The analysis of aggregates of therapeutic proteins is crucial in order to ensure efficacy and patient safety. Typically, the analysis is performed in the finished formulation to ensure that aggregates are not present. An important question is, however, what happens to therapeutic proteins, with regard to oligomerization and aggregation, after they have been administrated (i.e., in the blood). In this paper, the separation of whole blood, plasma, and serum is shown using asymmetric flow field-flow fractionation (AF4) with a minimum of sample pre-treatment. Furthermore, the analysis and size characterization of a fluorescent antibody in blood plasma using AF4 are demonstrated. The results show the suitability and strength of AF4 for blood analysis and open new important routes for the analysis and characterization of therapeutic proteins in the blood. . . . . . Keywords Whole blood Antibodies Plasma Serum Asymmetric flow field-flow fractionation (AF4) Fluorescence labelling Introduction Analyzing protein aggregates can be challenging due to the wide size range—from small oligomers to large sub-visible Protein-based drugs are a fast-growing sector within the phar- particles or even visible precipitation . The primary method maceutical industry and are nowadays used in the treatment of for determining size and amount of sub-visible aggregates is numerous diseases. Therapeutic proteins are complex with size-exclusion chromatography (SEC) in combination with respect to their large molecular size (cf. small-molecule drugs) suitable detectors. However, in recent years, SEC, although and their secondary and tertiary structures that must be main- being well established, has been questioned as it can give tained to function effectively as drug molecules. These intrin- erroneous estimates of the aggregate levels [3–5]. sic properties of proteins are, together with environmental Regulatory authorities, such as the US Food and Drug stress, part of the reasons that proteins are prone to aggregate Administration (FDA), now recommend conducting several in pharmaceutical processing, formulation, and during stor- complementary, orthogonal methods to verify the measure- age. The formation of protein aggregates can result in reduced ments. Analyses of protein aggregates in the finished formu- drug efficacy and/or immunogenicity, which compromises pa- lation are important but even more central would be to under- tient safety . Hence, control and elimination of protein ag- stand the aggregation potential after administration, including gregates are crucial part of the formulation of protein products. interactions with blood components . Analyzing the aggre- gate formation of protein therapeutics after drug administra- tion to the patient without tampering with the extracted sample has long been sought for and can only be achieved by analyz- Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-018-1127-2) contains supplementary ing blood either in vivo or ex vivo. material, which is available to authorized users. Blood plasma is a protein-rich solution in which white and red blood cells, as well as platelets, are suspended, and * Lars Nilsson serum is the remaining fluid after removal of the clot from email@example.com whole blood with principally the same composition as plas- 1 ma with the exception that the fibrinogens and clotting fac- SOLVE Research & Consultancy AB, Medicon Village, tors are absent. The protein concentration in plasma/serum 22381 Lund, Sweden 2 is approximately 60–80 mg/mL of which about 50–60% are Department of Food Technology, Engineering and Nutrition, Faculty albumins and 40% globulins (10–20% immunoglobulin G, of Engineering LTH, Lund University, 22100 Lund, Sweden Leeman M. et al. IgG) [7, 8]. The size distribution of blood components Materials and methods ranges from small molecules and ions (< 1 nm) to about 15 μm for white blood cells. Due to the complex nature Materials and the large size range of components in blood, such sam- ples are difficult to analyze and extensive sample pre- The salts used for carrier preparation (sodium chloride, di- treatment is generally included. Typical sample preparation sodium phosphate, potassium phosphate, potassium phos- steps involve centrifugation, extraction, and filtration [9, phate, potassium chloride, and sodium azide) were all analyt- 10]. This pre-treatment can, however, cause unintended ar- ical grade (Sigma-Aldrich, St Louis, MS, USA). The water tifacts such as unwanted loss of components, contamina- was purified on a Millipore Plus unit (Merck Millipore, tion, and protein aggregation. Therefore, it