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Rheological and molecular weight comparisons of approved hyaluronic acid products – Preliminary standards for establishing class III medical device equivalence Abstract: Hyaluronic acid (HA) of various molecular weights have been in use for the treatment of osteoarthritis (OA) knee pain for decades. Worldwide, these products are regulated as either as drugs or devices and in some countries as both. In the U.S. this class of products is regulated as Class III medical devices, which places specific regulatory requirements on developers of these materials under a Pre-Market Approval (PMA) process, typically requiring data from prospective randomized controlled clinical studies. In 1984 pharmaceutical manufacturers became able to file an Abbreviated New Drug Application (ANDA) for approval of a generic drug, thus establishing standards for demonstrating equivalence to an existing chemical entity. Recently, the first biosimilar, or “generic biologic”, was approved. Biosimilars are biological products that are approved by the FDA because they are ‘highly similar’ to a reference product, and have been shown to have no clinically meaningful differences from the reference product. For devices, Class II medical devices have a pathway for declaring equivalence to an existing product by filing a 510k application for FDA clearance. However, until recently no equivalent regulatory pathway was available to Class III devices. In this paper we consider the critical mechanical performance parameters for intra-articular hyaluronic products to demonstrate indistinguishable characteristics. Analogous to the aforementioned pathways that allow for a demonstration of equivalence, we examine these parameters for an existing, marketed device and compare molecular weight and rheological properties of multiple batches of a similar product. We propose that this establishes a scientific rationale for establishing Class III medical device equivalence. Keywords: Hyaluronic acid; rheology; molecular weight; medical device equivalence; generic; biosimilar

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1. Introduction Osteoarthritis (OA) afflicts a growing population of patients worldwide. As the most common form of chronic arthritis (up to 10% of the population), [1] the personal and societal impact of OA is increasing steadily due to aging demographics, and a more physically active patient population.[2] In the knee alone, 17% of the adult population over 40 has symptomatic OA, and by the year 2030 there are anticipated to be 3.5 million total knee arthroplasties a year.[2] There are currently no approved treatments that have clearly demonstrated their effectiveness in preventing, slowing or reversing the progression of OA, albeit there is some evidence that supports the use of intra-articular hyaluronans (HAs) in this role.[3,4] The standard of care for OA knee pain utilizes conservative non-pharmacologic (physical therapy, exercise, bracing, weight loss, and strengthening) and pharmacologic modalities (oral acetaminophen, NSAIDs, steroids, opioid and opioid-like, intra-articular injections of steroids or HAs), all of which address the symptoms of pain and loss in function, but not the root-cause. Intra-articular injections of steroids or HAs have been accepted in the U.S. as a standard treatment option for OA knee pain.[5] In this treatment modality high molecular weight hyaluronans are directly injected in to the joint space at concentrations close to that observed in the health joint with the primary aim of restoring the physical function of the synovial fluid. Recently, a comprehensive meta-analysis of pharmacologic treatments for OA knee pain concluded that intra-articular injections are amongst the most effective treatments available with effect sizes 2-3 times that of oral analgesics.[6] Furthermore, intra-articular HA effects were also reported to last longer than steroid injections and exhibited amongst the best safety profiles. In addition, the administration of HA has has also

