength close to that of the online
LS and RI detectors.

(i) Set the measurement temperature to 20 °C.

(ii) Wipe the measuring prism with ultrapure water to remove dust and contaminations.

(iii) Apply ~500 ?L sample, starting with pure solvent, and wait until the signal
has stabilised. It is of critical importance to wait until the displayed value has
stabilised; for denaturant-containing solutions, in particular, this may take some
time (i.e., 10–15 min).

(iv) Record the displayed RI value and continue with the next concentration.

(v) Correct the refractive index at each LDAO concentration by subtracting the refractive
index of solution A (₀
) to obtain ??=?n–₀.

(vi) Plot ? versus the detergent concentration in g/mL.

(vii) Perform a linear regression of the data using a spreadsheet program such as
MS Excel 45]. The slope provides the d/dc value in mL/g.

(B)
Use of the OPTILAB T-rEX in batch mode. At this point, it is necessary that the RI detector is connected to the HPLC pump
by a loop of 0.5–1 mL, bypassing any column or other detector, or to a syringe pump
providing a flow rate of 0.1–0.2 mL/min.

(i) Turn Purge on and flush the reference and measurement chambers with ultrapure water for ~10 min.

(ii) Turn Purge off and monitor the baseline. When the baseline signal is stable, zero the RI detector
by selecting Zero in the detector Main screen.

(iii) Prepare for data acquisition with the ASTRA software by opening a new experiment
using the template Batch (determine dn/dc).

(iv) Start data acquisition by running the experiment and introduce pure solvent as
first sample. Wait until the baseline signal is stable. Manual sample application
is preferred over use of an autosampler because the former is more flexible in terms
of volume applied and time allowed for signal stabilisation.

(v) During data collection in ASTRA, introduce the series of LDAO concentrations starting
with the lowest concentration. For each concentration, wait until the signal stabilises
and reaches a plateau before you apply the next sample.

(vi) After the sample with the highest LDAO concentration has been measured, inject
solution A (i.e., pure solvent) again to re-establish the baseline. When the baseline
is stable, stop data acquisition.

(vii) In the Baselines section, define a baseline by choosing the pure solvent blank. Left-click on the
left-hand side of the plot and drag a line from the blank plateau at the beginning
to the blank plateau at the end of the acquisition.

(viii) Define a peak for each plateau region, but do not select a peak for the blank
injection. In the table at the Refractive Index node, enter the concentration for each sample in the row entitled Concentration (g/mL).

(ix) Apply your settings by clicking OK; now, the d/dc value is provided in the Report section.

Determination of d/dc values as described above can be performed independently of all other steps.

System calibration (bench time 2 h, additional 4 h of automated run)

3| Equilibration of the system. Equilibrate the Superdex 75 10/300 column (dimensions 1.0 cm × 30 cm, total volume
~24 mL) and the detectors by running solution B at a flow rate of 0.5 mL/min for ~180 min.
Make sure that the RI detector is in purge mode to exchange the buffer in the reference
and measurement chambers.

4| Turn the UV lamp and the LS laser on and wait for at least 20 min before starting
data acquisition.

5| To ensure equilibration, verify that all three detectors (i.e., UV, LS, and RI)
show stable baseline signals towards the end of the equilibration period.

6| Disable Purge at the RI detector.

7| Configuration of ASTRA experimental settings. Use either one of the following two procedures.

(A) Create a new experiment in the ASTRA software using a template including data
collection of all three detectors (i.e., UV, LS, and RI) as well as the procedures
Interdetector Delay, Band Broadening, and Normalization.

(B) Create a new experiment template.

(i) Open a new experiment using the system template online.

(ii) Add a new detector by choosing Configuration???Edit Configuration in the Instruments section. In the Add line, choose Generic UV instrument.

(iii) Set the UV Response Factor to 1 (in general, you will find the right value for your detector in the detector
manual) and disable Band Broadening.