is highly desir- Darmstadt, Germany). The myoglobin, bovine serum albu- able to be able to analyze and characterize proteins in blood min, and immunoglobulin G reference samples were obtained with a minimization of change in conditions, i.e., maintain- from Sigma-Aldrich. The human serum (order number ing physiological pH and salinity and avoiding surfactants H4522) was obtained from Sigma-Aldrich. The plasma was and organic solvents. from rat and whole blood from mouse (kindly donated by In the FDA industry guidance on aggregate analysis, no Redoxis AB, Lund, Sweden). The FITC (fluorescein isothio- specific analytical method is recommended . However, cyanate)-labeled goat anti-human IgG antibody was obtained thorough assessment using qualified methods is requested from Capra Science Antibodies, Ängelholm, Sweden. The to eliminate the presence of aggregates and one referred to FITC loading was estimated to 6.2/antibody. the method is asymmetrical flow field-flow fractionation (AF4). AF4 is a method in which separation is achieved Method by applying an external field (cross flow) in a ribbon-like open channel without a stationary phase [11–13]. Due to The asymmetrical flow field-flow fractionation (AF4) analysis the absence of a stationary phase, several problems related was performed on an Eclipse II (Wyatt technology, Dernbach, to SEC are alleviated including minimization of non- Germany) in connection with a 1100-series LC-system specific protein adsorption, structural deformation at the consisting of an ERC-3415 vacuum degasser (ERC), a surface and high shear forces which may result in degra- G1311A pump, a G1329A auto sampler, a G1315A diode dation of analytes. Therefore, AF4 is a highly powerful array UV/VIS detector, and a G1321C fluorescence detector technique that is increasingly being used for the separation (Agilent Technologies, Santa Clara, CA, USA). A Dawn and characterization of biomacromolecules and pharma- Heleos II multi-angle light scattering (MALS) and Optilab t- ceutical molecules [14–16]. It has been proven to be a Rex differential refractive index (dRI) detector were connect- potential tool for studying biological structures such as ed on-line (Wyatt technology) after the channel. The UV de- proteins, antigens, and antibodies [17–21]. In previous tector was monitored at 250 and 280 nm, the fluorescence studies, field-flow fractionation (FFF) has been utilized detector was set to an excitation wavelength of 495 nm and for the separation and characterization of blood plasma monitoring the emission at 525 nm, and the MALS utilized a and lipoproteins [22–24]. Li et al. studied the possibility laser with 658 nm wavelength and measured scattered light of separating lipoproteins in blood plasma using symmet- with 17 detectors in the aqueous carrier liquid. The dRI detec- rical flow FFF, Mädorin et al. focused on the interaction tor operated at a wavelength of 658 nm. between a low molecular weight drug and plasma, and Data collection was performed by Astra 6.2 (Wyatt tech- quantitative analysis by the recovery of the drug after frac- nology). The asymmetrical flow field-flow fractionation chan- tionation by AF4, and the study by Park et al. study fo- nel was a Wyatt SC channel fitted with a 350-μmwide spacer. cused on comparing the plasma proteins (lipoproteins and For the analyses, a 10- or 100-kDa regenerated cellulose (RC) albumin) between patients and healthy persons using frit- membrane (Merck Millipore) was used. The carrier consisted inlet AF4. However, in all cases, the plasma samples were of phosphate-buffered saline (PBS), pH 7.4 with 3 mM sodi- prepared by some preparation methods (centrifugation and um azide (added to prevent microbial activity). Fractionation additives were added). was run at ambient temperature (approximately 22 °C) and all In this study, the purposes are to investigate the pos- experiments were repeated at least two times for sibility to utilize AF4 for high-resolution separation of reproducibility. proteins and other components in serum and plasma Performance testing of the AF4 separation as well as and furthermore to separate whole blood without sample checking the MALS-RI detection and molar mass determina- pre-treatment such as centrifugation and filtration. tion was done by analyzing solutions of myoglobin, bovine Furthermore, we investigate the feasibility of selectively serum albumin, and immunoglobulin G. For the MALS data separating and detecting a fluorescent antibody in the evaluation, the Zimm method was used and the dRI with a matrix. refractive index increment, dn/dc, of 0.185 mL/g