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been reported to provide a significant cost savings over the standard of care [7],as well as potentially delaying progression to a full knee replacement by on average 2.7 years.[8] HAs were first approved in 1987 as drugs in Italy and Japan for the treatment of OA knee pain. In the EU, Hyalgan® (sodium hyaluronate) was initially approved as a drug, but subsequently the majority of HAs have been approved as devices in the EU under a CE mark process, which has a significantly lower regulatory barrier for market entry as compared to the U.S. Class III medical device approval pathway. Accordingly, there are over 50 CEcleared HAs presently being marketed worldwide which has generated significant pricing competition for these products. The first HA approval (Hyalgan®) occurred in the U.S. in 1997 as a Class III medical device.[1] The regulation of HAs as devices in the U.S. was based upon the presumption that the primary mechanism of action in alleviating pain was due to the viscoelastic or mechanical properties of HAs in solution, i.e. lubrication, resistance to shear, and cushioning for the joint, rather than biological activity. However, the literature demonstrates that HAs can uniquely exhibit both mechanical and biological properties. HAs also exhibit unique viscoelastic properties with highly non-Newtonian characteristics that allow the synovial fluid to provide the fluid dynamic properties of lubrication, as well as resistance to compression and shear forces.[9,10] Healthy synovial fluid is primarily composed of HA of high molecular weight, but during the development of OA the concentration of HA drops and the average polymer length (normal molecular weight of 3-4 million Daltons [11]) degrades, which markedly impacts the viscoelastic properties of the synovial fluid. HA therapy is intended to temporarily restore the rheology of the synovial fluid towards its healthy state. A full treatment course of HA in the U.S. can range between 1-5 injections at a product cost of $570 - $1,112 per treatment (based on wholesale acquisition cost, WAC).[12] As noted previously, outside the U.S. because of extensive competition these prices are often Page 3 of 18

50%-80% less. To date the major barrier to entry for competitively priced equivalent HAs in the U.S. has been regulatory approval. An equivalent cost-effective alternative to the branded devices, similar to generic drugs, would provide better patient access and reduce health care expenditures. In 1984 the Drug Price Competition and Patent Term Restoration Act [13], also known as the Hatch-Waxman Act, first created legislation allowing for a generic drug approval that is comparable to a brand or reference listed drug product in dosage form, strength, quality and performance characteristics, as well as intended use. This law allowed for increased competitive pricing, and in the 10 year period 2004 through 2013 generic drug use has resulted in nearly $1.5 trillion in savings to the healthcare system.[14] Similarly, the Patient Protection and Affordable Care Act of 2010 set up an equivalent pathway for approval of biosimilar devices and the first biosimilar to Neupogen® (filgrastim) was recently approved (Zarxio®, Sandoz Inc.).[15] While standards of equivalence have been established for chemical entities and biologics, similar standards of mechanical equivalence have not been established for medical devices. We have proposed that the unique mechanical properties of HAs must drive the primary basis of documenting ‘equivalence’ of these Class III medical devices. In this study, we have focused on highly sensitive analytical methodologies to precisely compare the molecular weight distribution and rheology of various samples of HA sourced from a number of different suppliers, both in the U.S. and abroad. Specifically, we have compared a HA device currently cleared for use in the U.S., Supartz/Supartz FX®, (exU.S. branded names are Artz® or Artzal®) with a HA available in Japan (Adant®). Adant was specifically formulated in Japan by Meiji Seika Pharma to be a generic drug equivalent of Supartz/Supartz FX/Artz/Artzal (hereafter referred to as Supartz/Supartz FX®), and as such was designed to be chemically and biologically similar in composition and dosage form to Supartz/Supartz FX. In the U.S. Adant is being developed under the brand name GenVisc

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850 (hereafter referred to as GenVisc 850), but in addition to the historical ex-U.S. data demonstrating chemical and biological equivalence, one must also establish device performance equivalence for a U.S. filing. The physical properties of molecular weight distribution and rheology of the HA solutions provide unique and highly sensitive predictors of mechanical equivalence measures. Since this material is regulated as a medical device, it is appropriate to focus on these characteristics, particularly rheology. We hypothesized that if the variation of the mechanical properties in a blinded testing between various batches of the same product are indistinguishable from the differences between the two products then an acceptable documentation of device equivalence would be demonstrated. 2.

Materials and Methods

2.1.

Materials.