(iv) Add two Fluid connections and one Aux channel connection, representing real fluid connections and a data connection from the UV detector,
respectively. Proceed as when adding a new detector, but choose the Add???Browse command in the Connection section. For the first fluid connection, specify the Injector and the Generic UV instrument as Source and Destination Device, respectively. The second fluid connection must contain the Generic UV instrument and the miniDAWN TREOS as Source and Destination Device, respectively. For the Aux connection, the Generic UV instrument must be specified as Source Device and the LS detector as Destination Device. Additionally, specify the respective data port number for the Aux channel; in case
of doubt, check your computer’s settings for the correct port number.

(v) Change the LS device to miniDAWN TREOS if another LS device is specified.

(vi) Save the configuration as SEC_SLS_Calibration by choosing Save As Template.

8| Under Basic collection, set Duration to 60 min and Collection Interval to 0.125 s. Make sure to enable Trigger on Auto-Inject.

9| Save the experiment.

10| Create an acquisition method in ChemStation. From the Method menu, select Edit Entire Method. Enable Method Information and Instrument.

11| In the Method Information window, you can enter information about the intended use of the method (e.g., method
for system calibration), buffer composition (50 mM Tris, 50 mM NaCl, pH 7.4), SEC
column specification (Superdex 75 10/300), and flow rate (0.5 mL/min). This has no
influence on the experimental settings but helps in identifying the method later on.

12| Specify method settings in the respective dialogue as given in Table 2. The Stop Time must be the same as the Duration in ASTRA’s experimental settings; otherwise, data collection for the second and following
injections cannot be triggered by the injection signal. See Fig. 6 for an overview of the interplay between the ChemStation and ASTRA software packages.
Accept changes by clicking OK.

Table 2. Method settings in ChemStation for system calibration

Fig. 6. Scheme of control software. The main features of ChemStation and ASTRA and the menus
for controlling detectors, data acquisition, and data analysis are depicted together
with the interfaces between the two programs

13| Save your method as a new file with the name SEC_SLS_Calibration.

14| From the Sequence menu, select Sequence Table.

15| Fill the table with the information given in Table 3. The sample volume specified in the sequence table must be the same as the injection
volume in the method used. Otherwise, the sequence table setting overwrites the method
setting.

Table 3. Sequence table settings in ChemStation for system calibration

16| In the sequence menu, select Sequence Parameter.

17| Enter your name as operator and specify the prefix as SEC_SLS_Calibration, extended by the counter serving as file name, and the subdirectory in which your
data should be stored. If you omit this step, you will overwrite older files.

18| Save your sequence as SEC_SLS.

19| Transfer your BSA sample into a screw-top vial with septum, place it into the
sample tray, and make sure you put the tray back in place correctly.

20| Start the experiment in ASTRA. In the Experiment menu, select Run. Waiting for auto-injection signal will be displayed in the Collection plot. It is crucial to always start the sequence in ASTRA before starting the method in
ChemStation; otherwise, data collection cannot be triggered by the auto-injection
signal.

21| Start the run in ChemStation. Click the Start button to start the method. Select Yes when asked whether you want to save changes you made to the method.

22| After the run is completed, open your datasets in ASTRA. Choose the SEC_SLS_Calibration dataset and re-run it by selecting Run in the Manage option of the selected file.

23| Baseline correction. Go to Baselines and enable the LS 2 trace. Set your baseline by left-clicking in the blank region
at the beginning of the trace before (i.e., to the left of) the peak(s) and dragging
to the blank region at the end of the trace (i.e., to the right of the peak(s)). Click
Auto Baseline to transfer your selection to all other traces.

24| Visually inspect the baseline settings in all traces and change them if the displayed
baseline cuts peaks or proceeds below the background level of the signal. Click Apply to re-run the analysis sequence with new baseline settings.

25| Interdetector delay correction. Go to Interdetector Delay and select the main region of the BSA monomer peak at an elution time of ~19 min.
Click Determine Delays. The procedure provides the signal delays of the LS and RI detectors referenced to
the UV absorbance detector in millilitres.

26| Apply the value obtained for the UV–LS connection in the Volume row of the Fluid connection configuration for these two detectors. Click Apply.

27| To obtain the delay volume between LS and RI detectors, determine the difference
between the UV–LS and the UV–RI interdetector delays and insert this difference in
the Volume row of the Fluid connection between LS and RI detectors. Click Apply.