for Proteins and antibodies in serum, plasma, and whole blood—size characterization using asymmetrical flow... concentration determination to obtain molecular weight (MW). The AF4 separation method used a detector flow rate of 0.50 mL/min, giving a system pressure of approximately 3– 4 bar depending on detector configuration. Before injection was started, the system was allowed to stabilize crossflows and pressures for 2 min. Injection flow rate was 0.2 mL/min, injection time 1 min, and the focusing time 2 min; crossflow during injection and focusing was the same as used during elution (2.0 mL/min). During elution, the crossflow rate, Q , was 2.0 mL/min, which was kept constant for 4 min after the onset of elution, thereafter decaying according to Eq. 1 − = Q ¼ Q ∙2 ð1Þ c c;0 where Q is the volumetric crossflow rate at the onset of the c,0 decay, t is the time, and t is the decay rate (4 min in the present study). When the crossflow rate reached 0.15 mL/ min, it was kept constant for the remainder of the separation. Injection volume was 10 μL for all tests. Both blood serum, blood plasma, and whole blood were diluted 100-fold with the carrier (PBS) prior to injection onto the AF4-channel and the size separation. Assuming that the protein content of the se- rum or plasma is approximately 70 mg/mL, this gives that the protein concentration of the sample going onto the channel is approximately 700 μg/mL and the sample load on the AF4 channel is approximately 7 μg. For the tests with the whole blood, a 100-kDa molecular weight cutoff (MWCO) mem- brane was used to reduce the protein load on the channel by removing proteins with lower MW (such as serum albumin), which can exit the size separation channel through the membrane. Results and discussion Analysis of blood serum Blood serum was diluted 100-fold with phosphate-buffered sa- line (PBS) (10 μL serum to which was added 990 μLPBS) Fig. 1 AF4-UV-MALS-dRI fractograms and molecular weight. UV trace prior to AF4 separation. The dilution was in order to reduce the at 250 nm (green), dRI trace (blue), and LS at 90° trace (red). a Analysis viscosity of the solution. The elution time of the serum compo- of blood serum. Injection volume was 10 μL of a 100× diluted serum nent was compared to the elution time obtained when analyzing sample, i.e., corresponding to 0.1 μLserum. b Analysis of plasma. Injection volume was 10 μL of a 100× diluted plasma sample, i.e., the proteins myoglobin, bovine serum albumin, and immuno- corresponding to 0.1 μLplasma. c Analysis of 17 kDa myoglobin (red globulin G using identical AF4 conditions (Fig. 1). trace), 67 kDa bovine serum albumin (blue trace), and ~ 150 kDa The serum sample shows a broad and multi-modal size immunoglobulin G (green trace). The BSA and IgG samples contain dimers distribution as detected by the UV, MALS, and dRI detectors. The peak with the maximum at 4.8 min in the serum sample in the MALS give the molar mass at peak apexes as 70 kDa (at Fig. 1a corresponds well with the elution time of bovine serum albumin monomer peak in Fig. 1c(similarinsize tohuman 4.8 min) and 158 kDa (at 6.8 min). Two of the most abundant proteins in blood serum are expected to be serum albumin and serum albumin), and the peak at 6.8 min corresponds well with the elution time obtained when analyzing immunoglobulin G IgG (based on literature values , often given as approximate- ly 40 and 10 mg/mL, respectively). From these comparisons (monomer peak in Fig. 1c). Further, the molar mass data from Leeman M. et al. and based on the molecular weight data, we conclude that with utilized, which was labeled with the fluorescent marker FITC. the very high likelihood, the component eluting at 4.8 min is For reference, the fluorescently labeled antibody was analyzed mainly serum albumin and the components eluting around in PBS at a concentration of 100 μg/mL, sample volume 10 μL, 6.8 min are mainly IgG. to allow detection by UV, MALS, and dRI (Fig. 2a). The Obviously, given the huge number of different proteins that are to be expected to be present in blood serum, it can be expected that a large number of (similarly sized) proteins and other serum components are co-eluting with serum albumin and IgG. However, serum albumin and IgG are the most abun- dant protein and protein classes to be expected in blood and is likely the most significant contributors to the detected peaks. The identity of the serum components eluted after IgG (8– 11 min in Fig. 1a) is unknown but serum is known to contain proteins larger than IgG such as alpha-2-macroglobulin (720 kDa, ~ 3 mg/mL in serum) and IgM (950 kDa, ~ 