A total of 17 blinded samples of HAs were obtained, each from a unique batch from one of the HA products. Ten different batches of Supartz/Supartz FX (Seikagaku Corportation, Japan), five sourced from the U.S. supplier and five obtained from ex-U.S. sources were studied. In addition, five different batches of the GenVisc 850 (Tedec-Meiji Farma, S.A., Spain), were also studied. All of these samples had reported molecular weight ranges of 615-1,120 kDa. Finally, two additional HAs of higher molecular weight were included as controls, Ostenil® (TRB Chemedica International SA, Switzerland – approved in EU) had a reported molecular weight range of 1,600 kDa,[16] and Euflexxa® (Ferring Pharmaceuticals Inc. - approved in U.S.) with reported molecular weight of 2,400–3,600 kDa.[17]. All samples were received as manufactured as individually packaged sterile syringes. Each syringe for all devices contained 10 mg/mL solutions of sodium hyaluronate in physiological saline, in the presence of Dibasic Sodium Phosphate Dodecahydrate and Sodium Dihydrogen Phosphate Dihydrate. The syringes were anonymized by removing all

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labelling before shipment to the testing facility. Blind was broken only after initial analysis. Two specimens were provided for each batch of material. One randomly selected specimen was tested using GPC-MALLS, and the second specimen was used for rheology testing. In two cases (Euflexxa and Ostenil) only one syringe was received and samples initially underwent rheological testing from the first portion of the single syringe, before the syringe was closed and sent for determination of molecular weight distribution on the remaining portion. These two samples therefore had been open for a few days before testing (although resealed), unlike the other samples where the material was tested immediately upon opening. Previous stability studies have demonstrated that these products are stabile for up to 3 years at room temperature. All samples were tested before their expiry dates. 3.

Methods

3.1.

Rheology

A TA Instruments AR-G2 shear rheometer was used for the testing following conventional methods with a 4 cm, 2° stainless steel cone, 60 m truncation. The temperature was 37 °C throughout testing. The selected geometry was chosen to provide a balance between sensitivity and sample volume. Small amplitude oscillatory shear (SAOS) relies on the material under test being in its linear viscoelastic regime to generate meaningful complex modulus values. An expired sample of Supartz/Supartz FX was run initially using a torque sweep to verify that the oscillatory tests were being run in the linear viscoelastic regime, and the strain values selected accordingly (data not shown). Small Amplitude Oscillatory Shear (SAOS) frequency sweep was performed (10% strain, 100 Hz to 0.01 Hz) followed by a steady shear sweep from 0.001 s-1 to 100 s-1 over 5 min, with a termination at 3 rad/s to prevent excessive shearing of solution. Finally the shear rate ramp was reversed from the maximum shear rate obtained in the previous step to 0.001 sPage 6 of 18

1

over 5 min to accurately report the shear properties of the solution (and remove potential

issues with gelation, if they were present). A thin layer of 5 mPa.s silicone oil (Sigma Aldrich PN 317667, batch 655498MJ) was dribbled on to the exposed fluid surface to prevent evaporation during testing. This approach has been used routinely before at our laboratory for evaporation-prone aqueous-based systems. A number of authors have examined the rheological properties of HA in solution.[18] All report a strongly shear thinning response in steady shear, with a pronounced complex modulus crossover frequency in dynamic oscillation that is consistent with a dominant relaxation time indicative of a high molecular weight polymer in solution as previously reported.[18-20] This relaxation time governs the rate dependence of the fluid and is strongly sensitive to the high molecular weight components that control the behaviour of the solution.[21] This dominant relaxation time will be reported here as one of the relevant dynamic shear parameters. Since the shear data previously reported indicates a strong non-Newtonian response, it is a little harder to define a simple parameter for comparative purposes, or define the parameter that is most likely to be related to device performance. A common approach is to fit a relevant rheological model to the data, and a suitable model for the low shear rate viscosity plateau transitioning to a high shear-rate plateau observed here would be the so-called Crossmodel.[22] This model is similar in form to the Carreau-Yasuda model used previously for HA.[9] Data are fit to this model to generate characteristic parameters for the fluids where it is assumed that there is a low shear rate Newtonian region with a transition region towards a further (lower viscosity) Newtonian region at higher shear rates (which is not visible in these data). The Cross-model has the form: (𝜂−𝜂∞ ) (𝜂0 −𝜂∞ )