28| Band-broadening correction. Go to the Band Broadening procedure and select the BSA monomer peak; be careful not to choose a region affected
by other eluting species. Typically, one starts halfway up the leading edge until
the point where all detector signals have returned to the baseline. Choose the RI
detector as Reference Instrument and perform the fit by clicking Perform Fit.

29| Examine the fit. Without despiking or smoothing, the Instrumental Term, which represents the additional volume introduced by the measurement cell, should
not exceed 1 ?L. If despiking or smoothing has been performed before, this value might
be 1 ?L but should still be smaller than the Mixing Term, which accounts for the influence of the capillary. If the Instrumental Term is significantly larger and matching between the peaks is not good, repeat the fit
by using Reset and Apply seed values for Instrumental and Mixing Term.

30| If matching between the peaks is good, click OK to re-run the experiment with the new values.

31| Additionally, insert the determined Instrumental and Mixing Term in microlitres in the respective sections in the configuration sheets of the LS and
UV detectors.

32| Peak selection. Define the peak to be analysed around the maximum of the BSA monomer peak by left-clicking
into the chromatogram and dragging the borders around the region of interest. Make
sure that your selection does not contain any contamination from other eluting species,
particularly, the dimer fraction of BSA.

33| Normalisation. Select the Normalization procedure. Set Peak Name to Peak 1 and Radius to 3 nm. Normalization Type has to be standard and Radius Type rms. Choose Action Normalize.

34| Click Apply to use the new normalisation values.

35| Insert the normalisation values in the Normalization Coefficients section of the miniDAWN TREOS configuration in the row entitled New.

36| Save the experiment with all changes as template under SEC_SLS_online. Measurements used to determine PDC composition can be performed independently.

Measurement of OmpLA/LDAO complexes (bench time 45 min, additional 8 h of unattended
equilibration and data acquisition)

37| Determine the refractive index of solution E as described in step 2|.

38| Preparation of the system. Equilibrate the SEC column and the detectors as described in Steps 3|–6| but with
solution E, a flow rate of 0.4 mL/min, and for 210–240 min.

39| Configuration of ASTRA experimental template. Open a new experiment in ASTRA based on the template SEC_SLS_online (see step 36|).

40| Adjust Flow Rate to 0.4 mL/min, Duration to 80 min, and Collection Interval to 0.5 s. Make sure Trigger on Auto-Injection is enabled.

41| Additionally, the changes given in Table 4 regarding pump, sample, and buffer configuration must be applied.

Table 4. ASTRA settings for PDC measurements

42| Save the changed experiment template as SEC_SLS_PDC.

43| Generation of a sample set in ASTRA. For measurements comprising more than one injection or sample, it is convenient to
create a sample set instead of a single experiment in ASTRA. For this purpose, choose
Blank in the New section of the File menu.

44| Specify the experiment template and provide sample information. To this end, follow
either one of the following two procedures.

(A) Work with a general experiment template for all samples.

(i) Select the SEC_SLS_PDC template from step 42| as Default Experiment Template. This will be applied to all samples specified in the sample set. Sample information
is applied automatically.

(ii) Specify the position of your sample in the sampling tray in the field entitled
Well. Insert OMPLA_LDAO as Name under which data shall be saved and set the number of injections in the Inj field to 3. Check if the other settings are identical to the ones given in steps
40| and 41|.

(B) Work with separate experiment templates for each independent sample.

(i) Leave Default Experiment Template blank and choose the SEC_SLS_PDC template from step 42| for each sample in the Samples node individually. Thus, you can specify a different experiment template for each
sample.

(ii) Specify the position of your sample in the sampling tray in the field entitled
Well. Insert OMPLA_LDAO as the name under which data shall be saved and set the number of injections in the
Inj field to 3. Set the values of the other fields to the values given in steps 40| and
41| and select SEC_SLS_OMPLA as Template.

45| Save the sample set as OMPLA_LDAO.

46| Configuration of ChemStation settings. In the method SEC_SLS_Calibration (see step 13|), change Flow Rate, Stop Time, and Injection Volume to 0.4 mL/min, 80 min, and 50 ?L, respectively.