1 mg/ mL in serum) . Furthermore, there is the possibility that some of the detected components are smaller proteins that are aggregated or associated with other proteins, making their size larger (thereby eluting later) than the individual monomer protein would. Analysis of blood plasma Blood plasma was analyzed using the same settings as for the blood serum. The elution profile (Fig. 1b) is similar to that obtained for blood serum with components detected at similar elution time as serum albumin and immunoglobulin G. The most noticeable difference between the serum and plasma elu- tion profile is that there is a larger amount of components eluted in the elution time range from 3 to 6 min (higher intensity of the peak at 4–6 min in the plasma sample) in Fig. 1b. It may be speculated that this may be due to fibrinogen (340 kDa protein) which is expected to be present in plasma but should not be present in serum (removed by centrifugation when the blood has been clotted). This results show a higher resolution for sep- aration of blood plasma with FFF techniques, and more sensitive detection than the results from previous studies [22–24]. Fluorescently labeled antibody in PBS and plasma Both serum and plasma contain a wide variety of antibodies Fig. 2 AF4-UV-MALS-dRI-FL fractograms. Detected by UV at 250 nm (estimated as > 10 different antibodies . Of the immuno- (green trace), MALS (red trace), FL at 495/525 nm (orange trace), and globulin G, there are four classes (IgG1–IgG4), each class in dRI (blue trace). a Analysis of fluorescently labeled antibody in PBS. The turn consisting of a huge range of antibodies often differing injection volume was 10 μL of a 100 μg/mL solution (mass load = 1 μg). only very slightly in size and molar mass. To physically size b Analysis of fluorescently labeled antibody in blood plasma. The plasma was spiked for a concentration of 10 μg of fluorescently labeled antibody/ separate those is not feasible with AF4 due to insufficient res- mL plasma. The spiked plasma was then diluted 100× with PBS carrier olution. Thus, the antibodies eluting from the AF4 will elute as before analysis (sample volume was 10 μL, corresponding to a mass load a mixture of many antibodies. Therefore, to be able to detect of 1 ng of fluorescently labeled antibody on channel). c Analysis of and monitor one specific type of antibody, a selective detection fluorescently labeled antibody in PBS (green trace) and in blood plasma (red trace). The plasma was spiked for a concentration of 100 μgof is needed. Fluorescence detection can offer such a selective fluorescently labeled antibody/mL plasma. The spiked plasma was then detection if the antibody of interest is fluorescently labeled. diluted 100× with PBS carrier before analysis (sample volume was To investigate if a fluorescently labeled antibody could be mon- 10 μL, corresponding to a mass load of 10 ng of fluorescently labeled antibody on channel) itored in blood plasma, a goat antibody of the IgG type was Proteins and antibodies in serum, plasma, and whole blood—size characterization using asymmetrical flow... labeled antibody elutes at an elution time of 6.75 min similar to that of IgG in serum and plasma (6.8 min). It is noted that there is a shoulder on the peak (8–9 min) which is interpreted as the detection of (incompletely resolved) dimers. The fluorescently labeled antibody was spiked into the plas- ma for a concentration of 10 μg/mL plasma and analyzed. The fluorescence detector detects the labeled IgG (Fig. 2b) while the UV, MALS, and dRI detectors detect all the other components of the plasma. At the spiked concentration (10 μg/mL plasma), the amount of labeled IgG on the channel is 1 ng, which is much too low mass for the UV, MALS, or dRI detectors to detect. Comparing in more detail the elution profile of FICT-labeled IgG in PBS and in plasma reveals that there are differences as monitored by the fluorescence detector (Fig. 2c). The FICT- labeled IgG in PBS (green traces in Fig. 2c) has its apex at Fig. 3 Analysis of whole blood by AF4-UV-MALS-dRI. UV trace at 6.75 min and shows a tail on the main peak (at 8–9 min) 250 nm (green), dRI trace (blue), and LS at 90° trace (red). Injection volume was 10 μL of a 100× diluted blood sample, i.e., corresponding interpreted as a dimer (as noted above). In comparison, when to 0.1 μL whole blood the FICT-IgG is spiked into plasma (red trace in Fig. 2c), the peak shifts its apex to 6.9 min. Furthermore, the shoulder intensity (noise). Presumably, this is due to the elution of (dimer) is much more pronounced (higher intensity) when the large, very