1

= (1+(𝑘𝛾̇ )𝑚 )

(1)

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where 𝜂0 is the zero shear viscosity, 𝜂∞ is the “infinite” high shear rate viscosity, k is the “consistency” and m is the “rate index”. In the work here the model is fit to the high-to-low shear-rate data and is only fit above 0.2 rad.s-1. The most useful simple shear parameters reported here are from the Cross-model the zero shear viscosity and the consistency (which gives a measure of the onset of shear thinning for the fluid). The high shear plateau might also be of interest, but the shear rates used here were insufficiently high to reach this plateau. In all cases data is extracted using TA Instruments Rheology Advantage (V5.7). 3.2.

GPC-MALLS

Gel Permeation Chromatography (GPC), or more generally Size Exclusion Chromatography (SEC), is a ubiquitous technique for determining the molecular weight distribution of polymers and proteins by separating macromolecules based on their swollen size in solution. However, on its own the value of GPC is limited for determining the absolute molecular of complex polymers because it requires calibration against tightly controlled standards. Adding an additional detector to a SEC system, such as Multi-angle laser light scattering (MALLS), allows the system to directly probe the radius of gyration and molecular mass of the molecule eluted from the column using the Zimm method.[23] A Waters e2695 separations module was used with a Shodex OHPak SB-806M HQ (8.0mm x 300mm) column with two detectors, a Wyatt Optilab T-rEX Differential Refractive Index and a Wyatt DAWN HELEOS II MALLS Detector (ASTRA 5.3.4.20 software). This system is an 18-angle MALLS light scattering detector with a minimum detection angle at 22.5 º. Flow rate was 1.0 mL/min. of 0.1 M sodium nitrate with an injection volume of 100 μL. The column was held at 40 °C.

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4.

Results and Discussion

4.1.

Rheology

Rheological test data are shown in Figure 1, with collected SAOS and shear data shown in Figure 1 (a) and (b) respectively. Under blinded conditions only two samples were clearly distinguishable from the other samples. Once the blind was broken, these samples were determined to be Euflexxa and Ostenil. All of the samples demonstrated nonNewtonian behavior characteristic of HAs [19] analogous to that in synovial fluid.[24] The oscillatory data indicates a pronounced crossover from substantially viscous (G”>G’) at low frequencies to substantially elastic (G’>G”). The shear data are also consistent with previous studies and are characteristic of a shear thinning fluid, with a low-shear Newtonian (constant viscosity) plateau transitioning to a shear thinning region. In Figure 1 the data are presented as the average of the Supartz/Supartz FX and GenVisc 850 samples, with one standard deviation highlighted in a hatched region around the average curve. In both oscillatory and steady shear the GenVisc 850 and Supartz/Supartz FX groups appear to overlay across the frequency range and are indistinguishable, while the Ostenil and Euflexxa are clearly above the main grouping. The viscoelastic response of the Ostenil and Euflexxa exhibits a lower crossover frequency, consistent with higher molecular weights and a different shear response, with the Euflexxa in particular not exhibiting an obvious zero shear plateau at the shear rates investigated. Figure 1 NEAR HERE In more detail, the cross-over frequency for the Supartz/Supartz FX and GenVisc 850 groups (dark grey and black respectively in Figure 1) appear to be within one range of frequencies at approximately 80-150 rad/s. The Euflexxa and Ostenil samples both had lower crossover frequencies, consistent with their higher molecular weights at equivalent Page 9 of 18