47| Save the method under the name SEC_SLS_PDC.

48| Continue as described in steps 14| to 18| but make sure that the number of injections
in the sequence table now equals 3.

49| Transfer the OmpLA sample into a screw-top vial with septum, place it into the
sample tray, and make sure to put back the tray correctly.

50| Start data acquisition as described in steps 20| and 21| but by choosing Run in the Sample Set instead of the Experiment menu.

Data analysis can be performed independently of data acquisition.

Data processing (10-30 min per experiment)

51| In the File menu, choose Open and select the experiment file OMPLA_LDAO(001)[OMPLA_LDAO] from step 50| to load it into ASTRA.

52| From the Manage menu of the experiment, choose Add To Experiment and select the Analysis procedure Protein conjugate analysis. Move it by drag-and-drop into the analysis sequence so it is placed before the Fit Mass or Radius procedure.

53| Add an additional report for the protein conjugate. To do so, go to Add To Experiment and open the Result folder. Choose Report and click OK. A new report entry is created.

54| Double-click the newly added Report (untitled1) item. Expand the Template node and select the system report template protein_conjugate_detailed from the Report folder in the ASTRA installation folder. In the field entitled Description, name the report as Report PDC Characterisation.

55| Set the baseline as described in steps 23| and 24|. The selected baseline range
should span almost the entire elution time window to correct for fluctuations.

56| Select peaks around the maximum of the OmpLA/LDAO and the LDAO peaks at ~9 mL
and ~11 mL, respectively. The detailed procedure is described in step 32|. Make sure
your selection does not include parts of other peaks.

57| The table below the chromatogram displays both selected peaks. In the fields at
the LS Analysis node, the settings given in Table 5 should be displayed. If one or more values differ from the above ones, change them
accordingly.

Table 5. Settings for LS analysis in ASTRA

58| Additionally, insert the detergent’s d/dc value determined in steps1| and 2| in the field entitled Modifier d/dc (mL/g) at the Extended Parameters node of both peaks. Click Apply to re-run the analysis sequence with the changed settings.

59| In the Protein Conjugate Analysis section, inspect the Zimm plot and the values determined for the mass of the PDC
and the masses of the protein and the detergent for each data point of the selected
peak range.

60| Go to the Report section and select Report PDC Characterisation. The latter summarises all information regarding the configuration of your system,
including detector settings, peak settings used for analysis, and analysis results
both as weight- and number-averaged values of the molar mass of the PDC, the protein,
and the detergent and as z-averaged values of the radius of the complexes in both peaks. As additional information,
the protein fraction moment and the polydispersity are provided.

61| Check, in particular, the analysis range, the settings for the solvent refractive
index d/dc, and the absorbance values for correctness. If necessary, change settings in the
appending section and apply the changes to the experiment.

62| Save the analysed experiment.

(A) Choose Save. The analysed experiment is saved.

(B) Choose Save As Template. Changes in the analysis procedure and sample settings are saved as template and
can be applied to the other two data files of the measurement. Name the template SEC_SLS_OMPLA. Changes described in steps 52| to 54| and 58| are superseded.

All data files can be analysed independently. The experiment can be saved after any
step, and analysis can be continued later.

63| Analyse the other two datasets as described above.

(A) If alternative 62|(A) was chosen above, open the datasets OMPLA_LDAO(002)[OMPLA_LDAO] and OMPLA_LDAO(003)[OMPLA_LDAO] and repeat steps 51| to 62|.

(B) If you chose alternative 62|(B), open the datasets OMPLA_LDAO(002)[OMPLA_LDAO] and OMPLA_LDAO(003)[OMPLA_LDAO], go to Apply Template in the Experiment menu, and select the template SEC_SLS_OMPLA from step 62|(B). Baseline correction and peak selection have to be performed as
described in steps 23| and 55|, respectively. Repeat steps 59| to 62| as described
above.

Troubleshooting advice

The most common problems encountered in triple-detection SEC and data analysis, the
steps where they most likely appear, and possible solutions are summarised in Table 6.

Table 6. Troubleshooting advice