high MW components or particulates of whole sample is in plasma. The data is obtained on the same equipment, blood which is relatively few in number (such as entire blood analyzed next to each other, in duplicate, at two different occa- cells or fragments of them). sions, using the same conditions for both plasma and PBS. When Red blood cells have a size of approximately 0.5 × 8 μm the Ab-FICT is analyzed together with the plasma components, it and are outside the range of Brownian mode AF4 [27, 28]. As is evident that both the main peak shift, indicating that it is acting the intention of the analyses was not to separate these large as it was slightly larger during separation, and there is more components, it is not expected to have them properly separat- increase in the antibody having a size similar to that of an anti- ed. Rather, the objective was to test if it would be possible to body dimer. The conclusion is that interaction occurs between inject a blood sample with a minimum of sample pre- plasma components and the FICT-labeled IgG. Further investi- treatment and without removing any of the components. The gations are required to elucidate the nature of the interaction. only perturbations to the blood are that EDTA has been added (to prevent clotting) and that the sample was diluted with the Analysis of whole blood carrier (PBS) prior to injection (to reduce viscosity). No filter- ing, centrifugation, or addition of solvent modifiers has been Fresh blood from mouse was obtained in order to investigate done and the blood is analyzed in a PBS carrier which has an the capability of AF4 to separate an even more challenging ionic strength and pH very similar to blood. To the extent, if matrix than shown above (i.e., including blood cells). The whole blood can be analyzed by AF4, this test shows that it time between sampling from mouse and analysis was kept can be done; IgG and other proteins were eluted and the whole short (approximately 30 min) to minimize hemolysis. EDTA blood matrix did not cause the size separation to fail. was added to prevent clotting. The blood was diluted 100-fold However, the red blood cells due to their color could be with the carrier (PBS) immediately before the analysis and the followed visually as they were injected onto the channel and it blood was then directly injected onto the AF4-channel. For was noted that they stick to the membrane (see Electronic these analyses, a 100-kDa membrane was utilized to remove Supplementary Material (ESM) Fig. S1). After the analysis, lower MW proteins from the channel resulting in a lowering the channel was rinsed (pumping the PBS carrier through the of the protein load on the channel. Note that these separations channel at 0.5 mL/min, no crossflow) and after approximately were performed on a different membrane, giving different 1 h, no red traces could be visually observed in the channel. channel thicknesses and thereby different elution times com- This is interpreted as that either (a) the cells has been flushed pared to the above reported results. out or (b) the cells has been hemolysed and thereby lost their The fractogram (Fig. 3) shows a peak (at 6.0 min) that color (fragments perhaps being flushed out). Obviously, if coincides with that of IgG and a peak at 8–10 min. In contrast whole blood would be analyzed routinely, it would require to the data from serum and plasma, there is also a high abun- membrane type and chemistry, carrier composition, and rins- dance of larger sized components eluting in the time range ing protocols to be investigated and optimized. Furthermore, from 10 to 20 min of which the identity is unknown. The light the potential of clogging the flow lines, both in the scattering signal shows multiple narrow peaks with high Leeman M. et al. Open Access This article is distributed under the terms of the Creative autosampler and the detectors, is a concern, although no clog- Commons Attribution 4.0 International License (http:// ging was observed during these duplicate analyses. creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Conclusions The three most common types of blood samples (serum, plas- ma, and whole blood) were successfully size separated by References AF4. The ability to analyze the blood samples under close to native conditions (i.e., ionic strength, pH, no filtering, centri- 1. Frokjaer S, Otzen DE. Protein drug stability: a formulation chal- lenge. 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Analytical and Bioanalytical Chemistry – Springer Journals
Published: May 29, 2018
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