concentrations. Zero shear viscosity also qualitatively appears to reflect similar trends, with the Supartz/Supartz FX and GenVisc 850 samples superimposed, and the Euflexxa, and Ostenil appearing different. Using the median of each of these parameters (see Figure 2), plus the 95% confidence interval around the median indicates that the Euflexxa and Ostenil are significantly different from the other samples in both shear viscosity and crossover frequency. Figure 2 NEAR HERE Since crossover frequency is essentially the inverse of the longest relaxation time in the system, which in turn is a consequence of the highest molecular weight present, these data imply that rheology can sensitively separate the known high molecular weight samples from all of the other samples (the Ostenil and Euflexxa controls fall outside of the 95% confidence interval of the Supartz/Supartz FX and GenVisc 850 samples), and that the GenVisc 850 and Supartz/Supartz FX samples are essentially equivalent in these performance-based rheological tests. Student’s t-Test (Microsoft Excel 2013, two-tailed heteroscedastic analysis) suggests that the Supartz/Supartz FX and GenVisc 850 sample groups are drawn from the same population for the cross-over frequency (p=0.99), zero shear viscosity (p=0.78) and k-parameter (p=0.12). The observed variation within batches of the same product were as great as the variability between the different products. The higher standard deviations visible in the GenVisc 850 specimens may be a result of the smaller sample size or the slightly higher standard deviation noted in the concentrations of these samples. Although all specimens were well within the specifications for the Supartz/Supartz FX samples (9-11 mg/mL), and were statistically the same, the average concentration for the GenVisc 850 samples was 10.1±0.21 mg/mL, compared to Supartz/Supartz FX with 10.05±0.08 mg/mL.

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The MALLS data for the systems studied here are shown in Figure 3, in a normalized form to allow better comparison of the centres of the molecular weight distributions. The individual molecular weight distributions are averaged in this figure for the Supartz/Supartz FX and GenVisc 850 groups. Visually, the Euflexxa and Ostenil are substantially different from the other samples, with much higher molecular weights than the other two sample groups. The Euflexxa in particular has a substantially higher molecular weight peak (average M.W. of 3,000 kDa). Likewise, although closer to the test samples, the Ostenil also exhibits a higher molecular weight distribution (reportedly with an average M.W. of 1,600 kDa), and a much broader distribution. Figure 3 HERE The calculated molecular weight parameters from the distributions provided in Figure 3, are compared in Figure 4. The average Mn, Mw and Mz are reported and are all statistically the same for each of the parameters between the GenVisc 850 and Supartz/Supartz FX groups (p=0.44, 0.40 and 0.06 respectively). Likewise, the Ostenil and Euflexxa are clearly distinguished, and are consistent with the observations made for the rheological data. When the samples were examined under blinded conditions, all 15 test samples were indistinguishable. As was noted in the rheology data, after breaking the blind there appears to be slightly greater variation within the GenVisc 850 group, than within the Supartz/Supartz FX group. It should be remembered of course that the Mw and Mn are weighted averages of the overall distribution, and although very useful as a qualitative tool for characterizing the distribution, they do not necessarily capture the complete story in a quantitative measure of M.W. distribution. By visually examining after breaking the blind and clustering the results by source, Figure 3, one seems to observe slightly a broader molecular weight distribution in the GenVisc 850. However, this slight difference between the samples was not statistically

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significant. Supartz/Supartz FX on average appears to have a minor secondary peak at ~400 kDa, whereas the GenVisc 850 may have a slight “bump” in the distribution at over 2 MDa. In both cases, when the raw chromatograms are reviewed (not shown) these additional “bumps” in the data appear to be the result of one specimen slightly skewing the average distribution. Otherwise, these two sets of data are qualitatively coincident, as implied by the molecular weight parameters. Figure 4 NEAR HERE 5.

Conclusions

It is clear from both the rheological and molecular weight data that the two techniques have sufficient sensitivity and reproducibility to distinguish very similar HA products, GenVisc 850 and Supartz/Supartz FX from the markedly different Ostenil and Euflexxa controls. The statistical analysis using Student’s t-test (two-tailed, heteroscedastic) of the rheological data yields probabilities for the crossover and shear viscosity with p > 0.05, suggesting that the sample groups are statistically indistinguishable. Indeed, prior to breaking the blind there was no data to suggest a difference between the individual specimens in the 15 samples of GenVisc 850 and Supartz/Supartz FX and the intra-product batch variation was as great as the inter-product batch variation. In data not shown, this was confirmed in that both products were indistinguishable based upon the approved release specifications. Likewise, it was only after breaking the blind in the GPC-MALLS data that there was an indication that the GenVisc 850 samples might have a slightly broader standard deviation than the Supartz/Supartz FX specimens, which is in part supported by slight differences in the coefficient of variation (0.05 versus 0.02 for the GenVisc 850 versus the Supartz/Supartz FX). This may simply be due to the number of HA samples tested for each group (5 vs. 10). Page 12 of 18

Similarly, there was a suggestion in the data that the GenVisc 850 samples may have a very slightly higher molecular weight than the Supartz/Supartz FX samples, which might be qualitatively supported by a review of the average molecular distributions in Figure 4, but again none of the average molecular weight parameters were shown to be statistically different. Conversely, the Supartz/Supartz FX samples seemed to exhibit several batches with a lower M.W. distribution. Even so these slight variations are not visible in the rheology “performance test” and as previously noted, none of these differences were statistically significant. In this paper we examined samples of GenVisc 850, sourced from Tedec-Meiji Farma, S.A., and similar samples of Supartz/Supartz FX, manufactured by Seikagaku Corporation (Japan) and sourced from U.S. or ex-U.S. suppliers. The primary aim of this study was to establish a basis for demonstrating biophysical similarity of HAs analogous to the equivalence testing required for drugs (generics) and biologics (biosimilars). The secondary aim of this paper was to use these same biophysical parameters to examine the variation from batch to batch of the same product to establish acceptable tolerances for measuring these parameters. The rheological and GPC-MALLS data are complementary, with both approaches separating the known controls reliably, and both yielding similar relationships between the GenVisc 850 and Supartz/Supartz FX groups. The rheological analysis is probing the viscoelasticity and shear sensitivity of the molecules, and is thus sensitive to both the dominant molecular weight of the solution and to molecular interactions between the polymers. It is presumably these properties that will dominate the initial mechanical performance of the device in vivo. Rheology is advantageous for the purposes of providing HA-based device similarity because it is a simple, ubiquitous technique that is sensitive to both molecular weights and inter-molecular interactions. Therefore it provides a method for

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analysis that allows for an equivalency testing for polymer interactions, at the molecular level.

Neither generic drugs nor biosimilars have similar simple yet sensitive analytical

methodologies that can identify of the chemical structure, conformation and interactions of the molecules in solution in a manner to predict the equivalent mechanical performance of device product to its reference or brand standard. While there are clear regulatory pathways with standards for the equivalence of generic drugs and now biosimilars, such standards have not been established by the FDA for the HAs that are regulated as Class III medical devices in the U.S. The HA class of products seem to be uniquely positioned, having both mechanical and biological characteristics, to adopt a hybrid approach of demonstrating product equivalence that may also predict clinical performance and assure their safety and efficacy without extensive clinical testing. The biological characteristics have not been discussed here although similar analysis of comparability in biological response for GenVisc 850 have been performed in other studies, particularly for the 1995 approval in Japan of the product as a generic drug of Supartz/Supartz FX. However, as a device the most important feature that predicts mechanical performance in vivo would seem to be the viscoelastic properties of the products. Coupled with a detailed analytical chemistry comparison this would appear to be a reasonable profile of comparative standards to establish equivalence for a generic class III medical device approval analogous to drugs and biologics. In the study provided here the two devices, both the reference product Supartz/Supartz FX, and GenVisc 850, were indistinguishable and can therefore be considered equivalent in terms of device performance. This testing standard should be considered in future equivalence testing of these types of products. One potential concern with the similarity comparison outlined in this paper, which focussed on the performance of the device, is the impact of trace constituents in the

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composition that these performance parameters might not be sensitive to. Although the data is not provided here in the interests of brevity, the materials under discussion here have been extensively compared under a range of tests. Primarily these materials were proven to be comparable under both the European Pharmacopoeia (Ph. Eur.) and the Japanese Pharmacopoeia (JP), which place strict requirements on the hyaluronic acid characteristics, impurity profile (nucleic acid and proteins), and quantification of contaminants. In addition, a detailed range of methodologies focused on molecular characteristics, secondary structure, chemical structural determinations, and impurity profile established the similarity of products, in particular in the non-hyaluronate portions, were also performed. Hence, although this paper has emphasized the indistinguishable characteristics of M.W. dispersity and rheology through performance characterization of these devices, there is also wide range of safety and structural comparisons that must be applied to allow clinical use. 6.

Acknowledgements

The materials supplied for this study were provided by OrthogenRx, Inc. and the work was performed under GLP through a contract with OrthogenRx, Inc. 7.

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8.

Figures

Figure 1: (a) Collected SAOS and (b) shear data for all samples. Solid lines are G’, dotted lines are G”. Euflexxa (light grey) and Ostenil (moderate grey) data represent single runs. All GenVisc 850 samples are shown as black, all other samples shown as dark grey. Hatched/grey regions represent one standard deviation on either side of GenVisc 850 and Supartz/Supartz FX. Figure 2: Comparison of selected rheology data (crossover frequency, zero shear viscosity and k parameter from cross model). Box is one standard deviation, whiskers are 2.56 standard deviations (99% confidence), (*) represents the 1 and 99% percentiles. Note vertical axis is plotted as a logarithm for viscosity and k-parameter. Figure 3: Averaged molecular weight distributions for all samples. Euflexxa (light grey), Ostenil (moderate grey), GenVisc 850 (black) and Supartz/Supartz FX (dark grey). Grey and hatched regions represent one standard deviation on either side of GenVisc 850 and Supartz/Supartz FX respectively. Normalized to peak maximum to emphasize peak centers. Figure 4: Box plot comparisons of molecular weight data. Box is one standard deviation, whiskers are 2.56 standard deviations (99% confidence) and stars are the 1 and 99% percentiles indicating that underlying data overlaps in all cases.

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Figure 1

Supartz Genvisc 850 Euflexxa

Viscosity [Pa.s]

Modulus (G',G") [Pa]

Ostenil

Supartz G' Supartz G" Genvisc 850 G' Genvisc 850 G" Ostenil G' Ostenil G" Euflexxa G' Euflexxa G"

Frequency [rad/s]

Shear Rate [1/s]

Figure 2

150

50

0

100

Cross-over frequency [rad/s]

Genvisc 850

Supartz

Ostenil

Euflexxa 10

1

Genvisc 850

Supartz

Ostenil

Euflexxa

Supartz

Ostenil

Euflexxa

1

0.1

Genvisc 850

Cross-model k-parameter

100

Cross-model viscosity [Pa.s]

Figure 3

1.0

Relative signal

0.8

0.6

0.4

Genvisc 850 0.2

Supartz Ostenil Euflexxa

0.0 1000000 Molecular Weight [g/mol]

Figure 4

Genvisc 850 (25%~75%)

6

Supartz (25%~75%) Ostenil (25%~75%) Euflexxa (25%~75%) Range within 1.5IQR

Mn

Mw

Mz

Euflexxa

Ostenil

Supartz

Genvisc 850

Euflexxa

Median Line

Ostenil

10

6

Supartz

1.5x10

6

Genvisc 850

2x10

6

Euflexxa

2.5x10

6

Ostenil

Molecular Weight [g/mol]

3x10

6

Supartz

3.5x10

6

Genvisc 850

4x10